Inherent tissue homeostasis of the juvenile metaphysis provides a foundation for osteosarcoma development [RNA-seq]

Osteosarcomas typically arise from the metaphyseal cells of juvenile long bones, in contrast to most cancers that develop in aged individuals. Here, we show that the cyclin-dependent kinase inhibitor p21 is expressed in proliferating osteoblasts within the juvenile metaphysis in response to replication stress. Single-cell RNA sequencing focused on the juvenile metaphysis reveals a hierarchical differentiation trajectory of osteoblast-lineage cells, with active proliferation and DNA damage responses predominantly occurring in immature osteoblasts. We find that proliferation of p21+ osteoblasts is mediated by Hedgehog signaling associated with Indian hedgehog (IHH) expression in the growth plate. Consistently, the number of p21+ osteoblasts is markedly reduced following growth plate maturation or pharmacological inhibition of Hedgehog signaling. Induction of c-Myc—a known driver of replication stress and frequently amplified in early-stage osteosarcoma—enhances osteoblast proliferation specifically in juvenile mice, while concurrently augmenting replication stress responses including p53 activation. This c-Myc-driven proliferation remains dependent on Hedgehog signaling and is not sustained after growth plate maturation. Remarkably, functional inactivation of p53 enables continuous proliferation of c-Myc-induced osteoblasts, independent of IHH expression at the growth plate, leading to rapid and widespread metastasis to the lungs. These findings suggest that intrinsic DNA damage responses to replication stress, together with the spatiotemporal restriction of mitogen signaling, safeguard against oncogenic transformation in osteoblasts. Collectively, we uncover the unappreciated aspects of juvenile metaphyseal tissue homeostasis in mice, which may account for the characteristic age of onset, anatomical specificity, and mutational profile of human osteosarcomas.