Bidirectional regulation of bone formation by exogenous and osteosarcoma-derived Sema3A

International audienceSemaphorin 3A (Sema3A), a secreted member of the Semaphorin family, increases osteoblast differentiation, stimulates bone formation and enhances fracture healing. Here, we report a previously unknown role of Sema3A in the regulation of ectopic bone formation and osteolysis related to osteosarcoma. Human recombinant (exogenous) Sema3A promoted the expression of osteoblastic phenotype in a panel of human osteosarcoma cell lines and inhibited the ability of these cells to migrate and enhance osteoclastogenesis in vitro. In vivo, administration of exogenous Sema3A in mice after paratibial inoculation of KHOS cells increased bone volume in non-inoculated and tumour-bearing legs. In contrast, Sema3A overexpression reduced the ability of KHOS cells to cause ectopic bone formation in mice and to increase bone nodule formation by engaging DKK1/β-catenin signalling. Thus, Sema3A is of potential therapeutic efficacy in osteosarcoma. However, inhibition of bone formation associated with continuous exposure to Sema3A may limit its long-term usefulness as therapeutic agent. Osteosarcoma is an aggressive form of skeletal cancer, typically with childhood and adolescent onsets 1–3. Metastasis is a serious clinical manifestation of osteosarcoma 4,5. However, skeletal complications such as osteol-ysis and rapid production of ectopic " woven " bone cause skeletal fractures and pain 4,5. Thus, treatments aimed at reducing bone damage and halting metastasis would prove to be beneficial in terms of clinical outcomes and quality of life of osteosarcoma patients. Semaphorins belong to a highly conserved family of secreted and membrane-associated signaling proteins 6,7. Of the 8 different classes, only class 3 semaphorins (Sema3) are secreted 8–10. Type A Sema3 (Sema3A) is synthesized as a precursor protein and proteolytically processed into two active secreted forms of 95 and 65 kilodaltons 8–10. Secreted Sema3A is implicated in the migration of neuronal precursor cells and guided elongation and branching patterns of neurons 11–15. Sema3A, and its co-receptors Neuropilin (Nrp)1 and 2 16–18 , are expressed by a variety of human cancer cells. Engagement of Sema3A with Nrp1 and 2 and other receptors has shown to both enhance and reduce tumour cell motility and invasion in various preclinical models of cancer 19–24. Recent studies have reported that osteosarcoma tumours and cell lines overexpress both Nrp1 and Nrp2. Furthermore, Nrp2 expression is associated with poor prognosis and reduced overall survival in osteosarcoma patients 25–27 , and its knockdown in the human osteosarcoma cell line 143B inhibited tumour growth and reduced lung metastasis in mice 27. The Sema3A/Nrp axis plays a key role in the regulation of bone development and remodelling 28–30. Sema3A, Nrp1/2 and Plexin-A1 are highly expressed in hypertrophic chondrocytes during embryonic development and in blood vessels and nerves at later stages of bone development 31. Nrp1 and Nrp2 are expressed by osteoblasts and osteocytes, and mice deficient in Nrp2 exhibited bone loss due to enhanced osteoclast and reduced oste-oblast numbers 32. Recently, Hayashi and colleagues have reported that osteoblasts secrete Sema3A, and adult mice deficient in Sema3A had low peak bone mass mainly due to enhanced bone resorption and reduced osteo-blast differentiation 29,32. Furthermore, administration of Sema3A in mice enhanced osteoblast differentiation and bone formation, promoted fracture healing in osteoporotic rats and protected against bone loss due to oestrogen deficiency 29,31,33