Introduction:
Osteosarcoma (OS) is a highly malignant bone tumor with limited treatment options. The role of Apolipoprotein E (APOE) in OS remains unclear. This study explores the impact of APOE overexpression on OS, particularly its effects on ferroptosis and autophagy.

Material and methods:
APOE was identified as a key gene through weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network analysis of the GSE28424 dataset. APOE was overexpressed in OS cell lines to evaluate its effects on cell behavior. The role of autophagy was investigated using the autophagy inhibitor 3-MA. The involvement of ferroptosis and the mTOR/Stat3 signaling pathway was investigated utilizing Quantitative real-time reverse transcription PCR (qRT-PCR), Western blot (WB), and flow cytometry. A mouse xenograft model was employed to validate the in vitro results.

Results:
APOE overexpression significantly inhibited OS cell proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT), with 3-MA partially reversing these effects. APOE overexpression also inhibited the mTOR and Stat3 expression, enhancing autophagy, as shown by increased LC3B-1, LC3B-2, and Beclin1 expression. Additionally, APOE overexpression promoted apoptosis, associated with increased reactive oxygen species (ROS) and intracellular Fe²⁺ levels, and altered ferroptosis-related gene expression, including upregulation of TfR1 and downregulation of FPN, GPX4, and SLC7A11. In vivo, APOE overexpression in a mouse xenograft model resulted in significantly smaller tumors, with changes in autophagy and ferroptosis markers consistent with in vitro findings.

Conclusions:
APOE overexpression suppresses osteosarcoma growth by promoting ferroptosis and autophagy through the mTOR/Stat3 signaling pathway, highlighting its promise as a target for OS therapeutic intervention.