Mutant APC reshapes plasma membrane cholesterol-dependent Wnt proteolipid condensate structure-function and feedforward oncogenic ß-catenin signaling
Although the role of the Wnt pathway in colon carcinogenesis has been described previously, it has been recently demonstrated that Wnt signaling originates from highly dynamic nano-assemblies at the plasma membrane. However, little is known regarding the role of oncogenic APC in reshaping Wnt nanodomains. This is noteworthy, because oncogenic APC does not act autonomously and requires activation of Wnt effectors upstream of APC to drive aberrant Wnt signaling. Here, we demonstrate the role of oncogenic APC in increasing plasma membrane free cholesterol and rigidity, thereby modulating Wnt signaling hubs. This results in an overactivation of Wnt signaling in the colon. Finally, using the Drosophila sterol auxotroph model, we demonstrate the unique ability of exogenous free cholesterol to disrupt plasma membrane homeostasis and drive Wnt signaling in a wildtype APC background. Collectively, these findings provide a mechanistic link between oncogenic APC, loss of plasma membrane homeostasis and CRC development. Overall design: YAMC (Apc+/+), IMCE (Apc-/+) and IMCE Bcat (Apc-/+ Bcat-/+) mouse cell lines derived from the colonic epithelium were employed to elucidate the effect of oncogenic mutant APC on the expression of cholesterol homeostasis related genes. To achieve this, three independent biological replicates (n=3 per group) were utilized to establish the enrichment of multiple cholesterol homeostasis pathway genes in mutant APC and compared to WT APC (control) samples. Furthermore, the effect of aberrant dysregulation of Wnt signaling was also examined by comparing the dysregulation magnitude of cholesterol homeostatic genes across all cell lines,i.e., YAMC< IMCE<IMCE Bcat. Similarly, a colorectal cancer mouse model was utilized to examine the effect of mutant oncogenic APC on the expression of cholesterol homeostasis related genes in vivo. To achieve this, primary colonic crypts from the epithelium of heterozygous (AGC) and homozogous (AfGC) mutant APC mice (n=4 per group) were utilized to establish the enrichment of multiple cholesterol homeostasis pathway genes and compared to WT APC control mice (GC, n=4). Furthermore, the effect of aberrant dysregulation of Wnt signaling was also examined by comparing the dysregulation magnitude of cholesterol homeostatic genes across all mice genotypes (GC< AGC<AfGC).