RASGRP2 promotes doxorubicin chemoresistance and tumor progression in osteosarcoma via the Notch1-NICD-Hes1 axis.

BACKGROUND

Doxorubicin (DOX) resistance remains a major obstacle to favorable outcomes in osteosarcoma. The role of RASGRP2 in osteosarcoma chemoresistance is poorly understood.

METHODS

We analyzed RASGRP2 expression and prognostic value across pan-cancer datasets (TCGA, TARGET) and osteosarcoma cohorts (TARGET-OS, GSE21257, GSE126209). CCK-8, colony formation, wound healing, Transwell, and Western blot assays were employed to evaluate how RASGRP2 knockdown affects osteosarcoma cell proliferation, migration, invasion, and DOX sensitivity. We further established a tibial orthotopic xenograft model to assess the in vivo efficacy of combining RASGRP2 silencing with DOX. Immunohistochemistry was conducted on 53 clinical osteosarcoma samples to validate protein-level associations.

RESULTS

RASGRP2 was upregulated in multiple malignancies and osteosarcoma, correlating with advanced Huvos grade, metastasis, poor chemotherapy response, and unfavorable survival. Bioinformatic analyses revealed Notch and Wnt signaling enrichment in high-RASGRP2 tumors. DOX induced RASGRP2 upregulation in a dose- and time-dependent fashion. RASGRP2 knockdown suppressed proliferation, migration, and invasion, and enhanced DOX sensitivity in both cellular and animal models. Mechanistically, RASGRP2 promoted DOX resistance via the Notch1-NICD-Hes1 axis, as evidenced by knockdown and VPA-based rescue experiments and further confirmed by functional rescue with the specific Notch ligand Jagged-1. In the orthotopic model, RASGRP2 silencing reduced tumor burden, enhanced DOX efficacy, and reduced CD206-positive cell infiltration. In clinical samples, elevated RASGRP2 protein correlated with higher metastatic incidence (p = 0.003), reduced overall and progression-free survival (p < 0.0001 for both), and unfavorable chemotherapy response.

CONCLUSION

RASGRP2 is associated with DOX resistance and osteosarcoma progression through the Notch1-NICD-Hes1 signaling axis and represents a potential prognostic biomarker. Targeting RASGRP2 may offer a strategy to overcome chemoresistance in osteosarcoma.