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

ABSTRACT 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.