Background <p>In the therapeutic landscape of colorectal cancer (CRC), chemo-resistance poses a significant and prevalent obstacle that complicates treatment efficacy and patient outcomes. Over time, cancer cells can develop mechanisms to resist the toxic effects of chemo-therapy drugs, leading to reduced sensitivity or complete insensitivity to these agents. The enzyme Arylamine N-acetyltransferase 1 (NAT1) has emerged as a promising target in strategies aimed at overcoming this challenge. NAT1 is involved in the metabolism of various xenobiotics, including some chemotherapeutic agents. Understanding the complex interactions between NAT1 and chemotherapeutic agents, as well as the molecular mechanisms underlying chemo-resistance, is crucial for the development of novel therapeutic approaches. </p> Objective <p>This study aimed to assess the role of NAT1 in mediating chemo-resistance in CRC, with the goal of identifying novel strategies to overcome this clinical challenge.</p> Methods <p>We conducted a comprehensive analysis using various bioinformatics tools and in vitro experiments to evaluate the effect of NAT1 expression on chemo-resistance in CRC. Furthermore, we employed a multi-omics approach, including metabolomics and next-generation sequencing, to uncover the mechanisms by which NAT1 influences chemo-resistance. Additionally, we utilized single-cell RNA sequencing (scRNA-seq), the Cellchat assay, and western blot to explore the intercellular communication between tumor and endothelial cells in the context of anti-PD-1 therapy and NAT1’s impact.</p> Results <p>Our study reveals that decreased NAT1 expression in CRC tumor tissues, relative to adjacent normal tissues, is significantly associated with a poorer patient prognosis. Experimental data indicate that silencing NAT1 in CaCO2 and HCT116 cell lines results in heightened resistance to five chemotherapeutic agents: vinblastine, docetaxel, gemcitabine, vincristine, and daporinad. Additionally, NAT1 silencing increases the proportion of LGR5⁺ cells, which are known to be chemo-resistant. Our research further revealed that exposure to these five drugs induces a decrease in NAT1 expression within CRC cells. Mechanistic insights show that NAT1 knockdown triggers a metabolic reprogramming in CRC cells, shifting from oxidative phosphorylation and the tricarboxylic acid cycle to a preference for glycolysis. Furthermore, silencing of NAT1 in CRC cells leads to an up-regulation of VEGFA expression. Notably, the application of anti-PD-1 therapy was demonstrated to significantly disrupt the VEGFA-VEGFR axis signaling, an interaction critical between CRC cells and endothelial cells. This discovery underscores the potential of targeting the VEGFA pathway as a therapeutic approach to mitigate the adverse effects associated with NAT1 down-regulation in CRC.</p> Conclusion <p>Our study underscores the multifaceted role of NAT1 in modulating chemo-sensitivity, cellular metabolism, and angiogenesis in CRC. These findings position NAT1 as a compelling candidate for a biomarker and a potential therapeutic target, offering new avenues for CRC management.</p> Graphical abstract <p></p>