About the Project
Recent cancer genome sequence analyses suggest that somatic mutagenesis catalysed by the antiviral APOBEC3 (A3) cytosine deaminase enzymes is a major carcinogenic mechanism1. This field has exploded in the last three years, attracting great interest throughout the cancer research community and already a number of A3 inhibitor programmes have been initiated, both in academia and industry, with the aim of limiting acquired resistance by suppressing de novo mutagenesis during treatment2, 3. However, at present we know very little about A3-mediated genomic mutagenesis, which A3 enzymes are responsible in given cancers, how they are activated and whether A3 targeting by small molecule drugs or vaccination are feasible therapeutic approaches for cancer.
In our previous work, analysing gene expression and genomic alterations in human papillomavirus (HPV)-driven cancers, we uncovered a key role for one or more A3 enzymes and demonstrated A3-mediated mutagenesis of the PIK3CA proto-oncogene across multiple cancer types4. Transcript levels of two A3 family genes (APOBEC3A (A3A) and APOBEC3B (A3B)) are increased in HPV-infected keratinocytes and APOBECs target HPV DNA, which is localized in the nucleus5-9. This project will involve using a model of HPV-driven keratinocyte transformation to understand how A3 proteins become deregulated, leading to off-target activity against the host cell genomic DNA.
Hypothesis:
In normal cells, A3 enzymes are tightly regulated to avoid damage to genomic DNA. We hypothesize that in addition to mRNA changes, regulation of A3 proteins is also altered in HPV-infected cells, resulting in nuclear A3 activity necessary for editing of viral DNA but also increasing the risk of genomic mutagenesis.
Experimental Approach:
The aim of this project is to identify mechanisms by which A3 enzymes are regulated at the protein level in normal cells and to understand how this regulation is perturbed in HPV-infected cells. The high sequence homology between the seven human A3 genes has hampered development of specific antibodies against the individual proteins, thus post-translational A3 regulation has remained largely unexplored. To overcome this, we have used CRISPR/Cas9 gene editing to add epitope tags to the endogenous A3A and A3B genes in keratinocytes, allowing detection of the proteins by western blotting and immunofluorescence microscopy. This project is based on using these novel tools to investigate A3 protein regulation in detail. A3A and A3B expression and localization will be visualized under different conditions, including interferon stimulation and expression of HPV proteins. A3 binding proteins will be identified using mass spectrometry. The roles of selected binding proteins in regulating A3 enzymes and the functional importance of these interactions for preserving genomic integrity will be investigated.
The project will employ a variety of cell biology, biochemical and molecular biology techniques in addition to those listed above, including affinity purification, subcellular fractionation, cytosine deamination and DNA damage assays, retroviral gene transduction and CRISPR/Cas9 gene editing. There will also be opportunities to visit and learn techniques from our collaborators on this project, at the University of Cambridge and at the Francis Crick Institute in London.
Funding Notes
The Graduate Teaching Assistantship (GTA) provides a postgraduate research student with financial support in return for 96 hours per year of teaching. The stipend paid equals the full UK Research Council rate of £14,296 (rate for 2016/17) plus tuition fees at the home/EU rate. International applicants should make provision to meet the difference between Home /EU and International fees.
For further information on the Graduate Teaching Assistantship scheme go to: https://www.kent.ac.uk/scholarships/search/FNADGTA00001
References
1. Henderson, S. & Fenton, T. APOBEC3 genes: retroviral restriction factors to cancer drivers. Trends in molecular medicine 21, 274-284 (2015).
2. Harris, R.S. Molecular mechanism and clinical impact of APOBEC3B-catalyzed mutagenesis in breast cancer. Breast Cancer Res 17, 8 (2015).
3. Swanton, C., McGranahan, N., Starrett, G.J. & Harris, R.S. APOBEC Enzymes: Mutagenic Fuel for Cancer Evolution and Heterogeneity. Cancer Discov 5, 704-712 (2015).
4. Henderson, S., Chakravarthy, A., Su, X., Boshoff, C. & Fenton, T.R. APOBEC-Mediated Cytosine Deamination Links PIK3CA Helical Domain Mutations to Human Papillomavirus-Driven Tumor Development. Cell Reports 7, 1833-1841 (2014).
5. Kukimoto, I. et al. Hypermutation in the E2 gene of human papillomavirus type 16 in cervical intraepithelial neoplasia. Journal of Medical Virology 87, 1754-1760 (2015).
6. Vartanian, J.-P., Guetard, D., Henry, M. & Wain-Hobson, S. Evidence for Editing of Human Papillomavirus DNA by APOBEC3 in Benign and Precancerous Lesions. Science 320, 230-233 (2008).
7. Vieira, V.C. et al. Human papillomavirus E6 triggers upregulation of the antiviral and cancer genomic DNA deaminase APOBEC3B. mBio 5 (2014).
8. Wang, Z. et al. APOBEC3 Deaminases Induce Hypermutation in Human Papillomavirus 16 DNA upon Beta Interferon Stimulation. Journal of Virology 88, 1308-1317 (2014).
9. Warren, C.J. et al. APOBEC3A Functions as a Restriction Factor of Human Papillomavirus. Journal of Virology 89, 688-702 (2015).
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