TPX2 promotes osteosarcoma progression by conferring ferroptosis resistance through the AURKA/NRF2 signaling axis

Background Ferroptosis has emerged as a potential avenue for cancer therapy; however, the molecular mechanisms regulating ferroptosis resistance in osteosarcoma (OS) remain incompletely understood. This study aimed to investigate the role of targeting protein for Xklp2 ( TPX2 ) in OS progression and ferroptosis, focusing on the aurora kinase A ( AURKA ) / nuclear factor erythroid 2-related factor 2 ( NRF2 ) signaling axis. Methods OS-related genes were screened from GSE16088 and GSE42352, followed by functional enrichment, protein-protein interaction (PPI), and receiver operating characteristic (ROC) analyses. TPX2 expression and function in OS cells were evaluated via quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, cell counting kit-8 (CCK-8), colony formation, and flow cytometry. Ferroptosis was evaluated in U2OS cells by cellular sensitivity to erastin/RSL3, reactive oxygen species (ROS) accumulation, Fe 2+ , malondialdehyde (MDA) levels, glutathione (GSH) redox status, and solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) expression. TPX2 and AURKA interaction in U2OS cells was probed using rescue and co-immunoprecipitation (Co-IP) experiments. The stability and ubiquitination level of NRF2 protein were detected using a cycloheximide (CHX) tracing assay and ubiquitination assay. Potential AURKA phosphorylation sites on NRF2 were further assessed using sequence-based kinase scoring. Results TPX2 was abundantly expressed in OS and facilitated cell proliferation, clonogenicity, and G2-phase advancement. In U2OS cells, TPX2 depletion increased sensitivity to erastin and RSL3, ROS, Fe 2+ , and MDA levels and decreased GSH/GSSG, SLC7A11, and GPX4, which were partially reversed by ferrostatin-1. TPX2 interacted with AURKA, and rescue studies supported AURKA involvement in TPX2-dependent ferroptosis regulation. AURKA sustained NRF2 protein stability by reducing its ubiquitination and degradation. However, no NRF2 site reached the predefined AURKA scoring threshold. TPX2 overexpression had anti-ferroptotic effects, which were suppressed by NRF2 silencing. Conclusion TPX2 contributes to OS cell proliferation and ferroptosis resistance in U2OS cells through an AURKA/NRF2 -associated mechanism. Direct NRF2 phosphorylation by AURKA remains unproven, and the ferroptosis mechanism requires validation in additional OS models.