Cellular membranes serve as an epicentre combining extracellular and cytosolic components with membranous effectors, which together support numerous fundamental cellular signalling pathways that mediate biological responses. To execute their functions, membrane proteins, lipids and carbohydrates arrange, in a highly coordinated manner, into well‐defined assemblies displaying diverse biological and biophysical characteristics that modulate several signalling events. The loss of membrane homeostasis can trigger oncogenic signalling. More recently, it has been documented that select membrane active dietaries (MADs) can reshape biological membranes and subsequently decrease cancer risk. In this review, we emphasize the significance of membrane domain structure, organization and their signalling functionalities as well as how loss of membrane homeostasis can steer aberrant signalling. Moreover, we describe in detail the complexities associated with the examination of these membrane domains and their association with cancer. Finally, we summarize the current literature on MADs and their effects on cellular membranes, including various mechanisms of dietary chemoprevention/interception and the functional links between nutritional bioactives, membrane homeostasis and cancer biology.

Background: Increased rates of cholesterol synthesis have been recognized as an important aspect of the metabolism of transformed cells. However, precisely how cholesterol dysmetabolism affects membrane homeostasis is not yet fully understood. Since free cholesterol can be transported from the plasma membrane to other organelles in a dynamic and bidirectional fashion, there is an urgent need to determine to what extent endomembranes are affected by driver oncogene-mediated distortions in cholesterol homeostasis. Aim: Determine if cholesterol is increased in endomembranes, relative to the plasma membrane in Apc mutant cell lines and primary Apc null mouse colonocytes amd CRC samples. Methods: Three mouse epithelial isogenic wild type and mutant Apc colonic cell lines (YAMC (Apc +/+), IMCE (Apc +/-), IMCE βcat (Apc +/- + ΔN89 βcat) were used. In addition, primary colonocytes from Apc wildtype and null mice were examined. mRNA was sequenced using a TruSeq Illumina Stranded mRNA kit. Data analysis was conducted as follows: EdgeR was used to identify differentially expressed genes. Human CRC RNAseq data was obtained from a public reservoir and analyzed. A gene target list (~ 290 genes) related to organelle cholesterol transport was subsequently queried. Membranes from the endoplasmic reticulum (ER), mitochondria and lysosomes were assessed using fluorescent probes: ERTracker, MitoTracker and LysoTracker, respectively. Cells were fixed and stained with Filipin III and imaged using confocal microscopy. Fluorescence colocalization was calculated and statistical analysis was performed using a one-way ANOVA. Results: Apc mutant colonocyte cell lines and Apc null mouse models and human CRC samples exhibited differential expression of cholesterol trafficking genes including Acaa1b, Acaa2, Pcsk9, Stard5. Genes related to mitochondrial cholesterol metabolism, transport, and accumulation, e.g., Tspo, Ppargc1a and Cyp27a, were also found differentially expressed in the three models. Imaging experiments revealed that mitochondrial cholesterol was 17% higher in IMCE and IMCE βcat cell lines. Lysosomal cholesterol was 21% higher in IMCE cells. ER cholesterol levels were unaffected. Conclusions: Our preliminary findings indicate dysregulation of mitochondrial and lysosomal unesterified cholesterol levels in colonocytes expressing mutant APC. Citation Format: Monica Munoz-Vega, Alfredo Erazo-Oliveras, Michael L. Salinas, Xiaoli Wang, Jennifer S. S Goldsby, Robert S. Chapkin. Endomembranes accumulate unesterified cholesterol in Apc mutant models and humans with CRC as assessed by RNAseq and confocal microscopy. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P016.

Colorectal cancer (CRC) is the 3rd most common type of cancer in the U.S., and it is estimated it will account for an alarming 151,030 (8%) of new total cancer cases and 52,580 (9%) of total cancer deaths in 2022. Remarkably, dysregulation of the Wnt signaling pathway has been linked to ~90% of all colorectal cancer (CRC) cases. The vast majority (>80%) of sporadic CRC cases display mutations in Adenomatous Polyposis Coli (APC), a central Wnt signaling regulator. Loss of APC function causes aberrant stabilization of β-catenin (βcat), a crucial step in CRC initiation. Notably, attempts towards “drugging” this pathway still poses multiple hurdles due to poor tumor cell targeting, negative side effects associated with required long-term treatments and poorly understood mechanisms of action. Consequently, there is an urgent need to gather additional mechanistic insights associated with the Wnt pathway to develop novel therapeutic approaches. With respect to Wnt biology, two key plasma membrane receptors, LRP5/6 and Frizzled (Fzd) require lipid raft localization and nanoclustering for efficient signaling. Interestingly, various effectors that disrupt raft dynamics, alter LRP5/6-Fzd clustering, leading to reduced βcat stabilization. Thus, we examined the effect of mutant APC (mAPC) on plasma membrane lipid/protein-mediated interactions of Wnt-associated effectors in a CRC mouse model, human mAPC-expressing organoids and cell lines, and Drosophila. We show for the first time that mAPC significantly increased the levels of plasma membrane free cholesterol, a major component of lipid rafts known to selectively activate canonical Wnt signaling, in the intestinal epithelium including Lgr5+ colonic stem cells. Moreover, this change in plasma membrane cholesterol was associated with alterations in colonocyte membrane rigidity and raft organization. We subsequently examined the effect of mAPC on the interactions between Wnt signaling receptors/effectors in mAPC-expressing cell lines. mAPC significantly increased LRP6 and Fzd7 homo-clustering and LRP6-Fzd7 hetero-clustering as well as their interactions with key lipids, e.g., PIP2, resulting in the enhancement of Wnt signaling activation. Interestingly, the effects of mAPC were recapitulated by the addition of exogenous cholesterol to wild type colonocytes. Finally, we demonstrate that n-3 polyunsaturated fatty acids (n3-PUFA), i.e., DHA and EPA, reduces mAPC-driven colonic polyp formation in mice in part by reducing plasma membrane cholesterol, rigidity and LRP6-Fzd7 clustering in colonocytes. Collectively, these findings indicate that mAPC can directly perturb lipid homeostasis in the colon, thereby altering raft stability, LRP6-Fzd7 membrane nanoclusters and downstream Wnt signaling and supports the feasibility of using membrane therapy, i.e., dietary/drug-related strategies to target plasma membrane lipid composition, to reduce Wnt signaling and cancer risk. Citation Format: Alfredo Erazo-Oliveras, Mohamed Mlih, Mónica Muñoz-Vega, Eunjoo Kim, Venkat Thiriveedi, Michael L. Salinas, Xiaoli Wang, Jatin Roper, Jason Karpac, Robert S. Chapkin. A novel role of mutant APC and n3-PUFA in reshaping cholesterol-dependent Wnt pathway-associated proteolipid nanocluster organization and signaling in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P014.