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Phenotypic and molecular characterization of in vitro models of oncogenic HPV-induced disease progression.


Institute of Cancer and Genomic Sciences

About the Project

Human papillomaviruses (HPVs) are the cause of benign and malignant lesions of the cutaneous and mucosal surfaces of the skin. High-risk types (e.g. 16, 18, 31) are the cause of the majority of cervical and anogenital carcinomas, while infection with low-risk genital HPVs (e.g. 6, 11) causes genital warts. To maintain persistent infection, papillomaviruses target key host cell pathways to alter cell cycle control and cellular proliferation to create a favourable environment for productive virus infection. Using state-of-the-art primary cell-based models of oncogenic HPV establishment, we have shown that HPV induces transcriptional reprogramming of host cells by altering the epigenetic status of chromatin. Gene set enrichment analysis of differentially expressed genes shows that several cellular pathways associated with cell growth are targeted by the virus. Notably, gene sets associated with oncogenic pathways are signifcantly enriched in our model of HPV establishment, even though these cells are not yet transformed by HPV. However, whether these changes are early drivers of HPV-induced oncogenic transformation remains to be determined.
Phenotypic characterisation of HPV-genome containing cultures has demonstrated that long term culture of these cells results in the appearance of viral markers of disease progression such as viral integration into the host DNA and/or increased viral oncogene expression. These changes are coincident with increased cell growth and morphological changes that occur with oncogenic transformation but it is unclear whether these long term cultures are truly transformed or tumourigenic.
This project aims to fully characterise our in vitro models of HPV-induced disease progression on a phenotypic and molecular level. Low and high passage HPV18 genome-containing human keratinocytes will be analysed using a range of growth, invasion and migration assays. Alongside these assays, cells grown in 3-dimensional organotypic raft culture will be analysed to determine cellular differentiation and invasion potential. The expression of key genes that are differentially expressed by the virus upon initial establishment will be analysed after long term culture to determine whether these changes are sustained and/or increased in magnitude during HPV-driven disease progression. Finally, manipulation of gene expression by siRNA depletion or overexpression by lentivirus transduction will be used to determine whether gene expression changes that occur during HPV establishment and/or disease progression are required for virus replication and persistence.
Techniques to be used: This project will combine computational analysis of RNA-sequencing datasets with wet laboratory techniques including primary cell culture, real time PCR, western blotting, siRNA transfection and lentiviral transduction, organotypic raft culture (in vitro culture of ‘skin’ to analyse the HPV life cycle), immunofluorescence, invasion and migration assays.

Funding Notes

Self-funded only

References

1. Doorbar J, Quint W, Banks L, Bravo IG, Stoler M, Broker TR, Stanley MA. The biology and life-cycle of human papillomaviruses. Vaccine. 2012; 30: F55-70.
2. Moody CA, Laimins LA. Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Cancer. 2010; 10(8):550-60.
3. Roberts S., Pentland I., McCormack P., Parish J.L. Biology of Human Papillomavirus Life Cycle. In: DNA Tumour Viruses: Virology, Pathogenesis and Vaccines. Caister Academic Press (2018). https://doi.org/10.21775/9781910190791.02
4. Pentland I., Campos-Leon K., Cotic, M., Davies, K-J., Wood C.D., Groves I., Burley, M., Coleman N., Stockton, J., Noyvert, B., Beggs, A., West M.J., Roberts S., Parish J.L. Disruption of CTCF-YY1 dependent looping of the human papillomavirus genome activates differentiation-induced viral oncogene transcription. PLoS Biology. 2018; 16(10): e2005752
5. Roberts S., Evans D., Mehanna H., Parish J.L. Modelling HPV biology in oropharyngeal keratinocytes. (2019) Proceedings of the Royal Society B; Biological Proceedings. DOI: https://doi.org/10.1098/rstb.2018.0289

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