Abstract

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It is derived from the sympatho-adrenal lineage of the neural crest, during development of the sympathetic nervous system, and usually arises in the adrenal gland or sympathetic ganglia. Since >50% of diagnosed cases are metastatic, it is important to know the mechanisms underlying its dissemination. During metastasis, cells migrate, invade, and colonize local and distant organs. Focal Adhesion Kinase (FAK) and Integrin β1 are key regulators of focal adhesion formation and cell migration. FAK induces Integrin β1 activation, focal adhesion turnover, and consequently cell migration, and it has been shown to be upregulated in tumors. FAK binds several upstream receptors including Neogenin-1 (NEO1), which is involved in axonal guidance, angiogenesis, and neuronal cell migration. On the other hand, NEO1 binds to the Netrin family of ligands. Analysis of public databases of NB tumors reveals that high expression of NEO1 correlates with poor patient survival. The aim of this work is to evaluate the role of migrationrelated signaling pathways downstream of NEO1/Netrin-1 (main ligand of the Netrin family). To this end, we focused on FAK and Integrin β1 interaction/activation. Western blot in SK-N-SH (MYCN WT) and LAN-1 (MYCN high) NB cell lines indicated that NEO1 and Netrin-4 are expressed while Netrin-1 is only expressed in LAN-1. Also, NEO1 associated with Netrin-1, as shown by co-immunoprecipitation assays in both cell lines. To evaluate the contribution of NEO1 in cell migration, integrin β1 activation, and metastasis, stable NEO1 knockdown cells (shNEO1) were generated using shRNAs via lentiviral infection. Cell migration was decreased in shNEO1 cells, as shown in transwell assays using Netrin-1 as chemoattractant, which could be due to decreased sensibility to the chemotactic stimulus of the ligand. We also assessed FAK dependency by pharmacologic inhibition with the PF562,271 compound in a spheroid-based cell migration assay. To further evaluate the association between NEO1 with Integrin β1 and FAK, we performed co-immunoprecipitation assays. Our data show that NEO1 associates in a complex with Integrin β1 and FAK in SK-N-SH cells. Immunoprecipitation of activated Integrin β1 with a specific conformational antibody shows that activation of Integrin β1 is less in shNEO1 cells compared to control cells. In an in vivo analysis of metastasis, SK-N-SK shNEO1 and control cells were injected into flank of immunodeficient mice (NSG strain). Metastasis was evaluated via luciferase activity in several organs, revealing that NEO1 is important to NB metastases. Our work suggests that NEO1 is a tumor progression-promoting protein, with an active role in metastasis. We propose a possible mechanism whereby NEO1, via its interaction with Netrin-1, is involved in cell migration and metastasis of NB cells. NEO1 likely plays its role by associating with FAK, allowing its phosphorylation and complex formation with Integrin β1, inducing its activation. Our results provide further information on the potential use of NEO1 as a therapeutic target to reduce metastasis in neuroblastoma.