Abstract 4026: HPV16-positive and HPV16-negative head and neck squamous cell carcinomas display different patterns of expression of genes involved in the control of growth and EMT.

Up to 60% of oropharyngeal cancers and 25% of head and neck squamous cell carcinomas (HNSCC) are high-risk human papillomavirus (HPV) positive, primarily HPV16. Differences in risk factors, age of presentation and clinical behavior of these tumors indicate that HPV+ and HPV- tumors develop with different molecular mechanisms and are biologically distinct. Based upon a gene expression profile comparison of HPV- and HPV+ HNSCC specimens, obtained from the Medical University of South Carolina ENT/Head and Neck Surgery clinic, we identified 384 candidate genes differentially expressed (125 genes up-regulated and 223 down-regulated) in HPV+ in comparison with HPV- tumors. GO analysis of the microarray data indicated that gene expression changes in HPV+ tumors affected primarily proliferation and cell cycle control, while HPV- tumors showed changes in genes involved in EMT, cell motility, and angiogenesis. We selected five genes (c-MET, TP53, TGF-beta2, BRCA1 and SIX1) for further analysis in a set of 44 tumor and four normal (tonsil and uvula) samples. Seventeen tumor samples were from African American (AA) patients, and 27 from European Americans (EA). The prevalence of HPV infection was 30% in samples from AA patients, and 65% in those from EA patients. Most HPV+ tumor samples (20 of 24) were positive for HPV16 and a subset of the HPV16+ tumor samples (55%) expressed HPV16 E7, as documented by real-time RT/PCR using tumor-derived RNA. The prevalence of active HPV infections (where E7 was expressed) was 17% in AA and 50% in EA patients. These tumor samples were utilized to explore the expression of the selected genes by real-time RT/PCR. Data were normalized to normal human keratinocytes based on three reference genes derived from a panel of seven candidate reference genes, using Normfinder, BestKeeper and GeNorm. Normalized relative quantities for each gene of interest were then calculated using qbasePLUS (Biogazelle.) c-MET was overexpressed, when compared to control tissue, in all tumor samples but levels of c-MET were the highest in HPV- EA samples, and comparatively lower in HPV+ samples. TP53 was overexpressed in HPV+ samples; TGF-beta2 and Six1 were clearly overexpressed in all tumor samples, compared to control tissue samples; and BRCA1 levels were higher in HPV+ samples, compared to HPV- samples and controls. These data are consistent with our microarray results that indicate that HPV- tumors exhibit gene expression profiles indicative of activation of EMT. We are investigating the molecular interactions that lead to overexpression of SIX1, c-MET and BRCA1 in these tumors, and the functional consequences of this overexpression in terms of cell growth and EMT. Citation Format: Swati Tomar, Sangeeta Kowli, Diego Altomare, Christian Graves, Susannah Kassler, Natalie A. Sutkowski, Marion Boyd Gillespie, Saundra H. Glover, Kim E. Creek, Lucia A. Pirisi-Creek. HPV16-positive and HPV16-negative head and neck squamous cell carcinomas display different patterns of expression of genes involved in the control of growth and EMT. [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 4026. doi:10.1158/1538-7445.AM2013-4026