Diet therapy abates mutant APC and KRas effects by reshaping plasma membrane cholesterol nanodomains

Cholesterol-enriched plasma membrane domains are known to serve as signaling platforms in a diverse array of cellular processes. However, the link between cholesterol homeostasis and mutant APC-KRas-associated colorectal tumorigenesis remains to be established. Thus, we investigated the impact of Apc-Kras on 1) colonocyte plasma membrane cholesterol homeostasis, order, and receptor nanoclustering, 2) colonocyte cell proliferation, and 3) whether these effects are modulated by select membrane active dietaries (MADs). We observed that oncogenic APC-KRas increased membrane order by perturbing cholesterol homeostasis when cell proliferation is upregulated, in part by altering the expression of genes associated with cholesterol influx, export and de novo synthesis in mouse colorectal cancer (CRC) models and CRC patients. In addition, oncogeneinduced loss of cholesterol homeostasis altered Fzd7, LRP6, and KRas cluster structure/organization. Notably, we show that the combination of chemoprotective MADs, i.e., n-3 PUFAs and curcumin, reduced colonic membrane free cholesterol, order, receptor cluster size, cell proliferation, and the number of dysplastic foci in mutant APC-KRas models. This work highlights the dynamic shaping of plasma membrane organization during colon tumorigenesis and the utility of membrane-targeted cancer therapy. SIGNIFICANCE If current trends continue, the number of cancer cases diagnosed annually by 2050 is likely to double as a result of population aging. Therefore, if the healthcare community hopes to head off the coming storm, we need to get more serious about primary cancer prevention soon. Along these lines, although metabolic reprogramming is established as a hallmark of cancer, the contribution of lipid metabolism, especially cholesterol homeostasis, to colon tumorigenesis remains to be established. Thus, it is important to understand the impact of canonical cancer driver genes on cholesterol homeostasis and the functional properties of cholesterol in terms of modulating signaling pathways. In this study, we were able to demonstrate that oncogenic Apc and Kras increase plasma membrane order and protein, e.g., Wnt receptor and KRas, nanoclustering in colonocytes by modulating membrane free cholesterol levels during the early stages of tumorigenesis when colonic epithelial cell proliferation is upregulated. These changes, in part, were linked to the dysregulation of expression associated with cholesterol homeostatic genes. Notably, exogenous free cholesterol directly increased plasma membrane order and cell proliferation in mouse colonic organoids. As a means of membrane lipid therapy, the loss of plasma membrane homeostasis was normalized following administration of a chemoprotective diet (n-3 PUFAs + curcumin). Furthermore, n-3 PUFAs + curcumin reduced colonic membrane free cholesterol, order, receptor cluster size, cell proliferation, and the number of dysplastic foci in mutant APC-KRas mice. Collectively, these findings provide evidence that oncogenic Apc and Kras directly modulate the dynamic shaping of plasma membrane organization during colon tumorigenesis. Our findings demonstrate a novel biophysical link between plasma membrane cholesterol homeostasis, plasma membrane order, receptor nanoclustering, and plasma membrane-mediated signaling in the context of colon cancer has been described.