ZEB1 integrates stemness, differentiation blockade and immune suppression to orchestrate osteosarcoma aggressiveness

BACKGROUND: Osteosarcoma (OS) is a highly aggressive bone malignancy primarily affecting children and adolescents, with limited therapeutic options. The transcription factor ZEB1, a key regulator of epithelial-mesenchymal transition (EMT), promotes cancer stemness and tumor progression. METHODS: We investigated ZEB1 role in OS using genetic knockout and silencing in murine and patient-derived cellular OS models. Using in vitro functional assays, we assessed the effect of ZEB1 down-modulation on stemness (sarcosphere formation), anchorage-independent growth, chemotherapy sensitivity, and osteoblastic differentiation (osteogenic markers and matrix mineralization), while gene expression profiling provided hints on ZEB1 regulation of tumor cell transcriptional program. In vivo studies evaluated tumor growth and lung metastasis in ZEB1-deficient models, while ZEB1 restoration experiments mechanistically proved its role in OS aggressiveness. RESULTS: ZEB1 loss in OS mouse models markedly reduced stemness-associated gene expression and functional sarcosphere formation, impaired anchorage-independent growth, and increased sensitivity to chemotherapy. Furthermore, ZEB1 inhibition restored osteoblastic differentiation capacity, evidenced by upregulation of osteogenic markers and matrix mineralization. In vivo, ZEB1 deletion in mouse OS cells significantly hindered tumor growth and lung metastasis, accompanied by increased tumor differentiation and decreased infiltration of pro-tumoral macrophages. Gene expression profiling revealed suppression of oncogenic pathways and inflammatory mediators upon ZEB1 loss, while restoration of ZEB1 rescued OS aggressive phenotype. CONCLUSIONS: Overall, our findings position ZEB1 as a critical driver of OS aggressiveness by regulating stemness, cellular differentiation, oncogenic signaling, and immune infiltration, and highlight differentiation-based therapeutic strategies as a promising avenue for osteosarcoma treatment.