Interaction between FKBP10 and HSP10 activates the TGF-β/Smad pathway to promote the maintenance of osteosarcoma stemness.

Osteosarcoma is a primary malignant bone tumor with high incidence in adolescents. Its characteristics of high recurrence, high metastasis, and cancer stem cell (CSC)-mediated treatment resistance are major obstacles to improving patient prognosis. This study aimed to systematically investigate the role and molecular mechanism of FK506-binding protein 10 (FKBP10) in regulating osteosarcoma stemness and malignant progression. Clinical sample analysis showed that High expression of FKBP10 is negatively correlated with prognosis in osteosarcoma, and positively correlated with the expression of stemness markers SOX2 (SRY-box transcription factor 2) and Nanog (Homeobox transcription factor Nanog). Cellular experiments confirmed that FKBP10 dose-dependently upregulated the transcriptional and protein levels of stemness markers such as CD44 (CD44 molecule), CD105 (Endoglin), SOX2, and Nanog, expanded the populations of CD105+ and CD44+ stem cells, and enhanced the self-renewal (tumor sphere formation), clonogenic proliferation (colony formation), and invasion/migration abilities of osteosarcoma cells. Conversely, FKBP10 knockdown significantly inhibited these stemness characteristics and malignant phenotypes without affecting the proliferative activity of general tumor cells. Animal experiments demonstrated that FKBP10 knockdown remarkably reduced the volume of xenograft tumors in nude mice, decreased the frequency of CD105+ CSCs, and prolonged the survival time of tumor-bearing mice. At the molecular level, immunofluorescence staining and co-immunoprecipitation experiments confirmed that FKBP10 and heat shock protein 10 (HSP10) exhibited subcellular co-localization and direct interaction in osteosarcoma cells, with no mutual regulation of protein expression between FKBP10 and HSP10. HSP10 knockdown mimicked the effect of FKBP10 knockdown to inhibit osteosarcoma stemness. RNA sequencing and functional validation revealed that FKBP10 and HSP10 jointly regulated the expression of stemness markers and malignant phenotypes by activating the TGF-β/Smad pathway, and the regulatory effect of FKBP10 was completely dependent on this pathway. In addition, although HSP10 was involved in stemness regulation, it had no significant association with patient prognosis. This study is the first to clarify the core role of the "FKBP10-HSP10-TGF-β (Transforming growth factor-beta)/Smad (Sma- and Mad-related protein)" regulatory axis in maintaining osteosarcoma stemness, confirming that FKBP10 is a potential biomarker for osteosarcoma prognosis and a key target for targeted therapy. It provides a new theoretical basis and experimental foundation for the development of precision treatment strategies targeting CSCs in osteosarcoma.