AbstractAlternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGFxxx isoforms are produced via selection of the proximal 3’ splice site of the terminal exon. Use of an alternative distal splice site creates the anti-angiogenic VEGFxxxb proteins.
A bichromatic splicing-sensitive reporter was designed to mimic VEGF alternative splicing and used as a molecular tool to further investigate this alternative splicing event. The terminal exon and preceding intron of VEGF were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3’ splice site of the exon is used during splicing. The fluorescent output can be used to follow splicing decisions in vitro and in vivo.
Following successful reporter validation in different cell lines and altering splicing using known modulators, small pilot screens were undertaken to search for novel regulators of the splicing decision that creates pro-/anti-angiogenic VEGF isoforms. A larger screen was performed using a library of 1280 small molecules (LOPAC), all compounds of the library were pharmacologically active and have known biological targets. Alterations to reporter splicing were measured using a fluorescent plate reader to detect RFP and GFP expression. Compounds of interest were further validated using flow cytometry and assessed for effect on endogenous VEGF alternative splicing. In vitro angiogenesis assays were used to demonstrate anti-angiogenic effect. Anti-angiogenic activity and the effect on tumour growth were investigated in several in vivo models.
|Date of Award||10 May 2016|
|Supervisor||Sebastian Oltean (Supervisor) & David O Bates (Supervisor)|