PFKP promotes oncogenesis in osteosarcoma through PI3K/HIF-1α pathway-mediated H3K18la modification

The preferential utilization of aerobic glycolysis by cancer cells causes lactate accumulation, resulting in histone H3 lysine 18 lactylation (H3K18la), lactylation of other proteins, and alterations in gene expression. Given the rate-limiting actions of phosphofructokinase-platelet (PFKP) in glycolysis, its upregulation occurs in various cancer types; however, PFKP's role in osteosarcoma remains unclear. Here, we report that PFKP may promote osteosarcoma progression. Initial screening of three osteosarcoma GEO datasets identified PFKP as a major differentially expressed gene (DEG). Its upregulations in tumor over normal tissues and osteosarcoma cells compared to osteoblasts were demonstrated using TNMplot database, RT-qPCR, Western blot, and immunohistochemistry (IHC). Functionally, the knockdown of PFKP in osteosarcoma 143B and MG63 cells inhibited cell proliferation and migration, accompanied by reductions in lactate production, pan-lactylation, and H3K18la both in vitro and in vivo. Mechanistically, the enrichment of the PI3K/HIF-1α pathway was observed in PFKP-related pathways; inhibition of PI3K by LY294002 downregulated PFKP expression, phosphofructokinase activity, lactate production, pan-lactylation, and H3K18la in both 143B and MG63 cells; LY294002 abolished the exogenous lactate-induced upregulation of PFKP, as well as the PFKP-dependent increases in H3K18la, PI3K, and HIF-1α. Simultaneously, the reduction of PFKP and H3K18la mediated by 2DG significantly suppressed the protein expression of PI3K and HIF-1α. ChIP-qPCR revealed a significant enrichment of H3K18la at the promoters of PIK3CA and HIF1A. Furthermore, through analysis of the HERB and CTD databases and molecular docking simulations, we uncovered the binding of a small molecule compound quercetin to PFKP with the binding energy of -8.3 kcal/mol, indicating a stable interaction. The binding was confirmed by the Cellular Thermal Shift Assay (CETSA). Of importance, quercetin decreases PFKP expression, lactate levels, pan-lactylation, and H3K18la along with inhibition of cell proliferation and migration in 143B and MG63 cells. Collectively, we provide evidence supporting that PFKP promotes osteosarcoma cell proliferation and migration via facilitating lactate production, protein lactylation, and H3K18la via processes involving PI3K/HIF-1α. This study reveals that quercetin binds PFKP with high affinity and suppresses its oncogenic actions, highlighting the therapeutic potential of targeting PFKP and utilization of quercetin in osteosarcoma management.