Specific forms of TCF7L2 protein define the Wnt pathway dependent genetic program

Abstract

The TCF7L2 transcription factor is a critical regulator of Wnt pathway-dependent gene expression in the intestinal epithelia. Mutations in TCF7L2 have been previously reported in colorectal cancer, however their functional impact on tumour biology has not been investigated. The present work has determined that expression of the short form of TCF7L2 protein is not a product of alternative splicing; instead, the protein is predominantly expressed as the long form of the protein that is processed to the short form of the protein. I have identified discrete sequence-specific requirements in TCF7L2 by the identification of point mutations that abrogate conversion to the short form. Therefore, TCF7L2 specific processing is the principal mechanism dictating the ratio of long and short forms of the protein and the Wnt pathwaydependant transcriptional program. The consequence of altered expression ratios of long and short forms of TCF7L2 are biologically important, shown by a specific set of mutations in the TCF7L2 gene that are selected in some colorectal cancers; the mutations lead to the expression of a truncated version of TCF7L2 that is functionally identical to the processed, short form of the protein. Bioinformatics analyses have identified unique, molecular biomarkers and clinical characteristics of TCF7L2 mutant colorectal cancers that include microsatellite instability, exclusivity of BRAFV600E mutations, better prognosis and differential gene expression. Thus, specific TCF7L2 mutations classify a unique and discrete set of colorectal cancers. I further showed the outcome of TCF7L2 mutations and the altered ratio of long and short versions of the TCF7L2 protein – alteration of the Wnt pathway gene expression program with specific consequences on cell proliferation, clonogenic growth, cell cycle progression and survival. I also established experimental systems to study TCF7L2 expression levels using organoid models. The technology consisted of organoids cultured in media containing precise activities of essential growth factors, obviating issues endemic to the use of commercially sourced, impure growth factor preparations. I focused on the two essential organoid media growth factors, R-spondin1 and Gremlin-1, that were produced from bacterial expression and optimised for organoid culture. I further investigated whether R-spondin1 could be further engineered to increase its potency in organoid growth media. My strategy was to introduce mutations that specifically increase its affinity for its cognate receptor LGR5. LGR5 expression is specific to stem cells whereas the closely related LGR4 receptor is expressed by more differentiated cell types in the intestinal epithelia. My engineering was successful, producing a mutant version of R-spondin1 that displayed a 6-fold increased binding affinity for LGR5 and a significantly reduced ability to bind LGR4. My work has defined a unique colon cancer subset defined by specific TCF7L2 mutations and developed a system for analysing the consequence of the mutations in vitro. Unravelling the recircuitry imposed by mutant TCF7L2 protein will reveal therapeutic vulnerabilities and pave the way for developing treatment options.