Introduction

Colorectal cancer (CRC) is the 3^rd most common type of cancer in the U.S. and accounts for an alarming 153,020 (8%) of new estimated total cancer cases and 52,550 (9%) of estimated total cancer deaths in 2023. In the vast majority (>80%) of human sporadic CRC cases, sequencing data indicate the presence of mutations in the adenomatous polyposis coli (APC) gene. This “gatekeeping” gene is a key regulator of Wnt signaling and loss of its function induces aberrant stabilization of β-catenin (βcat), a crucial step in CRC initiation. Notably, attempts to target this pathway using drugs still pose multiple hurdles.

From a functional perspective, Wnt factors organize to form specialized PM domains. Dysregulation of Wnt domain structure can promote oncogenic Wnt signaling. Here, we describe an intricate Wnt signaling-associated mechanism involving oncogenic truncated APC and the loss of PM cholesterol homeostasis, which alters the organization of Wnt signaling nanoassemblies (biomolecular condensates) and drives aberrant Wnt signaling and CRC tumorigenesis.

Methods

To comprehensively assess the role of oncogenic truncated APC in reshaping PM cholesterol, proteolipid hierarchical organization and downstream Wnt signaling, mouse, fly, and human orthogonal models were used. Second, we combined total internal reflection fluorescence (TIRF) with direct stochastic optical construction microscopy (STORM) and wide-field fluorescence lifetime imaging microscopy combined with fluorescence resonance energy transfer (FLIM-FRET) for the analysis of proteolipid membrane nanoclustering within Wnt macromolecular condensates. Third, we assessed whether free cholesterol alone can recapitulate some of the phenotypes associated with oncogenic truncated APC-induced loss of PM homeostasis. Fourth, we conducted experiments to show that stabilization of βcat increases PM free cholesterol and rigidity, thus linking downstream to upstream Wnt signaling and further highlighting the non-autonomous nature of mutant APC in CRC.

Results

We report that oncogenic APC increases PM free cholesterol liquid ordered (Lo) domains, thereby promoting membrane rigidity and lipid raft stability in various CRC mouse and human models expressing different forms of truncated APC. Additionally, we show that APC-driven dysregulation of PM homeostasis modulates Wnt receptor (Fzd7 and LRP6) nanoscale proteolipid organization and their interactions with key Wnt signaling effectors in both mouse and human colonocytes. Finally, using the Drosophila sterol auxotroph model, we demonstrate the unique ability of exogenous free cholesterol to disrupt PM homeostasis and drive Wnt signaling in a wildtype APC background. Collectively, these findings provide a mechanistic link between oncogenic APC, loss of PM homeostasis, and CRC development.

Conclusion

We describe a critical role for mutant APC in shaping PM cholesterol-enriched domains, membrane rigidity, Wnt receptor nanoassemblies and their interactions with key lipid and protein effectors within Wnt condensates. These findings corroborate the concept that PM cholesterol and PM-associated Wnt signaling are essential drivers of CRC development in the presence of mutant APC. To our knowledge, this is the first evidence directly linking the interplay between mutant APC, the levels of free cholesterol in the PM, membrane order, aberrant Wnt signaling and cholesterol homeostatic pathways in cancer. We also document the unique ability of exogenous cholesterol to disrupt PM homeostasis and drive Wnt signaling autonomously in Drosophila intestine and cultured cells. In contrast, cholesterol-lowering drugs suppress these phenotypes. Altogether, from a translational perspective, these findings suggest that therapeutic approaches aimed at restoring PM cholesterol homeostasis and proteolipid spatiotemporal dynamics could prove an effective strategy to reduce cancer risk.