Host cell reprogramming by oncogenic human papillomavirus

Viral infections are estimated to cause over 1 in 10 of all cancers, resulting in over 1.3 million deaths a year, worldwide. One of the most significant contributors to this virally-induced cancer burden are human papillomaviruses (HPV); HPV infections cause almost half of these cancers, including cancers of the uterine cervix and genitals, and oral cancers in the mouth and throat. While the causal link between HPV and the development of cancer is well established, the specific changes that occur in HPV infections that drive the formation of HPV tumours are not well understood. It is important to understand these changes so that new therapies to treat HPV infections and cancers, and diagnostic tools for early disease detection, can be developed.

There are hundreds of distinct types of HPV, but only a very small number of these are classified by the World Health Organisation as carcinogenic. HPV type 16 causes the majority of cervical and oral cancers whereas HPV type 18 is the second most common cause of cervical cancer but is rarely found in oral cancers. The reasons for this disparity are unknown but we hypothesise that HPV types 16 and 18 differentially manipulate host cells at different anatomical sites resulting in the observed differences in clinical outcome.

All viruses manipulate their host cell environment to enhance production of infectious progeny. We have shown that establishment of HPV infection in the natural host cells significantly alters the expression of a subset of host cell genes. This HPV-driven gene expression 'reprogramming' is fundamental to persistence of HPV infection, a significant risk factor for cancer development. Importantly, many of the gene expression changes that occur early in the natural history of persistent HPV infections increase the growth rate and life span of the infected cells, and therefore are very likely contribute to disease progression.

The experiments described in this proposal are designed to answer long-standing questions about how HPV16 and 18 behave in a normal infection of the oral cavity or uterine cervix. We will fully characterise the molecular basis of HPV-induced gene expression changes and determine which of these changes are important for viral persistence and disease progression at each body site. Using cancer cell lines and clinical samples of HPV-associated tumours, we will explore whether novel virus-specific changes are required for cancer cell growth and sustained in HPV-driven tumours. These experiments will provide much needed novel insight into the normal life cycle of HPV infections at clinically relevant body sites and identify virus-specific events that drive disease progression.

Persistent oncogenic human papillomavirus (HPV) infection causes cancers within the anogenital and oropharyngeal tracts. The fundamental link between HPV and cancer is established, but the molecular changes that drive disease progression are poorly defined. Importantly, the natural history of HPV infection is different between body sites; disease progression in the uterine cervix follows defined premalignant phases that have not been identified in the oropharynx. In addition, oncogenic HPV types behave differently at specific anatomical sites. While HPV16 causes the majority of disease at both sites, HPV18 is the second most common type in cervical cancers but only rarely found in oropharyngeal tumours. The reasons for these differences are unknown.

We hypothesise that HPV16 and 18 differentially induce transcriptional reprogramming of host cells at different anatomical sites, defining niche-specific persistence and disease progression. To test this, we have developed physiological models of the HPV life cycle. Primary keratinocyte cultures from the tonsil and anogenitals are transfected with HPV16 or 18 genomes and persistent episome replication established. We show that HPV18 induces targeted transcriptional reprogramming of host cells by redistribution of an important cellular transcriptional regulator, CCCTC binding factor (CTCF) resulting in altered epigenetic chromatin status. We will use our models of oncogenic HPV infection and innovative methods including RNA-Seq, ChIP-Seq, ATAC-Seq and 4C to unravel HPV type- and anatomical site-specific nuances in host cell manipulation, HPV biology and disease progression. Key pathways will be analysed in our HPV life cycle models and tumour sections to determine their role in HPV persistence and disease progression. Consolidation of our findings will identify virus-specific events important in HPV-driven disease, which are likely to be clinically useful, and answer important questions about HPV biology.

The work in this proposal focusses on understanding the fundamental biology driving difference in the behaviour and pathogenesis of oncogenic HPV at specific anatomical sites. We will study global transcriptional reprogramming of primary keratinocytes, the natural host of HPV, taken from the tonsil and anogenital tract and use state-of-the-art methods to study HPV types 16 and 18. The results will provide a step-change in our understanding of HPV biology and HPV-driven disease progression, but more broadly to our understanding of the host cell transcription regulation and carcinogenesis. The main beneficiaries will be:

THE ACADEMIC COMMUNITY
To our knowledge we are the only grouping addressing the specific questions raised in our proposal and, with our combined expertise, we are ideally placed to answer these long-standing and important questions. Therefore, the results obtained will benefit those interested in the HPV life cycle and HPV-induced host cell reprogramming. Our study will also be beneficial to those interested in other persistent DNA viruses and CTCF-mediated epigenetic control of host transcription. CTCF is mutated and dysregulated in many cancers and human disease and therefore our study will also benefit those interested in CTCF function in healthy and disease states.
Establishment of state-of-the-art methods (e.g. 4C, ATAC-Seq) will benefit our local academic community, as these methods will be shared with our colleagues at the University of Birmingham and further afield. The academic community will also benefit from the training of a postdoctoral scientist in the use of such methods to study the HPV life cycle, gene expression and chromatin structure, as well as more general training in project management and communication skills.

CLINICIANS
By identifying molecular events that contribute to HPV-driven carcinogenesis, we will benefit clinicians interested in HPV-associated disease. The proposed work will facilitate immediate engagement with Prof. Mehanna, consultant Head and Neck Surgeon and Director of InHanse (University of Birmingham) and future studies will take advantage of the highly integrated clinical academic environment at the University of Birmingham and within Birmingham Health Partners (BHP).

STAFF WORKING ON THE PROJECT
The postdoctoral scientist working on this project will develop a skill set highly desirable for future employment. With guidance from Parish and Roberts, the researcher will gain project management skills, obtained from running a research project, which are transferrable to many other disciplines in addition to the scientific employment sector. The researcher will also develop their communication and supervision/teaching skills that will be transferable to any management discipline.

SOCIETY
The immediate impact on society will be in the development of a highly skilled academic researcher. The proposed work could also have an economic impact in the long term through the development of novel biomarkers and therapies for HPV-driven disease.