PDK1-dependent metabolic reprogramming regulates stemness and tumorigenicity of osteosarcoma stem cells through ATF3

Osteosarcoma stem cells (OSCs) are characterized by their self-renewal and multilineage differentiation abilities, contributing to osteosarcoma malignancy. The Warburg effect describes cancer cells' preference for glycolysis over mitochondrial oxidative phosphorylation (OXPHOS) for energy production. Unlike differentiated cancer cells, cancer stem cells exhibit unique and diverse metabolic properties depending on the context. This study investigated the metabolic reliance of OSCs and related genes through in silico analyses of clinical osteosarcoma specimens and in vitro and in vivo genetic and pharmacological analyses. Glycolysis and OXPHOS pathways were more active in OSCs than in non-OSCs at single-cell resolution. Pyruvate dehydrogenase kinase 1 (PDK1), a key enzyme balancing glycolysis and OXPHOS, was upregulated in OSCs and correlated with poor prognosis in patients with osteosarcoma. Genetic inhibition of PDK1 via RNA interference reduced OSC stemness, glycolysis, and heterotopic tumor formation. Pharmacological inhibition of PDK1 mirrored these genetic effects and repressed orthotopic tumor burden and pulmonary metastasis. Activating transcription factor 3 (ATF3) was identified through screening as a downstream factor of PDK1-regulated OSC properties. ATF3 overexpression reversed the stemness reduction caused by PDK1 deficiency through, at least in part, activating the TGF-β/Smad pathway without affecting the metabolic balance. ATF3 expression, glycolysis, and stemness were significantly induced by wild-type PDK1 overexpression but not by a kinase-dead PDK1 mutant in OSCs. Pharmacological inhibition of glycolysis counteracted the upregulation of ATF3 expression and increased stemness in OSCs by PDK1 overexpression. These findings indicate that PDK1 fine-tunes metabolic balance to govern OSC stemness and tumorigenicity through, at least in part, modulating ATF3/TGF-β/Smad pathway, suggesting a potential therapeutic approach for targeting OSCs in osteosarcoma.