ANXA3 activates HIF-1α/VEGF Signaling Via the PI3K/AKT/mTOR Pathway to Promote Osteosarcoma Stem Cell-Like Phenotype

BACKGROUND: Aberrant activation of HIF‑1α contributes to the maintenance of CSC phenotypes in various malignancies. Our previous work revealed that ANXA3 is up‑regulated in OS cells. Here, we further investigated how ANXA3 regulates CSC‑like properties of OS cells through the HIF‑1α/VEGF signaling pathway. METHODS: Lentiviral vectors were used to modulate ANXA3 expression in OS cell lines. The expression of stemness‑associated genes was examined by qPCR and Western blotting, and sphere‑formation assays were performed to evaluate CSC self‑renewal capacity. Migration, invasion, and clonogenic survival under cisplatin treatment were assessed using Transwell and colony‑formation assays. An orthotopic intratibial implantation model in NOD-SCID mice was established to evaluate the effects of ANXA3 on tumor growth, metastasis, and CSC‑related features. Potential signaling mechanisms were analyzed by Western blotting and ELISA, and pathway‑specific inhibitors were applied to dissect the PI3K/Akt/mTOR-HIF‑1α/VEGF cascade. RESULTS: ANXA3 expression was markedly elevated in osteosarcoma spheres compared with parental cells. ANXA3 knockdown down‑regulated stemness‑related genes, impaired sphere formation, and increased cisplatin sensitivity, whereas ANXA3 overexpression produced opposite effects. In vivo, ANXA3 silencing significantly suppressed tumor growth and pulmonary metastasis. Mechanistically, ANXA3 activated the PI3K/Akt/mTOR pathway, thereby enhancing HIF‑1α/VEGF expression. Knockdown of PI3K or HIF‑1α abrogated ANXA3‑induced stemness and chemoresistance, confirming the signaling axis mediates ANXA3‑driven oncogenic effects. CONCLUSION: ANXA3 plays a critical role in maintaining CSC‑like traits in osteosarcoma by up‑regulating the HIF‑1α/VEGF pathway in a PI3K/Akt/mTOR‑dependent manner. Targeting ANXA3 or its downstream signaling components may represent a promising therapeutic strategy for reversing CSC phenotypes and overcoming chemoresistance in osteosarcoma.