Transcriptional and translation control of thymidylate synthase (TYMS) is poorly understood, particularly in response to chemotherapeutic drugs such as 5-Fluorouracil (5-FU) and its derivatives. The current study addressed this gap by demonstrating a biphasic response in TYMS protein levels upon 5-FU treatment. Indeed, we observe an initial reduction within the first few hours, followed by a marked increase at 24 h. These changes occurred independently of transcriptional regulation, as TYMS mRNA levels remained stable during the early phase and showed only a moderate increase later. We further showed that neither autophagy nor proteasomal degradation contributed to this dynamic, but instead it is driven by a change in its translation. Using thermal proteome profiling, we identified SIN1, a key component of the mTORC2 complex, as a key regulator of TYMS protein levels. Our functional studies revealed that SIN1 depletion negatively alters TYMS levels and dynamics and sensitizes cancer cells to 5-FU-mediated cell death. Moreover, our data show that SIN1 protein is not only required for stress mediated increase in P53 activity, but also for TYMS translation. These findings uncover a novel mechanism controlling TYMS protein levels and suggest that targeting SIN1 may represent a promising strategy to enhance the therapeutic efficacy of 5-FU-based treatments.
Supplementary Information: The online version contains supplementary material available at 10.1186/s12964-025-02640-y.