TYMS Enhances Colorectal Cell Antioxidant Capacity Via the KEAP1-NRF2 Pathway to Resist Ferroptosis

<b>Purpose:</b> Thymidylate synthase (TYMS) is a key regulatory enzyme in DNA synthesis. We identified the biological effect and molecular mechanisms of TYMS in colorectal cancer (CRC). <b>Methods:</b> We employed western blot and immunohistochemistry for the assessment of TYMS expression in CRC samples. MTT and colony assay were carried out to illuminate the effect of TYMS on the proliferation of CRC cells. Xenograft models were constructed to evaluate the consequences of TYMS overexpression on CRC <i>in vivo</i>. Metabolomics was utilized to analyze the alterations in cellular molecular metabolites subsequent to TYMS overexpression. The impact of TYMS on NRF2 localization and KEAP1 expression was explored by means of western blot. The expression levels of GSH, ROS, MDA, and <i>PTGS2</i> mRNA were measured to assess ferroptosis. <b>Results:</b> TYMS expression in CRC tumor tissues was upregulated compared to adjacent non-cancerous tissues. Cells overexpressing TYMS displayed enhanced proliferative capabilities. Metabolomic analysis revealed that overexpression of TYMS was associated with elevated levels of GSH within cells and a decrease in the lipid peroxidation product, 4-hydroxyhexenal. ROS detection assays further demonstrated a significant enhancement in cellular antioxidant capacity due to TYMS overexpression. Overexpression of TYMS downregulated KEAP1 expression and promoted NRF2 translocation into the nucleus. Consequently, transcription of downstream antioxidant genes was upregulated, enhancing cellular antioxidant capacity, reducing ROS levels, diminishing lipid peroxidation products, and heightening resistance to ferroptosis induced by erastin. Additionally, our study indicated that the TYMS inhibitor 5-fluorouracil (5-FU) exhibited favorable drug synergism with erastin. <b>Conclusion:</b> TYMS was overexpressed in CRC, which was correlated with poor prognosis of CRC patients. TYMS enhanced the antioxidant capacity of CRC cells via the KEAP1-NRF2 pathway, thereby increasing resistance to erastin-induced ferroptosis.