LF3 exerts anti-osteosarcoma effects through Wnt signaling inhibition-coupled ferroptosis induction via HO-1/ACSL4...

Osteosarcoma, the most common primary malignant bone tumor, predominantly affects adolescents and is characterized by high aggressiveness and early metastatic propensity. Chemoresistance and recurrence remain major clinical challenges, highlighting the urgent need for novel therapeutic targets and molecular mechanisms.

Bioinformatics analysis of the GEO dataset (GSE42352) revealed a negative correlation between Wnt target gene expression and ferroptosis‑related gene signatures. The expression of Wnt and ferroptosis pathway genes/proteins, as well as autophagy and pyroptosis markers, was validated in 143B and MG63 cells by qRT‑PCR and Western blotting. ROS, Fe²⁺, and lipid peroxidation levels were measured using DCFH‑DA, FerroOrange, and Liperfluo, respectively. Mitochondrial morphology was assessed by transmission electron microscopy (TEM). Cell proliferation, migration, and invasion were evaluated by live/dead staining, wound healing assay, and Transwell invasion assay, respectively. Functional roles of HO‑1 and ACSL4 were validated via siRNA‑mediated knockdown in 143B cells. An osteosarcoma xenograft model was established; tumor expression of ACSL4, HO‑1, and Ki67 was examined by IHC, and serum ferritin levels were measured by ELISA.

Bioinformatics analysis of the GEO dataset (GSE42352) revealed a negative correlation between Wnt target gene expression and ferroptosis‑related gene signatures, suggesting that ferroptosis may be involved. In 143B and MG‑63 cells, LF3 (40 μM) downregulated Wnt target genes (AXIN2, BMP4, SMAD6, LEF1) and TCF4 protein expression, significantly inhibiting cell proliferation, migration, and invasion. LF3 treatment induced ROS elevation, lipid peroxidation, and characteristic mitochondrial morphological changes, independent of apoptosis, autophagy, or pyroptosis. Mechanistic studies demonstrated that LF3 activated ferroptosis through upregulation of HO‑1 and ACSL4/LPCAT3. In an osteosarcoma xenograft model, LF3 reduced tumor volume by approximately 60%, accompanied by elevated serum ferritin and tumor 4‑HNE levels; these effects were partially reversed by DFO co‑treatment. No systemic toxicity was observed.

These findings establish LF3 as a dual-targeting agent that overcomes osteosarcoma through coordinated Wnt suppression and ferroptosis activation via the HO-1/ACSL4 axis, providing a novel therapeutic strategy for chemoresistant osteosarcoma.