Targeted Inhibition of NOTCH2 and Importin‐β Promotes Osteogenic Differentiation of Osteosarcoma Cells

Osteosarcoma (OS) is a common malignant bone tumor, frequently associated with impaired osteogenic differentiation of tumor cells. Recent studies have suggested that the NOTCH signaling pathway plays a crucial role in maintaining tumor cell stemness and may influence their differentiation status. This study investigates the role of NOTCH2, a key receptor in the NOTCH family, in regulating osteogenic differentiation in OS. By analyzing public datasets, we compared the expression patterns and functional relevance of NOTCH1-4 in OS and identified NOTCH2 as the most significant. Using MG-63 and Saos-2 OS cell lines, we found that NOTCH2 silencing suppressed cell proliferation, invasion, and stem-like properties, while promoting osteogenic differentiation under inductive conditions. This was accompanied by increased expression of osteogenic markers. Further experiments demonstrated that Importazole, an Importin-β inhibitor, blocked the nuclear translocation of NOTCH2. Treatment with Importazole alone inhibited OS cell proliferation and invasion, reduced stem-like features, and enhanced osteogenic differentiation. When combined with NOTCH2 knockdown, Importazole exerted a synergistic effect, further inhibiting tumor progression and promoting differentiation. In vivo, xenograft models confirmed that the combination treatment more effectively suppressed tumor growth and induced osteoblast-like characteristics compared to either intervention alone. These findings indicate that NOTCH2 is a critical regulator of OS cell behavior, and that targeting NOTCH2 - especially in combination with Importazole - may offer a promising therapeutic strategy to promote differentiation and suppress tumor progression in OS.