Inhibition of the mitochondrial antioxidant response blocks ex vivo lung colonization in pediatric osteosarcoma

Osteosarcoma (OS) is the most common type of bone cancer affecting children and adolescents. Pulmonary metastasis is the primary cause of OS mortality, yet treatments have not improved patient outcomes in four decades. This gap emphasizes the urgent need to identify and vet novel anti-metastatic therapies. When OS tumor cells spread to the lung, they are subjected to oxidative stress caused by high levels of reactive oxygen species (ROS) secreted from microvascular endothelial cells and alveolar macrophages. This stress is further amplified by limited antioxidant capacity and other redox-imbalancing features of the lung microenvironment. We previously demonstrated that metastatic OS cells upregulate NFE2-like BZIP Transcription Factor 2 (NRF2), promoting cell survival in this stressful environment. In addition to being a master regulator of redox balance, NRF2 also influences mitochondrial function by controlling the expression of thioredoxin system members, including the antioxidant enzyme peroxiredoxin 3 (PRDX3) located in the mitochondrial matrix. Therefore, we hypothesize that PRDX3 upregulation promotes metastatic cell survival during the early stages of lung colonization, and disrupting this response will impair adaptation to the lung microenvironment. We evaluated thiostrepton (TS), a Streptomyces -derived thiopeptide antibiotic with demonstrated PRDX3-inhibitory activity in preclinical and clinical cancer studies, to determine its effect on metastatic OS tumor cells. To mimic the oxidative stress of the lung microenvironment in vitro, we treated cells with tert -butyl hydroperoxide (tBHP), which chemically induces oxidative stress. Through cell viability assays we showed that subtoxic levels of TS sensitize metastatic OS cells to tBHP-induced oxidative stress. In addition, we determined that PRDX3 is upregulated when metastatic OS cells are subjected to oxidative stress via confocal microscopy. Through protein immunoblots, we demonstrated that TS acts by dimerizing PRDX3, inactivating the catalytic site of the enzyme. Furthermore, we found that combining TS with the chemotherapeutic agents doxorubicin or etoposide, which are commonly used to treat pediatric pulmonary OS metastasis, produced a synergistic increase in cell death compared to either drug alone. Lastly, we studied TS as a single agent therapy using the pulmonary metastasis assay, an ex vivo lung explant system, and found that treatment significantly decreased OS lung tumor burden. Our findings provide promising preclinical data supporting the use of TS as a possible anti-metastatic therapeutic for pediatric OS .