The majority of colon tumors are driven by aberrant Wnt signaling in intestinal stem cells, which mediates an efficient route toward initiating intestinal cancer. Natural lipophilic polyphenols and long-chain polyunsaturated fatty acids (PUFAs) generally suppress Wnt- and NF-κB- (nuclear factor-κ light-chain enhancer of activated B-cell) related pathways. However, the effects of these extrinsic agents on colonic leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5⁺) stem cells, the cells of origin of colon cancer, have not been documented to date. Therefore, we examined the effect of n-3 PUFA and polyphenol (curcumin) combination on Lgr5⁺ stem cells during tumor initiation and progression in the colon compared with an n-6 PUFA-enriched control diet. Lgr5-EGFP-IRES-^creERT2 knock-in mice were fed diets containing n-6 PUFA (control), n-3 PUFA, n-6 PUFA+curcumin or n-3 PUFA+curcumin for 3 weeks, followed by 6 azoxymethane (AOM) injections, and terminated 17 weeks after the last injection. To further elucidate the effects of the dietary bioactives at the tumor initiation stage, Lgr5⁺ stem cells were also assessed at 12 and 24 h post AOM injection. Only n-3 PUFA+curcumin feeding reduced nuclear β-catenin in aberrant crypt foci (by threefold) compared with control at the progression time point. n-3 PUFA+curcumin synergistically increased targeted apoptosis in DNA-damaged Lgr5⁺ stem cells by 4.5-fold compared with control at 12 h and maximally reduced damaged Lgr5⁺ stem cells at 24 h, down to the level observed in saline-treated mice. Finally, RNAseq analysis indicated that p53 signaling in Lgr5⁺ stem cells from mice exposed to AOM was uniquely upregulated only following n-3 PUFA+curcumin cotreatment. These novel findings demonstrate that Lgr5⁺ stem cells are uniquely responsive to external dietary cues following the induction of DNA damage, providing a therapeutic strategy for eliminating damaged Lgr5⁺ stem cells to reduce colon cancer initiation.

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 theDrosophilasterol 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 link between oncogenic APC, loss of plasma membrane homeostasis and CRC development.

Bis-indole-derived compounds including 1,1-bis(3′-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) analogs bind both orphan nuclear receptors 4A1 (NR4A1) and NR4A2, and DIM-3,5 compounds act as dual receptor inverse agonists and inhibit both NR4A1- and NR4A2-regulated responses. Chromatin immunoprecipitation assays show that β1-integrin and the methyltransferase gene G9a are regulated by both NR4A1 and NR4A2 acting as cofactors for Sp1- and Sp4-dependent gene expression. DIM-3,5 treatment results in the loss of one or more of these nuclear factors from the β1-integrin and G9a promoters. Single-cell and RNAseq analyses show that both receptors regulate common (<10%) and unique genes in SW480 colon cancer cells; however, functional enrichment analysis of the differentially expressed genes converges to several common pathways and gene ontology terms.

Early-life microbial colonization is essential for gut and immune development. Human milk oligosaccharides (HMOs) support the growth of Bifidobacterium infantis (BI). Here, we studied the individual and combined effects of BI and HMOs on the immune and colon transcriptomes and on serum and cecal metabolome. Germ-free mice were randomly assigned to four groups (10–14/group: HMO, BI, BI + HMO, and control [no HMO or BI]). HMO and BI + HMO groups received 5 mg/day each of 2′-fucosyllactose, lacto-N-tetraose, and 3′-sialyllactose for 14 days. BI and BI + HMO received BI ATCC 15,697 (1 × 10 ⁹ CFU/day) on days 1, 4, and 9. Mono-colonization with BI increased monocytes, macrophages, B cells, CD4 ⁺ T cells, and Treg cells in mesenteric lymph nodes (MLN) relative to controls. In the spleen, BI alone increased B cells, dendritic cells, Th17 cells, and ILC3 cells, and enriched serum amino acid metabolism pathways. Additionally, BI influenced colonocyte gene expression and modulated serum metabolites that regulate circadian rhythms. BI + HMO increased MLN Th17 cells and spleen monocytes compared with HMO alone. Collectively, the results of this study highlight the complex interplay among host-microbe-diet interactions and emphasize the importance of considering these interactions when designing strategies to modulate infant health during early life. IMPORTANCE: Early life immune and gut microbiome development are shaped by human milk (HM). One of the most important drivers of these processes is the human milk oligosaccharides (HMOs). Bifidobacterium infantis (BI) possesses a unique enzymatic system that enables efficient HMO uptake and intracellular metabolism, providing a competitive advantage over other microbial species in the breastfed infant gut. To date, the potential direct and synergistic effects of BI and HMO have not been fully explored. The knowledge generated herein identified the independent and synergistic effects of HMOs and BI on gut immune response, serum and cecal metabolites, and colonic gene expression.