Autophagy Protease <scp>ATG4D</scp> Facilitates Proliferation and Malignancy of Osteosarcoma Cells

Osteosarcoma is one of the most prevalent malignant tumors in pediatric cancer, with survival rates remaining stagnant for over a decade, particularly among patients with metastatic disease. Thus, identifying novel therapeutic targets is critical for improving clinical outcomes. The ATG4 family (ATG4A, ATG4B, ATG4C, and ATG4D) encodes proteases essential for autophagy, a process implicated in cancer progression and drug resistance. However, the role of ATG4 proteins in osteosarcoma remains unclear. This study showed that silencing ATG4 family members using small interfering RNA (siRNA) induced G1-phase cell cycle arrest and promoted cell death in osteosarcoma cells. Among them, ATG4D knockdown significantly impaired cell migration and invasion. Consistently, stable knockdown of ATG4D via short hairpin RNA (shRNA) reduced cell motility and tumorsphere formation. Moreover, ATG4D depletion enhanced autophagic markers, including LC3B-II puncta and p62 protein levels, and sensitized osteosarcoma cells to starvation and chemotherapy-induced cell death. In vivo, osteosarcoma cells harboring ATG4D-targeting shRNA exhibited reduced tumor growth and elevated apoptosis in xenografted mice compared to control cells. Clinically, ATG4D protein expression was elevated in osteosarcoma tissues compared to normal bone cells, with higher levels correlating with poor overall survival, particularly in patients older than 10 years or with tumors located in the lower limbs. These findings suggest that ATG4D may serve as a potential diagnostic biomarker and therapeutic target for osteosarcoma.