Abstract

Introduction: Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It arises during development of the sympathetic nervous system. Neogenin-1 (NEO1) is a transmembrane receptor involved in axonal guidance, neuronal cell migration and cell death. It has been proposed as a key factor to target NB metastasis, although there is controversy about its function. NEO1 binds Netrin (NTN) family, where NTN4 are the one most studied. Materials and methods: NTN4 in NB histological cuts were evaluated via IHC. Stable NEO1 and NTN4 knock-down cells were generated using shRNAs through lentiviral infection. Cell death analysis were performed using TUNEL and immunofluorescence against cleaved-Caspase-3. Cell migration was evaluated via Transwell assays using rhNTN4 as a chemoattractant and cell adhesion molecule. Co-immunoprecipitation of NEO1/NTN4 and Laminin γ1 (LMγ1) was evaluated with specific antibodies against NEO1. CAM assay was performed by using shNEO1 and control cells and measuring Alu sequence via Q-PCR. Results: We show that NTN4 is broadly expressed in tumor, stroma and blood vessels of NB patient samples. Accordingly, our analyses in NB cell, SK-N-SH, revealed that knocking-down NEO1 reduces cell migration, whereas silencing of endogenous NTN4 induced cell death. Conversely, overexpression of NEO1 resulted in higher cell migration in the presence of NTN4, and increased apoptosis in the absence of ligand. Likewise, cell death induced after NTN4 knock-down was rescued when NEO1 was transiently silenced. NTN4 was shown to act as a cell adhesion molecule required for the migration induced by NEO1 in SK-N-SH neuroblastoma cells via the binding with LMγ1. In vivo analysis showed that NEO1 and endogenous NTN4 are involved in tumor extravasation and metastasis. Discussion: NEO1-induced cell death is abolished in presence of NTN4 and NEO1 promotes cell migration through NTN4 and LMγ1 ternary complex. Our data collectively demonstrate that endogenous NTN4/NEO1 maintain NB growth via both pro-survival and pro-migratory molecular signaling.