Background: Advanced gastric cancer (GCa) remains highly lethal due to the lack of effective therapies. Identifying promising therapeutic targets and developing effective treatment against GCa are urgently needed. Here, we investigated whether Autophagy-related gene 4B (ATG4B) could be a potential therapeutic target against GCa and identified marine natural product Azalomycin F4a (Am-F4a) as a potent ATG4B inhibitor and a potential anticancer agent.
Methods: The expression of ATG4B in clinical GCa tumor specimens were examined by immunoblotting and interrogation of public databases. The association between ATG4B expression and patient’s survival was assessed. FRET, surface plasmon resonance, transmission electron microscopy analysis, enzyme activity assay and compute docking were used to assess the binding and inhibition of ATG4B by Am-F4a. RNA interference, inhibitors, CCK-8 cell viability assay, colony formation, apoptosis assay, wound-healing, transwell invasion assay, western blotting and Patient-derived organoids were used to assess the role of ATG4B in GCa cells. GCa cell-derived xenograft models, patient-derived xenografts and orthotopic metastasis xenografts were used to evaluate the anti-tumor growth and anti-metastasis effects of Am-F4a alone or in combination with 5-FU in vivo.
Results: ATG4B was highly upregulated in GCa tumors. Its high expression was associated with patient’s poor prognosis. knockdown of ATG4B significantly inhibited GCa cell survival and tumor growth. Am-F4a was identified as a novel and potent ATG4B inhibitor. Am-F4a effectively inhibited GCa cell autophagy and growth via targeting ATG4B both in vitro and in vivo. Besides, both pharmacological inhibition and knockdown of ATG4B significantly suppressed GCa cell migration and invasion. Am-F4a potently blocked the metastasis of primary GCa tumors and effectively sensitized tumors to chemotherapy.
Conclusion: These findings indicate that ATG4B is a potential novel therapeutic target against GCa and that the natural product Am-F4a is a novel ATG4B inhibitor that can be further developed for treatment of GCa.