Microenvironment-induced CREPT expression by cancer-derived small extracellular vesicles primes field cancerization

Cancer local recurrence increases the mortality of patients, and might be caused by field cancerization, a pre-malignant alteration of normal epithelial cells. It has been suggested that cancer-derived small extracellular vesicles (CDEs) may contribute to field cancerization, but the underlying mechanisms remain poorly understood. In this study, we aim to identify the key regulatory factors within recipient cells under the instigation of CDEs. In vitro experiments were performed to demonstrate that CDEs promote the expression of CREPT in normal epithelial cells. TMT-based quantitative mass spectrometry was employed to investigate the proteomic differences between normal cells and tumor cells. Loss-of-function approaches by CRISPR-Cas9 system were used to assess the role of CREPT in CDEs-induced field cancerization. RNA-seq was performed to explore the genes regulated by CREPT during field cancerization. CDEs promote field cancerization by inducing the expression of CREPT in non-malignant epithelial cells through activating the ERK signaling pathway. Intriguingly, CDEs failed to induce field cancerization when CREPT was deleted, highlighting the importance of CREPT. Transcriptomic analyses revealed that CDEs elicited inflammatory responses, primarily through activation of the TNF signaling pathway. CREPT, in turn, regulates the transduction of downstream signals of TNF by modulating the expression of TNFR2 and PI3K, thereby promoting inflammation-to-cancer transition. CREPT not only serves as a biomarker for field cancerization, but also emerges as a target for preventing the cancer local recurrence. Overall design: To investigate the role of CREPT in CDEs-treated non-malignant epithelial cells, we utilized two mammary epithelial cell lines, namely MCF10A and NMuMG, and employed the CRISPR-Cas9 system to knockout the CREPT gene in these cells, resulting in the generation of four cell lines: MCF10A, MCF10AKO, NMUWT, and NMUKO. Then we performed gene expression profiling analysis using data obtained RNA sequencing of these four cell lines under conditions with or without CDEs treatment. Comparative analysis of gene expression profiles based on RNA-Seq data in the cells with and without CDEs treatment. Subsequently, the differences in the expression profile changes between the cells with and without CREPT expression were evaluated.