Unfavorable neuroblastoma prognostic factor NLRR2 inhibits cell differentiation by transcriptional induction through JNK pathway

The novel human gene family encoding neuronal leucine rich repeat (NLRR) proteins were identified as prognostic markers from our previous screening of primary neuroblastoma (NB) cDNA libraries. Of the NLRR gene family members, NLRR1 and NLRR3 are associated with the regulation of cellular proliferation and differentiation, respectively. However, the functional regulation and clinical significance of NLRR2 in NB remain unclear. Here, we evaluated the differential expression of NLRR2, where high expressions of NLRR2 were significantly associated with a poor prognosis of NB (P = 0.0009), in 78 NBs. Enforced expression of NLRR2 in NB cells enhanced cellular proliferation and induced resistance to retinoic acid (RA)-mediated cell growth inhibition. In contrast, knockdown of NLRR2 exhibited growth inhibition effects and enhanced RA-induced cell differentiation in NB cells. After RA treatment, NLRR2 expression was increased and correlated with the upregulation of c-Jun, a member of the activator protein-1 (AP-1) family in NB cells. Moreover, the expressions of NLRR2 and c-Jun were suppressed by treatment with a JNK inhibitor, which ameliorated the promoter activity of the NLRR2 gene while knockdown of c-Jun reduced NLRR2 expression. We then searched AP-1 binding consensus in the NLRR2 promoter region and confirmed c-Jun recruitment at a consensus. Conclusively, NLRR2 must be an inducible gene regulated by the JNK pathway to enhance cell survival and inhibit NB cell differentiation. Therefore, NLRR2 should have an important role in NB aggressiveness and be a potential therapeutic target for the treatment of RA resistant and aggressive NB.