Transsynaptic cerebellin 4-neogenin 1 signaling mediates LTP in the mouse dentate gyrus.

Five decades ago, long-term potentiation (LTP) of synaptic transmission was discovered at entorhinal cortex→dentate gyrus (EC→DG) synapses, but the molecular determinants of EC→DG LTP remain largely unknown. Here, we show that the presynaptic neurexin­ligand cerebellin-4 (Cbln4) is highly expressed in the entorhinal cortex and essential for LTP at EC→DG synapses, but dispensable for basal synaptic transmission at these synapses. Cbln4, when bound to cell-surface neurexins, forms transcellular complexes by interacting with postsynaptic DCC (deleted in colorectal cancer) or neogenin-1. DCC and neogenin-1 act as netrin and repulsive guidance molecule-a (RGMa) receptors that mediate axon guidance in the developing brain, but their binding to Cbln4 raised the possibility that they might additionally function in the mature brain as postsynaptic receptors for presynaptic neurexin/Cbln4 complexes, and that as such receptors, DCC or neogenin-1 might mediate EC→DG LTP that depends on Cbln4. Indeed, we observed that neogenin-1, but not DCC, is abundantly expressed in dentate gyrus granule cells, and that postsynaptic neogenin-1 deletions in dentate granule cells blocked EC→DG LTP, but again did not affect basal synaptic transmission similar to the presynaptic Cbln4 deletions. Thus, binding of presynaptic Cbln4 to postsynaptic neogenin-1 renders EC→DG synapses competent for LTP, but is not required for establishing these synapses or for otherwise enabling their function.

Neogenin-1 distinguishes between myeloid-biased and balanced Hoxb5+ mouse long-term hematopoietic stem cells.

Hematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with single cell transplants and lineage tracing suggest that adult HSCs are diverse in their reconstitution and lineage potentials. However, prospective isolation of these subpopulations has remained challenging. Here, we identify Neogenin-1 (NEO1) as a unique surface marker on a fraction of mouse HSCs labeled with Hoxb5, a specific reporter of long-term HSCs (LT-HSCs). We show that NEO1+Hoxb5+ LT-HSCs expand with age and respond to myeloablative stress in young mice while NEO1-Hoxb5+ LT-HSCs exhibit no significant change in number. Furthermore, NEO1+Hoxb5+ LT-HSCs are more often in the G2/S cell cycle phase compared to NEO1-Hoxb5+ LT-HSCs in both young and old bone marrow. Upon serial transplantation, NEO1+Hoxb5+ LT-HSCs exhibit myeloid-biased differentiation and reduced reconstitution while NEO1-Hoxb5+ LT-HSCs are lineage-balanced and stably reconstitute recipients. Gene expression analysis reveals erythroid and myeloid priming in the NEO1+ fraction and association of quiescence and self-renewal-related transcription factors with NEO1- LT-HSCs. Finally, transplanted NEO1+Hoxb5+ LT-HSCs rarely generate NEO1-Hoxb5+ LT-HSCs while NEO1-Hoxb5+ LT-HSCs repopulate both LT-HSC fractions. This supports a model in which dormant, balanced NEO1-Hoxb5+ LT-HSCs can hierarchically precede active, myeloid-biased NEO1+Hoxb5+ LT-HSCs.

Identification of NEO1 as a prognostic biomarker and its effects on the progression of colorectal cancer.

BACKGROUND: Due to the high morbidity and poor clinical outcomes, early predictive and prognostic biomarker identification is desiderated in colorectal cancer (CRC). As a homologue of the Deleted in Colorectal Cancer (DCC) gene, the role of Neogenin-1 (NEO1) in CRC remained unveiled. This study was designed to probe into the effects and potential function of NEO1 in CRC. METHODS: Online databases, Gene Set Enrichment Analysis (GSEA), quantitative real-time PCR and western blotting were used to evaluate NEO1 expression in colorectal cancer tissues. Survival analysis was performed to predict the prognosis of CRC patients based on NEO1 expression level. Then, cell proliferation was detected by colony formation and Cell Counting Kit 8 (CCK-8) assays. CRC cell migration and invasion were examined by transwell assays. Finally, we utilized the Gene Set Variation Analysis (GSVA) and GSEA to dig the potential mechanisms of NEO1 in CRC. RESULTS: Oncomine database and The Cancer Genome Atlas (TCGA) database showed that NEO1 was down-regulated in CRC. Further results validated that NEO1 mRNA and protein expression were both significantly lower in CRC tumor tissues than in the adjacent tissues in our clinical samples. NEO1 expression was decreased with the progression of CRC. Survival and other clinical characteristic analyses exhibited that low NEO1 expression was related with poor prognosis. A gain-of-function study showed that overexpression of NEO1 restrained proliferation, migration and invasion of CRC cells while a loss-of-function showed the opposite effects. Finally, functional pathway enrichment analysis revealed that NEO1 low expression samples were enriched in inflammation-related signaling pathways, EMT and angiogenesis. CONCLUSION: A tumor suppressor gene NEO1 was identified and verified to be correlated with the prognosis and progression of CRC, which could serve as a prognostic biomarker for CRC patients.

Neogenin-1 Promotes Cell Proliferation, Motility, and Adhesion by Up-Regulation of Zinc Finger E-Box Binding Homeobox 1 Via Activating the Rac1/PI3K/AKT Pathway in Gastric Cancer Cells.

BACKGROUND/AIMS: Neogenin-1 (Neo1) has been reported to be involved in diverse physiology and pathology functions, including cell proliferation, differentiation and migration. The present study aimed to explore the functional role of neogenin-1 (Neo1) in gastric cancer (GC), together with underlying mechanisms. METHODS: Neo1 expression was analyzed by qRT-PCR and Western blot analysis in both human GC cell lines and normal gastric epithelial cell line. Neo1 was respectively overexpressed or silenced by transfection with pcDNA3.1 or siRNA, and then the cells were incubated with or without different concentrations of cisplatin, transforming growth factor (TGF)-ß1, and/or inhibitors of Rac-1 and PI3K. Thereafter, cell viability, invasion, and adhesion were measured by CCK-8, wound healing and adhesion assays, respectively. The expression levels of key factors involved in epithelial mesenchymal transition (EMT) and the PI3K/AKT pathway were analyzed by Western blot analysis. RESULTS: The results showed that the Neo1 level was significantly increased in GC cell lines, with the highest level in SGC-7901 cells. Overexpression of Neo1 significantly reduced the GC cell sensitivity to cisplatin and increased the cell viability, motility and adhesion ability, and while silencing of Neo1 showed contrary results. Moreover, overexpression of Neo1 dramatically downregulated the E-Cadherin level and upregulated the levels of N-Cadherin and Vimentin. In addition, the data revealed that Neo1 positively regulated the expression of Zinc finger E-box-binding homeobox 1 (ZEB1) by activating the Rac1/PI3K/AKT pathway. CONCLUSIONS: Neo1 could promote cell proliferation, motility, and adhesion by up-regulation of ZEB1 via activating the Rac1/PI3K/AKT pathway in GC cells.

Neogenin1 is a Sonic Hedgehog target in medulloblastoma and is necessary for cell cycle progression.

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.

Predictive capacity for local disease control of neogenin-1 (NEO1) transcriptional expression in patients with head and neck squamous cell carcinoma.

PURPOSE: To analyze the predictive capacity for local disease control of the transcriptional expression of neogenin-1 (NEO1) gene in patients with head and neck squamous cell carcinoma (HNSCC). METHODS/PATIENTS: A retrospective study was performed on tumor biopsies from 107 patients with HNSCC treated surgically. The transcriptional expression of NEO1 was determined by RT-PCR. NEO1 transcriptional expression value was categorized according to local disease control by recursive partitioning analysis. RESULTS: Lower NEO1 transcriptional expression was associated with worse local control after surgical treatment. Patients with lower NEO1 expression (n = 25, 23.4%) had a 5-year local recurrence-free survival of 61.8% (95% CI: 42.1-81.5%), while patients with higher NEO1 expression (n = 82, 76.6%) had a 5-year local recurrence-free survival of 85.6% (95% CI: 77.6-93.6%), (P = 0.003). According to the result of multivariable analysis, patients with lower NEO1 expression had a 2.7-fold increased risk of local tumor recurrence (95% CI: 1.0-7.0, P = 0.043) compared to patients with higher NEO1 expression. CONCLUSIONS: HNSCC patients with a lower transcriptional expression of NEO1 have a significantly higher risk of local recurrence after surgical treatment.

The Netrin-1-Neogenin-1 signaling axis controls neuroblastoma cell migration via integrin-ß1 and focal adhesion kinase activation.

Neuroblastoma is a highly metastatic tumor that emerges from neural crest cell progenitors. Focal Adhesion Kinase (FAK) is a regulator of cell migration that binds to the receptor Neogenin-1 and is upregulated in neuroblastoma. Here, we show that Netrin-1 ligand binding to Neogenin-1 leads to FAK autophosphorylation and integrin ß1 activation in a FAK dependent manner, thus promoting neuroblastoma cell migration. Moreover, Neogenin-1, which was detected in all tumor stages and was required for neuroblastoma cell migration, was found in a complex with integrin ß1, FAK, and Netrin-1. Importantly, Neogenin-1 promoted neuroblastoma metastases in an immunodeficient mouse model. Taken together, these data show that Neogenin-1 is a metastasis-promoting protein that associates with FAK, activates integrin ß1 and promotes neuroblastoma cell migration.

The Netrin-4/Laminin γ1/Neogenin-1 complex mediates migration in SK-N-SH neuroblastoma cells.

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It arises during development of the sympathetic nervous system. Netrin-4 (NTN4), a laminin-related protein, has been proposed as a key factor to target NB metastasis, although there is controversy about its function. Here, we show that NTN4 is broadly expressed in tumor, stroma and blood vessels of NB patient samples. Furthermore, NTN4 was shown to act as a cell adhesion molecule required for the migration induced by Neogenin-1 (NEO1) in SK-N-SH neuroblastoma cells. Therefore, we propose that NTN4, by forming a ternary complex with Laminin γ1 (LMγ1) and NEO1, acts as an essential extracellular matrix component, which induces the migration of SK-N-SH cells.

The Netrin-4/ Neogenin-1 axis promotes neuroblastoma cell survival and migration.

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.

Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness.

Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1, the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1lox/lox mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes.