Abstract 1828: Proteomic analysis of the effect of E6 star expression on cellular pathways in HPV positive SiHa and HPV negative C33A cervical carcinoma cells
Authors: Evans Whitney , Filippova Maria , Aragon Robert , Filippov Valeri , Reeves Mark E. , Duerksen-Hughes Penelope 
Published: August 2015

High-risk types of the human papillomavirus (HR-HPV) are the causative agents of nearly all cases of cervical cancer, as well as a significant number of head, neck, penile, vulvar and anal cancers. Like many other viruses with small genomes, HPV (∼8 kb) utilizes numerous mechanisms to increase the capacity of its genome to encode the proteins necessary for successful completion of its infectious life cycle, including alternative splicing. Studies over the past few decades have focused intensively on the activities and roles of E6 proteins from HR-HPVs during the process of cellular transformation, clearly implicating E6 as a major transforming agent. In contrast, the role of the smaller splice isoform, E6*, in the carcinogenic process has not yet been established. In a recent study, we demonstrated that the over-expression of E6* reduces tumor growth by SiHa (HPV16 positive) and C33A (no HPV) cells in nude mice, suggesting that therapies emulating the actions of E6* may be of medical benefit. Furthermore, tumor growth inhibition by E6* was greater in tumors derived from HPV positive cells than in tumors derived from HPV negative cells. This difference implies that E6* interferes with the oncogenic activity of the full-length protein as well as by acting through HPV-independent mechanisms. The goal of this study is to determine the pathways affected by E6* that may lead to the observed reduction in tumor formation in xenograft models. To elucidate how E6* may affect the levels of cellular proteins and thereby orchestrate pathway regulation, in both E6 positive and negative environments, SiHa pFlag, SiHa pE6*, C33A pFlag, and C33A pE6* cells were created and their differential protein expression examined using mass spectrometry and Ingenuity Pathway Analysis (IPA) software. Lysates of these cells were reduced, alkylated, trypsinized, and TMT labeled, and the labeled peptides were analyzed using an LTQ-Orbitrap Velos mass spectrometer. Proteins were quantified by TMT tags and identified by comparison against the human library using Proteome Discoverer Software. 322 proteins were detected as differentially expressed using a 1.3 fold-change cut-off value. Further analysis by IPA revealed that E6* induced changes in apoptosis and death receptor signaling pathways in both HPV- and HPV positive cells, while other pathways, such as those involving mitochondrial dysfunction and TNFR1 signaling, were more profoundly affected in HPV negative cells. Our study provides several promising leads for future experiments and analyses, specifically in the context of human cancers, and carries with it the exciting possibility of replicating the anti-oncogenic activity of E6* in such a way as to provide therapeutic benefit. Future work will involve more detailed examination of our preliminary results and comparing these observations with those obtained from actual tumors derived from these cells.

Citation Format: Whitney Evans, Maria Filippova, Robert Aragon, Valeri Filippov, Mark E. Reeves, Penelope Duerksen-Hughes. Proteomic analysis of the effect of E6 star expression on cellular pathways in HPV positive SiHa and HPV negative C33A cervical carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1828. doi:10.1158/1538-7445.AM2015-1828