PDF file, 106K, Phosphorylation of Sp1 is important for its transcriptional efficiency to induce MYCN. EGF-mediated phosphorylation of Sp1 was cancelled by MEK1/2 inhibitor and dephosphorylating agent. The Sp1 inhibitor mithramycin cancels MYCN induction by EGF. Pretreatment with dephosphorylating agent CIAP cancels MYCN induction and inhibits Sp1 recruitment to the MYCN promoter upon EGF stimulation.
ARTICLE ABSTRACTNeuronal leucine-rich repeat protein-1 (NLRR1), a type-1 transmembrane protein highly expressed in unfavorable neuroblastoma, is a target gene of MYCN that is predominately expressed in primary neuroblastomas with MYCN amplification. However, the precise biological role of NLRR1 in cell proliferation and tumor progression remains unknown. To investigate its functional importance, we examined the role of NLRR1 in EGF and insulin growth factor-1 (IGF-1)–mediated cell viability. We found that NLRR1 positively regulated cell proliferation through activation of extracellular signal–regulated kinase mediated by EGF and IGF-1. Interestingly, EGF stimulation induced endogenous MYCN expression through Sp1 recruitment to the MYCN promoter region, which was accelerated in NLRR1-expressing cells. The Sp1-binding site was identified on the promoter region for MYCN induction, and phosphorylation of Sp1 was important for EGF-mediated MYCN regulation. In vivo studies confirmed the proliferation-promoting activity of NLRR1 and established an association between NLRR1 expression and poor prognosis in neuroblastoma. Together, our findings indicate that NLRR1 plays an important role in the development of neuroblastoma and therefore may represent an attractive therapeutic target for cancer treatment. Cancer Res; 72(17); 4587–96. ©2012 AACR.