Everolimus destabilizes thymidylate synthase via suppressing its O-GlcNAcylation and sensitizes HER2-negative breast cancer to fluorouracil.

5-Fluorouracil (5-FU) and its prodrugs are widely used drugs for chemotherapy in various cancers. However, their effectiveness in breast cancer is limited. mTORC1 pathway, as a known mediator of therapy resistance in breast cancer, presents a compelling target for combination approaches. This study investigated the mTORC1 inhibitor everolimus as a sensitizer to 5-FU and capecitabine in breast cancer, exploring thymidylate synthase (the direct target of 5-FU) as a predictive biomarker and targeted mechanism. Our results demonstrate that everolimus significantly enhances 5-FU efficacy in HER2-negative breast cancer in vivo and in vitro. Mechanistically, everolimus downregulates thymidylate synthase (TYMS) by inducing its proteasomal degradation through a ubiquitination-independent way involving downregulation of O-GlcNAc transferase (OGT) and a reduced O-GlcNAcylation of TYMS, which destabilizes TYMS homodimer. Overexpression of OGT reversed TYMS degradation. Importantly, this combination strategy was effective in refractory breast cancer patients, and decreased levels of TYMS and OGT were observed in breast cancer patient specimens collected before and after everolimus-containing treatment. In conclusion, our study reveals that everolimus sensitizes breast cancer to fluoropyrimidines by destabilizing TYMS through modulation of its O-GlcNAcylation. These findings support a promising combination strategy to improve the therapeutic efficacy of 5-FU and capecitabine in breast cancer.