High dietary cholesterol promotes epithelial-to-mesenchymal transition and invasion in colorectal cancer via the Wnt/Snai2 axis of transcriptional regulation

Introduction: Colorectal cancer (CRC) progression is frequently influenced by diet and lifestyle factors, and these variables can affect clinical outcomes. We have previously reported that modulation of cholesterol levels can facilitate epithelial-to-mesenchymal transition (EMT) and invasion in CRC. However, the exact signaling mechanisms driving this process have not yet been identified. In this study, we sought to further characterize the invasive phenotype associated with high cholesterol in vivo , and to investigate transcriptome-level changes that occur in response to a high cholesterol diet, in search of mechanistic insights. Methods: We generated a variable-diet APC fl/fl CDX-Cre ER CRC mouse model, in which the mice were fed either a high cholesterol diet (2% cholesterol), a low cholesterol diet (1% cholesterol), or a chow diet (0% cholesterol) for 6-8 weeks. Tumor growth was induced by intraperitoneal tamoxifen injection. Four weeks after injection, colon tissue samples were collected. Formalin-fixed sections were stained with hematoxylin and eosin (H+E), and examined for histological indicators of invasion by a clinical pathologist in a blinded manner. The epithelial markers claudin-1, Zo-1, and E-cadherin were stained by multiplex immunofluorescence. Frozen colon samples from chow diet-fed and high cholesterol-fed mice were submitted for bulk RNA sequencing. KEGG pathway enrichment analysis of differentially expressed genes was performed using the limma package in R. Results: H+E-stained colon tumor sections from mice fed the high cholesterol diet showed markedly more extensive desmoplastic reactions and increased cancer invasiveness compared to the chow diet-fed mice, with low cholesterol-fed mice displaying an intermediate phenotype. Immunofluorescence revealed that expression and junctional co-localization of claudin-1, Zo-1, and E-cadherin were consistently downregulated in the tumor cells of high cholesterol-fed mice compared to mice fed either the low cholesterol or chow diet. Expression of genes involved in the Wnt signaling pathway was found to be significantly upregulated in high cholesterol-fed mice relative to chow diet-fed mice, as was expression of the EMT transcription factor Snai2 (Slug). Conclusions: High dietary cholesterol promotes an EMT-like phenotype in CRC, which involves a loss of epithelial gene expression and activation of Wnt signaling. Notably, high cholesterol increases expression of the transcription factor Snai2 (Slug), which is known to be induced by Wnt signaling and to suppress expression of Cldn1, Zo1, and E-cadherin. These findings indicate that high cholesterol promotes EMT in CRC via a Wnt-dependent mechanism of transcriptional regulation, which could potentially increase the risk of invasion and metastasis in CRC patients affected by high cholesterol.