Glycosylated steroidal saponins suppress HNSCC progression by targeting the RAS-ERK pathway

Head and neck squamous cell carcinoma (HNSCC) is characterized by high incidence, pronounced invasiveness and metastasis, and poor prognosis, underscoring an urgent need for novel and effective therapeutic strategies. Natural steroidal saponins serve as promising anti-tumor leads, yet prior work only reports their downstream ERK suppression, lacking upstream regulators and systematic glycosylation-related structure-activity relationship (SAR) analysis. This study focused on diosgenin and its two glycosylated derivatives. Firstly, in vitro cell proliferation, apoptosis and invasion assays combined with an in vivo subcutaneous xenograft model were performed to verify anti-HNSCC activities of the candidate compounds. Subsequently, core genes were screened via network pharmacology and machine learning, and validated by molecular docking, pull-down and Western blotting. All three compounds exhibit significant anti-HNSCC activity in vitro and vivo, which increases progressively with the number of glycosyl groups. Combined network pharmacology and machine learning analyses screened out 11 core genes, based on which a stable diagnostic prediction model was constructed. Molecular docking verified that the three saponins can stably bind to and suppress EPHB4 and ADAM17. The Raf1-RBD pull-down assay further confirmed that these saponins markedly reduce the level of intracellular active RAS and specifically downregulate ERK phosphorylation, ultimately restraining the malignant progression of HNSCC. This work reveals the RAS-MAPK inhibitory axis by targeting EPHB4 and ADAM17 of glycosylated steroidal saponins for the first time and highlights glycosylation as a critical activity-boosting moiety, supporting RAS-targeted lead discovery and structural optimisation against HNSCC.