Specific viral infections are thought to cause ~15 to 20% of human cancers. Human papillomaviruses (HPVs) are small DNA tumor viruses that infect, persist in and cause proliferative lesions in the epithelial cells of the skin, ectoderm-derived mucosae and their adnexa. Mucosal high-risk (HR) HPV types are as- sociated with most, if not all, carcinomas of the uterine cervix, many anogenital cancers and ~25% of head and neck cancers (HNC). HR HPV type 16 is the most common HPV;it also is found in 90 to 95% of HPV-positive HNCs. In persistent infection, HPV genomes are maintained as circular, unintegrated plas- mids in the nuclei of the infected cells. In clinical cancer specimens, the viral genome often is disrupted and integrated in the cellular genome. The integrated HR HPV fragments in cervical cancer and derived cell lines express the viral transforming genes, E6 and E7, from chimeric virus-cell mRNAs while the down- stream early genes that are required for viral replication and regulate viral gene expression are abrogated. Relatively little is known about the gene structure and integration of HPV-16 in HNC and limited information is available about the integration process itself. Over the past period, we have developed a quantitative colony immortalization assay, isolated sets of isogenic clones of primary human keratinocytes of tonsillar, cervical and foreskin origin 'immortalized'by extrachromosomal HPV-16 genomes, and designed and tested methods that allow us to follow HPV-16 persistence and integration in culture. Using these assays and reagents, we will analyze HPV-16 virus-cell interactions as a model of HNC carcinogenesis with emphasis on the process of viral integration. Specifically, we will: (i) Characterize HPV-16 integration status and gene expression in HNC tumors in comparison with isogenic primary human keratinocyte clones 'immortalized'by replicating or integrated HPV-16 plasmid genomes. The purpose is to characterize virus-cell interactions in HNC tumor tissues and to de- termine to what extent HPV integration in culture shares underlying features with integration in HPV- associated HNC and thus can serve as a model to study the process in culture. (ii) Determine the stability of HPV-16 persistence in 'immortalized'primary keratinocyte clones and quantitate the rates of viral integration in long-term culture. Furthermore, these experiments will identi- fy keratinocyte clones capable of long-term HPV plasmid persistence for use in Aim 3. (iii) Test potential approaches to eliminating persistent HPV plasmids from keratinocyte clones with stably replicating plasmid genomes. These studies will determine if the treatment eliminates ex- trachromosomal HPV plasmids from cells and whether it leads to unintended induction of faster-dividing in- tegrants. These results will have direct impact on potential therapeutic strategies to eliminate persistent HPV infection before the development of cancer.
We propose to study the role of HPV as a major cause in the development of cancers of the head and neck. Head and neck cancer (HNC) is a major cause of cancer morbidity and mortality in elderly;other known risk factors for the development of HNC are cigarette smoking and excessive alcohol consumption. While HNC in the general population has been decreasing, the numbers of newly diagnosed HNC cases among veterans, both recently retired and elderly, are on the rise. The research thus directly addresses high priority subjects in the Department of Veterans Affairs: chronic diseases in elderly veterans and etiology of cancer. Human papillomavirus infection has been linked to head and neck cancer by association, but little is known about the way these viruses contribute to tumor development. The proposed studies focus on the progression of HPV-infected cells to cancer and the changes in the virus-cell interactions in the process, with the ultimate objective of developing diagnostic biomarkers and future therapeutic interventions.
