Primary Supervisor: Dr Vicki Smith, Institute of Metabolism and Systems Research
Secondary supervisor: Dr Victoria Heath
PhD project title: Investigation of PBF as a novel regulator of endothelial cell motility and angiogenesis
University of Registration: University of Birmingham
Angiogenesis, the formation of new blood vessels, is a process that is critical throughout life, from embryonic development to old age, particularly in reproduction, wound healing and tissue repair. Dysregulated angiogenesis is also a key contributing factor to several age-related diseases, such as cancer and cardiovascular disease.
Pituitary tumor-transforming gene (PTTG)-binding factor (PBF) is a transmembrane glycoprotein that has mostly been studied in epithelial tumours, given its upregulation in multiple solid cancers and its pro-tumourigenic activity, such as the stimulation of cell proliferation, migration, invasion and genetic instability. Its primary function remains unknown; however recent data reveal PBF as a phosphoprotein with a physiological role in cellular signalling and motility. PBF is currently being investigated in our lab as a potential signalling node linking RTK/MAPK, Src and Rho GTPase pathways with cell adhesion and motility.
Analyses of global and single cell transcriptomics data show that within normal tissue the highest levels of PBF expression are found in blood vessels, and in multiple tissues PBF is highly expressed in endothelial cells (ECs). Interestingly, and in contrast with its pro-tumourigenic functions, a mouse model of human trisomy 21 (Tc1) revealed PBF as one of 4 novel genes associated with reduced angiogenesis and tumour growth in Down Syndrome. This study suggested that 3 copies of PBF, JAM-B, ERG and ADAMTS1 within blood vessels all contributed to the inhibition of VEGF-induced angiogenesis and likely regulate it via a common signalling pathway. Despite these observations, PBF remains entirely uncharacterised in ECs. This project will now determine the role of PBF in EC function, particularly in cell migration, and in the regulation of angiogenesis.
PBF will be characterised in ECs in vitro using human umbilical vein endothelial cells (HUVECs) and mouse primary endothelial cells. PBF expression will be manipulated to determine the effect of PBF on multiple steps of angiogenesis through assessing EC invasion, migration, proliferation and formation of vessel structures. PBF knockout mice, phospho-null mice and wild-type controls will be utilised to study ex vivo PBF expression and localisation in highly vascularised tissues and, importantly, to assess the effect of PBF deletion or loss of PBF phosphorylation on angiogenesis.
Potential molecular mechanisms of PBF regulation of EC function/angiogenesis will subsequently be explored. Initially, we will investigate putative relationships between PBF, JAM-B, ERG and ADAMTS1, both through direct interactions and also through evaluating shared signalling pathways.
As a transactivating protein, PBF’s interacting partner PTTG can upregulate VEGF, FGF2, their receptors and MMP-2 to induce angiogenesis. PTTG expression and subcellular localisation will be determined in ECs with altered PBF expression along with its downstream targets. PTTG knockdown will determine whether it mediates any effects of PBF.
Finally, Src and Rho GTPase signalling is known to regulate EC function and angiogenesis. Src-mediated regulation of PBF phosphorylation will be assessed to determine the importance of this modification. Crucially, both recent and future novel findings from our current BBSRC-funded study on PBF and Rho GTPase signalling will feed directly into this project.
Overall, this project will characterise a novel endothelial cell protein, which is highly expressed in the vasculature of multiple tissues. A functional role for PBF in angiogenesis will be investigated and the molecular mechanisms elucidated, significantly increasing our knowledge of the regulation of this fundamental process.
Institute of Metabolism and Systems Research
- Tumour angiogenesis is reduced in the Tc1 mouse model of Down's syndrome.
- Reynolds LE et al. Nature. 2010 Jun 10;465(7299):813-7. doi: 10.1038/nature09106
- Pro-invasive Effect of Proto-oncogene PBF Is Modulated by an Interaction with Cortactin. Watkins RJ et al. J Clin Endocrinol Metab. 2016 Dec;101(12):4551-4563. doi: 10.1210/jc.2016-1932.
- Manipulation of PBF/PTTG1IP phosphorylation status; a potential new therapeutic strategy for improving radioiodine uptake in thyroid and other tumors. Smith VE et al. J Clin Endocrinol Metab. 2013 Jul;98(7):2876-86. doi: 10.1210/jc.2012-3640.
BBSRC Strategic Research Priority: Integrated Understanding of Health: Ageing & Regenerative Biology
Techniques that will be undertaken during the project:
- Cell culture and manipulation (viral transduction, siRNA transfection)
- Expression and localisation studies (quantitative PCR, Western blotting, immunofluorescence, immunohistochemistry)
- Imaging techniques
- Functional assays (matrix invasion assays, scratch wound assays, proliferation assays)
- Angiogenesis analysis (tube formation assays, aortic ring sprouting assays)
Contact: Dr Vicki Smith, University of Birmingham