PRMT1-Catalyzed NUSAP1 Methylation Enhances Notch2 Signaling and 5-FU Resistance in Gastric Cancer

5-Fluorouracil (5-FU) resistance remains a significant challenge in the treatment of gastric cancer, limiting its clinical efficacy. Our study identifies NUSAP1, a nucleolar and spindle-associated protein, as a key driver of 5-FU resistance in gastric cancer. Proteomic analyses of 5-FU-resistant gastric cancer cell lines revealed that NUSAP1 is significantly upregulated, and functional studies demonstrated its essential role in promoting resistance, proliferation, migration, invasion, and tumor growth. Mechanistic investigations revealed that NUSAP1 undergoes asymmetric dimethylation (ADMA) at R418 and R422, mediated by PRMT1, with the R422 site being critical for its function. NUSAP1 interacts with the PEST domain of Notch2 through its R422 site, inhibiting Notch2 ubiquitination and stabilizing its expression, thereby activating the Notch2 signaling pathway. This pathway is closely linked to gastric cancer progression and chemoresistance. Inhibition of PRMT1 or mutation of the R422 site abrogated NUSAP1’s ability to stabilize Notch2 and regulate downstream signaling. These findings unveil a novel mechanism by which NUSAP1 promotes 5-FU resistance in gastric cancer and highlight the therapeutic potential of targeting the NUSAP1-Notch2 axis or PRMT1 in overcoming chemoresistance.