UBE2S promotes 5-fluorouracil resistance in colorectal cancer by activating non-canonical NF-κB/TYMS/TK1 signaling

Background Chemoresistance to 5-fluorouracil (5-FU) remains a formidable barrier to the successful treatment of colorectal cancer (CRC). This resistance is largely driven by the metabolic rewiring of the pyrimidine synthesis pathway, specifically failure to suppress key enzymes. We aimed to investigate the role of the ubiquitin-conjugating enzyme E2S (UBE2S) as a master regulator of 5-FU resistance in colorectal cancer. Methods A 5-FU-resistant HCT116 cell model (HCT116R) was established through stepwise dose escalation. Cell viability was assessed via MTT assays following 5-FU treatment. Molecular mechanisms were explored using Western blot, qPCR, and cell proliferation assays. Functional validation was performed through overexpression, siRNA, and CRISPRi (CRISPR interference)-mediated knocldown assays. In vivo efficacy was validated using a zebrafish xenograft model, and pharmacological inhibition was tested using the IKK inhibitor BMS-345541 and NF-κB inhibitor JSH-23. Results HCT116R cells exhibited significantly higher viability than parental HCT116 cells following 24 h of 80 µM 5-FU treatment, as well as 48 h of treatment across a range of 10 to 80 µM. UBE2S was significantly upregulated in HCT116R cells at both the mRNA (9.45-fold) and protein levels. Furthermore, thymidylate synthase (TYMS) and thymidine kinase 1 (TK1), which bridge the de novo and salvage nucleotide synthesis pathways, showed significantly increased mRNA and protein expression in HCT116R cells. siRNA-mediated knockdown of UBE2S in HCT116R cells and UBE2S overexpression in HCT116 cells significantly regulated the mRNA and protein levels of both TYMS and TK1. Additionally, the use of CRISPRi-mediated UBE2S knockdown of HCT116R stable cell lines, or treatment with the inhibitors BMS-345541 and JSH-23, disrupted this metabolic defense and synergistically enhanced 5-FU-induced cytotoxicity. In zebrafish xenografts, UBE2S depletion markedly suppressed tumor growth and restored 5-FU sensitivity. Conclusions Our findings reveal a novel UBE2S-dependent non-canonical NF-κB signaling that governs CRC chemoresistance. By coordinating the TYMS/TK1 "double-lock" defense through NF-κB signaling, UBE2S serves as a potent prognostic biomarker and a high-value therapeutic target for overcoming fluoropyrimidine resistance in clinical settings.