Regulation of 5‐fluorodeoxyuridine monophosphate‐thymidylate synthase ternary complex levels by autophagy confers resistance to 5‐fluorouracil

5‐Fluorouracil (5‐FU) is a cornerstone drug used to treat colorectal cancer (CRC). However, the prolonged exposure of CRC cells to 5‐FU results in acquired resistance. We have previously demonstrated that levels of the 5‐fluorodeoxyuridylate (FdUMP) covalent complex with thymidylate synthase (FdUMP‐TS) and free‐TS (native enzyme) are higher in 5‐FU‐resistant CRC cells than in the parental cell line (HCT116). Accordingly, resistant cells may have an efficient system for trapping and removing FdUMP‐TS, thus imparting resistance. In this study, using a model of 5‐FU‐resistant CRC cells generated by repeated exposure, the role of autophagy in the elimination of FdUMP‐TS in resistant cells was investigated. The resistant cells showed greater sensitivity to autophagy inhibitors than that of parental cells. Autophagy inhibition increased 5‐FU cytotoxicity more substantially in resistant cells than in parental cells. Furthermore, autophagy inhibition increased FdUMP‐TS protein accumulation in resistant cells. Our findings suggest that resistance to 5‐FU is mediated by autophagy as a system to eliminate FdUMP‐TS and may guide the use and optimization of combination therapies involving autophagy inhibitors.