Wnt signalling is a critical developmental pathway and associated aberrant signaling drives oncogenesis including colorectal and bone cancer. β-catenin, a central protein forms a range of protein-protein interactions(PPIs), fundamental to driving the transcription of oncogenes inducing proliferation and tumour formation. To further elucidate these interactions, we have sought to i) identify inhibitors of β-catenin PPIs and ii) employ a proximity dependent labelling technique (MiniTurbo) to characterize novel PPIs.
MiniTurbo-Mutant Biotin ligase BirA is genetically fused to β-catenin and upon addition of Biotin, tagging is induced of proteins within 20nm in as quick as 10 minutes enabling streptavidin pull down and tagging to Mass spectrometry. CTNNB1 gene was successfully inserted into the Miniturbo plasmid construct using SLICE cloning. DH5alpha bacteria were transformed with plasmid and single colony selected with Ampicillin. Following plasmid purification, it was added with Retroviral components for viral transfection and production in HEK293 cells. U2OS adenocarcinoma cells transduced and selected using puromycin. Validated with western blot and immunofluorescence. 24-hour application of MSAB derivatives following induction of Miniturbo with Doxycycline. Streptavidin pulldown was carried out and samples were subjected to Mass Spectrometry following on-bead trypsin digestion. Proteomic analysis was carried out to elucidate novel PPI.
-MSAB derivatives influence levels of β-catenin and Wnt target genes at protein and transcriptional level. -Successful introduction and selection of the miniturbo plasmid into U2OS cells. Western blots and immunofluorescence verification. -Novel Proteomic networks of Beta-catenin protein interactions in U2OS cells. The results illustrate the dynamic PPI network of β-catenin in a 2D cancer model and how those interactions are modulated in the presence of small molecule inhibitors for therapeutic indications.
Targeting β-catenin directly for Oncotherapy has been difficult with very little success. Further characterization and understanding of the dynamic β-catenin interactome may pave an avenue for addressing this challenge.
Ewing and Baud Lab Group/University of Southampton.
University of Southampton (50%)/Kerkut Trust Charitable organization (50%).
P. Skipp: Financial Interests, Institutional, Ownership Interest: TopMD Precision Medicine Ltd. All other authors have declared no conflicts of interest.