NFS1 activates PI3K/AKT/mTOR signaling to upregulate GPX4 expression and enhance ferroptosis resistance in osteosarcoma

Osteosarcoma continues to exhibit poor survival outcomes due to chemoresistance and metastasis, with metabolic reprogramming and ferroptosis resistance being key features of tumor heterogeneity, yet their upstream regulators remain poorly defined. NFS1, a cysteine desulfurase essential for iron-sulfur cluster biogenesis, protects multiple cancers from ferroptosis, but its role in osteosarcoma is unknown. In this study, we performed a transcriptomic meta-analysis and found that NFS1 expression was significantly upregulated in osteosarcoma tissues, with further elevation in metastatic lesions, and high NFS1 expression correlated with poor overall survival. Genome‑wide CRISPR screening data revealed a marked NFS1 dependency in osteosarcoma cell lines. Functionally, NFS1 promoted cell proliferation, migration, and invasion, whereas its knockdown suppressed these phenotypes. Using single‑cell RNA sequencing data from 27 osteosarcoma specimens, we applied a multi‑algorithm glycolytic scoring framework and observed NFS1 enrichment in highly glycolytic malignant cells, along with an association with PI3K/AKT/mTOR pathway activation. Mechanistically, NFS1 selectively enhanced PI3K, AKT, and mTOR phosphorylation without altering total protein levels, and upregulated GPX4, a central ferroptosis suppressor, leading to elevated ferroptosis resistance scores in NFS1‑high malignant cells. Collectively, these findings identify a previously unrecognized NFS1-PI3K/AKT/mTOR-GPX4 regulatory axis in osteosarcoma, linking metabolic reprogramming to ferroptosis resistance, and suggest that NFS1 functions as an oncogenic driver, as well as a promising prognostic biomarker and therapeutic target in osteosarcoma.