Neogenin-1 (NEO1) is a transmembrane receptor involved in axonal guidance, angiogenesis, neuronal cell migration and cell death, during both embryonic development and adult homeostasis. It has been described as a dependence receptor, because it promotes cell death in the absence of its ligands (Netrin and Repulsive Guidance Molecule (RGM) families) and cell survival when they are present. Although NEO1 and its ligands are involved in tumor progression, their precise role in tumor cell survival and migration remain unclear. Public databases contain extensive information regarding the expression of NEO1 and its ligands Netrin-1 (NTN1) and Netrin-4 (NTN4) in primary neuroblastoma (NB) tumors. Analysis of this data revealed that patients with high expression levels of both NEO1 and NTN4 have a poor survival rate. Accordingly, our analyses in NB cell lines with different genetic backgrounds 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. Increased apoptosis was prevented when utilizing physiological concentrations of exogenous Netrin-4. Likewise, cell death induced after NTN4 knock-down was rescued when NEO1 was transiently silenced, thus revealing an important role for NEO1 in NB cell survival. In vivo analysis, using the chicken embryo chorioallantoic membrane (CAM) model, showed that NEO1 and endogenous NTN4 are involved in tumor extravasation and metastasis. Our data collectively demonstrate that endogenous NTN4/NEO1 maintain NB growth via both pro-survival and pro-migratory molecular signaling.

The canonical Sonic Hedgehog (Shh)/Gli pathway plays multiples roles during central nervous system (CNS) development. To elucidate the molecular repertoire of Shh mediators, we have recently described novel transcriptional targets in response to Shh pathway modulation. Among them, we were able to identify Neogenin1 (Neo1), a death dependence receptor, as a new direct Shh downstream regulator in neural precursor proliferation. As appropriate Shh signaling is required for cerebellar growth and alterations cause Shh-driven medulloblastoma (MB), here we have addressed the role of the Shh/Neogenin1 interaction in the context of cerebellar development and cancer. We demonstrate that the Shh pathway regulates Neogenin1 expression in mouse models that recapitulate the Shh MB subtype. We show that the canonical Shh pathway directly regulates the Neo1 gene acting through an upstream sequence in its promoter both in vitro and in vivo in granule neuron precursor cells. We also identified and characterized a functional Gli-binding site in the first intron of the human NEO1 gene. Gene expression profiling of more than 300 MB shows that NEO1 is indeed upregulated in SHH tumors compared to the other MB subgroups. Finally, we provide evidence that NEO1 is necessary for cell cycle progression in a human MB cell line, because a loss of function of NEO1 arrests cells in the G2/M phase. Taken together, these results highlight Neogenin1 as a novel downstream effector of the Shh pathway in MB and a possible therapeutic target.Medulloblastoma (MB), a primitive neuroepithelial tumor, is the most common malignant childhood primary central nervous system (CNS) tumor. Current treatment protocols encompassing surgical resection, chemotherapy and radiotherapy contributed to a better prognosis of MB patients. However, approximately one-third of the MB patients remain incurable and recurrence remains frequent.

Neogenin-1, a transmembrane receptor involved in axonal guidance, angiogenesis, neuronal cell migration, and cell death, is activated when bound to the Netrin and RGM ligand families during development and adult homeostasis. While Neogenin-1 and its ligands have been shown to be involved in tumoral progression, little is known about the specific function of the proteins and the cell signaling events that occur in cancer cells. Here, we demonstrate the exclusive presence of Neogenin-1 and Netrin-4 in SK-N-SH and SH-SY5Y neuroblastoma cells. Our research elucidates the function of Neogenin-1 and its autocrine ligand Netrin-4 in cell migration, proliferation, survival, and metastasis, the cellular aspects of cancer. To evaluate the contribution of Neogenin-1, the protein was knocked down using shRNAs (shNeo1) and overexpressed. We also knocked down Netrin-4 with shRNA (shNTN4) and performed gain of function studies using a human recombinant of this protein. Cell migration was analyzed via wound healing and transwell assays using increasing concentrations of Netrin-4. In addition, we evaluated cell proliferation and cell apoptosis after treatments. We also assessed metastasis and tumoral progression via chick chorioallantoic membrane (CAM) assay and quantitative-PCR to evaluate presence of human Alu sequences in genomic DNA extracted from chick embryonic lungs. We observed that shNeo1 and shNTN4 cells migrate less in transwell assays, possibly due to their insensibility to the chemotactic stimuli of Netrin-4. shNeo1 cells migrated less in wound healing assays as well. We detected increased cell migration when overexpressing Neogenin-1, but observed that cells were insensible to the Netrin-4 stimulus when overexpressing only the intracellular domain of Neogenin-1. We found less proliferation of shNTN4 cells in comparison to control cells, indicating that endogenous expression of Netrin-4 is required. This may be the case if Netrin-4 acts as a survival factor via its receptor Neogenin-1. Additionally, we observed increased cell death when knocking down Netrin-4 and that exogenous Netrin-4 was able to reduce this effect. Also, when Neogenin-1 is overexpressed, cell death increases and Netrin-4 is able to rescue cells from apoptosis. Finally, in vivo analysis showed that the weight of primary tumors in shSCR (control), shNeo1, and shNetrin-4 cells are similar. However, shNeo1 and shNTN4 did not produce secondary tumors. Metastasis analysis indicated that shNeo1 and shNTN4 metastasize less than control cells. Overall these results demonstrate that Neogenin-1 is involved in cell migration via Netrin-4 in neuroblastoma cells, and that endogenous Netrin-4 is important for cell survival, as it reduces apoptosis induced by Neogenin-1. We demonstrate that the Neogenin-1/Netrin-4 signaling complex modulates metastasis of neuroblastoma cells: Netrin-4 induces cell migration when bound to Neogenin-1, and conversely mediates apoptosis when not bound to Neogenin-1. Thus, endogenous Netrin-4 maintains the cells in a pro-survival and pro-migratory state. Our results shed light on the cellular mechanics of Netrin-4 and its receptor, Neogenin-1, which provides further information on their potential use as therapeutic targets to reduce metastasis in neuroblastoma. Citation Format: Andrea Villanueva, Natalie Andrea Espinoza, Luis Solano, Luis Milla, Veronica Palma. Neogenin-1 promotes cell migration, survival, and metastasis through Netrin-4 in neuroblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B50.