Anti-tumor Mechanisms of HPV16 E6*

Abstract

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. Based on previous studies, we proposed that E6* had the potential to reduce tumor formation in vivo. To test this prediction, we injected HPV16+ SiHa and HPV16- C33A cervical cancer cells overexpressing E6* into nude mice and monitored tumor growth over several weeks while comparing them with control tumors lacking E6* overexpression. E6* was found to reduce growth in both SiHa- and C33A-derived tumors. These findings were followed up by in vitro experiments showing that E6* binds to full-length E6 and decreases the growth rate in SiHa cells. Subsequently, we sought to find the cellular pathways most influenced by E6* in attenuating SiHa and C33A tumor growth. Thus, proteomic analysis was performed on both cell lines, revealing that the β -Integrin pathway in SiHa cells and the mitochondrial dysfunction and oxidative phosphorylation pathway in C33A cells were the most significantly altered. Proteomic data was confirmed using immunoblot, microscopy, and flow cytometry techniques. These findings will assist in our quest for understanding the fundamental driving forces behind HPV-mediated and non-HPV-mediated cervical cancers and introduce novel ideas for small molecule inhibitors. Our studies provide several promising leads for future analyses, specifically in the context of human cancers, and carry with them the exciting possibility of replicating the anti-oncogenic activity of E6* in such a way as to provide therapeutic benefit