Forced expression of NR4A3 induced the differentiation of human neuroblastoma-derived NB1 cells.

Nuclear receptor subfamily 4, group A, member 3 (NR4A3) is a member of the NR4A subgroup of orphan nuclear receptors, implicated in the regulation of diverse biological functions, including metabolism, angiogenesis, inflammation, cell proliferation, and apoptosis. Although many reports have suggested the involvement of NR4A3 in the development and/or progression of tumors, its role varies among tumor types. Previously, we reported that DNA hypomethylation at NR4A3 exon 3 is associated with lower survival rate of neuroblastoma (NB) patients. As hypomethylation of this region results in reduced expression of NR4A3, our observations suggested that NR4A3 functions as a tumor suppressor in NB. However, the exact mechanisms underlying its functions have not been clarified. In the present study, we analyzed public databases and showed that reduced NR4A3 expression was associated with shorter survival period of NB in two out of three datasets. An in vitro study revealed that forced expression of NR4A3 in human NB-derived cell line NB1 resulted in elongation of neurites along with overexpression of GAP43, one of the differentiation markers of NB. On the other hand, siRNA-mediated knockdown of NR4A3 suppressed the expression level of GAP43. Interestingly, the forced expression of NR4A3 induced only the GAP43 but not the other molecules involved in NB cell differentiation, such as MYCN, TRKA, and PHOX2B. These results indicated that NR4A3 directly activates the expression of GAP43 and induces differentiated phenotypes of NB cells, without affecting the upstream signals regulating GAP43 expression and NB differentiation.