Abstract

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.