Targeting β-catenin to inhibit the Wnt pathway in colorectal cancer cell lines

Thesis

In colorectal cancer (CRC), one of the post prevalent cancers, the Wnt signalling pathway is upregulated due to mutations in the key players Adenomatous polyposis coli (APC) and CTNNB1 (β-catenin). While intensive research efforts are being placed in blocking the Wnt pathway, no inhibitors are yet in clinical use. In this project, different strategies were assessed to target β-catenin protein-protein interactions (PPIs) with the aim to inhibit the Wnt pathway in CRC cell lines.

Through 2x2 statistical analyses of known microarray gene expression and mutation data and preliminary immunofluorescence imaging of β-catenin localisation in CRC lines, low E-cadherin expression was associated with a more nuclear β-catenin subcellular localisation (Chapter 3).

Three different approaches to target the Wnt pathway were described: firstly, small molecule inhibitors against the β-catenin/B cell lymphoma 9 (BCL9) interaction site were tested in vitro (Chapter 4). The compounds exhibited non-specific killing on a large panel of CRC cell lines and fibroblasts. Furthermore, the compounds were also shown to be albumin dependent, limiting their further development. In Chapter 5, the Intracellular Antibody Capture (IAC) methodology to identify intracellular single domain antibodies (sdAb) against β-catenin is described. Through a large variable-heavy (VH) single domain library screen, three sdAb against β-catenin were identified, but subsequentially shown to be non-specific binders. Lastly, a previously described llama single-domain antibody fragment (VHH) was further characterised in SW480 CRC cells (Chapter 6). The VHH was shown to inhibit T-cell factor (TCF)-mediated Wnt pathway transcription and the expression of the Wnt target genes ASCL2 and AXIN2. The VHH binding site was refined to the first three Armadillo repeats of β-catenin.

Overall, this work explores different routes of Wnt signalling inhibition by targeting β-catenin PPIs and discusses their advantages and potential limitations for further development (Chapter 7).

Authors: Colling, LA

• DOI: 10.5287/ora-ganzjax84
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English