SLC30A9-mediated mitochondrial zinc homeostasis drives osteosarcoma chemoresistance by suppressing the mtDNA-cGAS-STING pathway.

AIMS: To investigate the role of mitochondrial zinc (Zn) homeostasis in chemoresistance of osteosarcoma.

MATERIAL AND

METHODS

Zinc levels were measured in osteosarcoma cells treated with methotrexate (MTX). The expression of SLC30A9 was analyzed in chemoresistant cells. SLC30A9 knockdown and overexpression osteosarcoma cell lines were constructed for functional studies, assessing mitochondrial zinc levels, mitochondrial function, and MTX sensitivity. Subcellular localization of SLC30A9 and its zinc efflux function were determined by immunofluorescence staining and mitochondrial zinc detection. The opening of the mitochondrial permeability transition pore (mPTP), release of mitochondrial DNA (mtDNA), activation of the cGAS-STING pathway, NF-κB signaling, and NDRG1 transcription were analyzed. Xenograft mouse models were established to evaluate the effect of SLC30A9 knockdown combined with a STING agonist.

KEY FINDINGS: MTX treatment elevated mitochondrial zinc levels, leading to mitochondrial dysfunction. SLC30A9 was highly expressed in chemoresistant osteosarcoma cells. Knockdown of SLC30A9 caused abnormal mitochondrial zinc accumulation, mitochondrial dysfunction, and reversed MTX resistance. SLC30A9 was localized to mitochondria and mediated zinc efflux; its loss triggered mtDNA release into the cytosol via mPTP, activating the cGAS-STING pathway, which in turn promoted NF-κB-driven transcription of the pro-apoptotic molecule NDRG1. In vivo, SLC30A9 knockdown combined with STING-agonist3 enhanced the chemotherapeutic effect of MTX.

SIGNIFICANCE: SLC30A9 maintains a low-zinc environment and mitochondrial integrity, thereby inhibiting mtDNA-mediated cGAS-STING pathway activation. Targeting SLC30A9 to disrupt this protective mechanism provides a novel therapeutic strategy for overcoming chemoresistance in osteosarcoma.