Mechanisms of human papillomavirus and host gene transcriptional deregulation in cervical carcinogenesis

摘要

Cervical malignancy is the fourth most common cause of cancer-related mortality in women worldwide; infection with high-risk human papillomavirus (HRHPV) is responsible for over 500,000 cases of cervical carcinoma each year, approximately 90% of which are squamous cell carcinomas (SCCs). Over half of all HPV-positive cervical SCCs are caused by the deregulated expression of HPV16 oncogenes E6 and E7 in proliferating basal cells of the cervical squamous epithelium. The major risk factor associated with cervical neoplastic progression is integration of HRHPV into the host genome, which is detected in $~$85% of HPV16-positive cervical carcinomas. The work presented in this doctoral thesis sought to provide insights into our understanding of the process of HPV16 integration as well as to elucidate mechanisms that deregulate both virus and host gene expression following integration.The W12 cell model system used in this project is a polyclonal cervical keratinocyte line generated by explant culture of a low-grade cervical squamous intraepithelial lesion (LSIL) that arose following natural infection with HPV16. The W12 clones were isolated in the absence of selective pressure, and as such represent the range of integration events that occur in a pre-malignant lesion at the early stages of carcinogenesis, prior to integrant selection. Despite identical genetic backgrounds, expression levels of oncogenes E6 and E7 varied up to 16-fold between the W12 clones. Expression of HPV oncogenes is ultimately determined by transcription factor binding to the non-coding long control region (LCR) of the viral genome. The initial result of this study found that genomic mutations affecting transcription factor binding at the LCR of the W12 clones was not a cause of differential viral expression, concluding that epigenetic control may be at play.Higher levels of virus expression per template were associated with increased levels of histone post-translational modification (PTM) hallmarks of transcriptionally active chromatin and reduced levels of repressive hallmarks. There was greater abundance of the active/elongating form of the RNA polymerase-II enzyme (RNAPII-Ser2P), together with CDK9, the component of positive transcription elongation factor-b (P-TEFb) responsible for the Ser2 phosphorylation. The changes observed were functionally significant, as cells with higher HPV16 expression per template showed greater sensitivity to depletion and/or inhibition of histone acetyl transferases and CDK9, as well as reduced sensitivity to histone deacetylase inhibition. Employing next generation sequencing data available for five representative W12 clones, the sites of HPV16 host integration were identified. The three-dimensional (3D) structure of the nucleus and physical interactions between stretches of the genome over long distances (i.e. enhancer and promoters) are known to exert an additional level of gene regulation. Identification of 3D virus-host interactions in the W12 clones employing the newly developed and unique 'Sequence Capture of Regions Interacting with Bait Loci Hi-C' (SCRiBL-Hi-C) protocol showed that both short- ($~$50 kb), and long-range ($~$1 Mb) interactions occur during the early stages of carcinogenesis.Together, the data in this thesis indicate that transcription and subsequent expression of the HPV16 genome is controlled by multiple layers of epigenetic regulation.