Apc truncation enforces optimal Wnt signalling during early intestinal tumourigenesis

Abstract

Colorectal cancer (CRC) development is frequently initiated by inactivation of tumour suppressor gene adenomatous polyposis coli (APC), resulting in hyperactivation of the Wnt/ {beta}-catenin signalling pathway. The level of Wnt pathway activation is determined by the number of functional {beta}-catenin binding site repeats retained within the truncated APC protein. Accumulating evidence indicates that acquisition of the second APC mutational event is non-random and selects for mutations that confer a just right level of Wnt activity, suggesting that tumour initiation requires Wnt signalling to be maintained within a specific range. In this study, we investigated how specific Apc mutations modulate Wnt pathway activity and downstream cellular programs. Using CRISPR/Cas9 technology, we generated mouse intestinal organoid cultures harbouring distinct alterations in the number of {beta}-catenin binding repeats. We demonstrate a direct correlation between the number of retained repeats and Wnt signalling activity. Notably, organoids retaining higher numbers of functional repeats were subjected to selective pressure, resulting in the acquisition of additional mutations that lead to increased Wnt activity, consistent with the just right model. Transcriptomic analysis by RNA sequencing revealed that clones with distinct {beta}-catenin binding capacity exhibit differential transcriptional programs, including graded changes in cell cycle activity and a metabolic shift associated with progressive loss of {beta}-catenin binding repeats. Together, these findings reveal functional and metabolic heterogeneity among Apc- mutant cells, support the existence of selective pressures for optimal Wnt signalling during tumor initiation, and identify potential metabolic vulnerabilities that may be exploited for therapeutic intervention including interception of premalignant lesions.