SHIP2-SRC-β-catenin signaling axis sustains thymidylate synthase expression and promotes fluoropyrimidine resistance.

Fluoropyrimidine-based chemotherapies, including 5-fluorouracil (5-FU) and floxuridine (FuDR), are widely used in cancer treatment, but their efficacy is limited by adaptive resistance driven by TYMS upregulation. The upstream mechanisms controlling TYMS expression remain poorly defined. Here, we identify INPPL1 (SHIP2) as a critical regulator of TYMS expression and fluoropyrimidine response in breast cancer cells. We show that SHIP2 enhances basal and drug-induced TYMS expression at the transcriptional level independently of its phosphatase activity. Mechanistically, SHIP2 increases SRC levels and nuclear accumulation of {beta}-catenin, driving TYMS expression. Inhibition of SRC or {beta}-catenin suppresses TYMS induction and restores sensitivity to FuDR. Importantly, SHIP2 rewires TYMS regulation from a P53-dependent program to a {beta}-catenin-driven pathway, enabling sustained TYMS expression under chemotherapeutic stress. Consistent with this model, differential sensitivity to SHIP2 depletion correlates with baseline TYMS levels across cell lines. Analysis of patient cancer datasets reveals that high INPPL1 expression correlates with increased TYMS levels and poor clinical outcomes. These findings identify SHIP2 as a non-canonical regulator of TYMS and a potential therapeutic target to overcome fluoropyrimidine resistance.