Gentisic acid prevents colorectal cancer metastasis via blocking GPR81-mediated DEPDC5 degradation

•Gentisic acid (GA) acts as an inhibitor of GPR81 in CRC.•Inhibition of CRC metastasis by GA is due to the suppression of GPR81-mediated mTOR signaling.•GA inhibits mTOR signaling by preventing GPR81/CMA-mediated DEPDC5 degradation. Metastasis driven by epithelial-mesenchymal transition (EMT) remains a significant contributor to the poor prognosis of colorectal cancer (CRC), and requires more effective interventions. GPR81 signaling has been linked to tumor metastasis, while lacks an efficient specific inhibitor. Our study aimed to investigate the effect and mechanism of Gentisic acid on colorectal cancer (CRC) metastasis. A lung metastasis mouse model induced by tail vein injection and a subcutaneous graft tumor model were used. Gentisic acid (GA) was administered by an intraperitoneal injection. HCT116 was treated with lactate to establish an in vitro model. MC38 cells with mCherry fluorescent protein were injected into tail vein to investigate lung metastasis ability in vivo. GA was administered by intraperitoneal injection for 3 weeks. The therapeutic effect was evaluated by survival rates, histochemical analysis, RT-qPCR and live imaging. The mechanism was explored using small interfering RNA (siRNA), Western blotting, RT-qPCR and immunofluorescence. GA had a therapeutic effect on CRC metastasis and improved survival rates and pathological changes in dose-dependent manner. GA emerged as an GPR81 inhibitor, effectively suppressed EMT and mTOR signaling in CRC induced by lactate both in vivo and in vitro. Mechanistically, GA halted lactate-induce degradation of DEPDC5 through impeding the activation of Chaperone-mediated autophagy (CMA). CMA-mediated DEPDC5 degradation is crucial for lactate/GPR81-induced CRC metastasis, and GA may be a promising candidate for metastasis by inhibiting GPR81 signaling. [Display omitted]