IADR Abstract Archives
HPV16 E5 Contributes to EGFR Overexpression and Activation in HNSCC
Objectives: A recent study showed that the HPV16 E5 oncogene plays a role in epidermal growth factor receptor (EGFR) overexpression in transformed HPV 16-positve anal epithelial cells (i.e. AKC2), however little research has been done looking at the role of E5 in HPV16-positive head and neck squamous cell carcinomas (HNSCC) cell lines. The goal of this research project was to detect E5 expression in HNSCC cell lines and investigate its role in EGFR overexpression and downstream EGFR signaling pathways.
Methods: HPV16 E5 expression was analyzed in two HNSCC cell lines (SCC90 and SCC47) using real-time PCR techniques. AKC2 cells were used as a positive control. SCC90 and AKC2 cells were transfected using E5-targeted siRNAs as well as negative control silent siRNAs. HPV16 E5 knockdown efficiency was evaluated 72-hours post transfection using real-time PCR. Cell proliferation was evaluated 72 hours post-transfection using standard cell counts. Total and phosphorylated EGFR and protein kinase B (AKT) protein expression were measured using western blot analysis and quantified using imageJ analysis.
Results: E5 RNA expression was 40- and 55-times higher in SCC90 and SCC47 cell lines, respectively, compared with AKC2 cells. Compared with SCC90 cells transfected with control siRNAs, transfection of SCC90 cells with E5-targeted siRNAs led to: 1) 85% reduction in E5 RNA expression; 2) 60% reduction in cell proliferation; 3) decreases in total EGFR and phosphorylated EGFR protein expression levels; and 4) decreased expression of phosphorylated AKT.
Conclusions: Our data show that similar to HPV 16-positive anal cell lines, E5 is expressed in HNSCC cell lines. E5 plays a role in EGFR overexpression and activation of downstream pathway proteins in HNSCC cells. Finally these data suggest that E5 could be a good therapeutic target for HNSCC.
Methods: HPV16 E5 expression was analyzed in two HNSCC cell lines (SCC90 and SCC47) using real-time PCR techniques. AKC2 cells were used as a positive control. SCC90 and AKC2 cells were transfected using E5-targeted siRNAs as well as negative control silent siRNAs. HPV16 E5 knockdown efficiency was evaluated 72-hours post transfection using real-time PCR. Cell proliferation was evaluated 72 hours post-transfection using standard cell counts. Total and phosphorylated EGFR and protein kinase B (AKT) protein expression were measured using western blot analysis and quantified using imageJ analysis.
Results: E5 RNA expression was 40- and 55-times higher in SCC90 and SCC47 cell lines, respectively, compared with AKC2 cells. Compared with SCC90 cells transfected with control siRNAs, transfection of SCC90 cells with E5-targeted siRNAs led to: 1) 85% reduction in E5 RNA expression; 2) 60% reduction in cell proliferation; 3) decreases in total EGFR and phosphorylated EGFR protein expression levels; and 4) decreased expression of phosphorylated AKT.
Conclusions: Our data show that similar to HPV 16-positive anal cell lines, E5 is expressed in HNSCC cell lines. E5 plays a role in EGFR overexpression and activation of downstream pathway proteins in HNSCC cells. Finally these data suggest that E5 could be a good therapeutic target for HNSCC.