Paracrine inhibition of osteoblast differentiation induced by neuroblastoma cells.

The aim of our study was to investigate whether the defective function of osteogenic cells induced by neuroblastoma might play a role in the development of skeletal metastases. This mechanism has been extensively demonstrated for multiple myeloma, in which the blockage of osteoblast differentiation has been ascribed to the inhibitors of canonical Wingless pathway (Wnt), namely Dickkopf 1 (Dkk1). Our purpose was to verify if neuroblastoma cells derived from bone marrow metastases (SH-SY5Y, LAN1) or primaries (NB100, CHP212) hamper the differentiation of mesenchymal stem cells (hMSCs) into osteoblasts in a paracrine manner, and to test whether this ability depends on Dkk1 activity. We found that all neuroblastoma cells increased the proliferation of hMSCs collected from pediatric-aged donors, with a corresponding decrease in osteoblast differentiation markers, including alkaline phosphatase (ALP), analyzed as gene expression, enzymatic activity and number of ALP-positive colony forming units, osteoprotegerin (OPG) release, OPG and osteocalcin gene-expression. Dkk1 mRNA and protein were detectable in all cell lines, and the use of neutralizing anti-Dkk1 antibody reversed the effects induced by SH-SY5Y cells. Taken together, our results confirm that neuroblastoma hinders osteoblastogenesis, and that Dkk1 release seems to play a crucial role in blocking the differentiation of osteoprogenitor cells, though the ability to promote osteoclast activation remains an essential requirement for the development of skeletal metastases. Finally, our findings suggest that strategies regulating Wnt signaling and Dkk1 activity could be considered for adjuvant therapies in neuroblastoma metastasizing to the skeleton.