Netrin-1 and UNC5B Cooperate with Integrins to Mediate YAP-Driven Cytostasis.

Opposite expression and pro- or anti-cancer function of YAP and its paralog TAZ/WWTR1 stratify cancers into binary YAPon and YAPoff classes. These transcriptional coactivators are oncogenic in YAPon cancers. In contrast, YAP/TAZ are silenced epigenetically along with their integrin and extracellular matrix adhesion target genes in neural and neuroendocrine YAPoff cancers (e.g., small cell lung cancer, retinoblastoma). Forced YAP/TAZ expression induces these targets, causing cytostasis in part through Integrin-αV/ß5, independent of the integrin-binding RGD ligand. Other effectors of this anticancer YAP function are unknown. Here, using clustered regularly interspaced short palindromic repeats (CRISPR) screens, we link the Netrin receptor UNC5B to YAP-induced cytostasis in YAPoff cancers. Forced YAP expression induces UNC5B through TEAD DNA-binding partners, as either TEAD1/4-loss or a YAP mutation that disrupts TEAD-binding (S94A) blocks, whereas a TEAD-activator fusion (TEAD(DBD)-VP64) promotes UNC5B induction. Ectopic YAP expression also upregulates UNC5B relatives and their netrin ligands in YAPoff cancers. Netrins are considered protumorigenic, but knockout and peptide/decoy receptor blocking assays reveal that in YAPoff cancers, UNC5B and Netrin-1 can cooperate with integrin-αV/ß5 to mediate YAP-induced cytostasis. These data pinpoint an unsuspected Netrin-1/UNC5B/integrin-αV/ß5 axis as a critical effector of YAP tumor suppressor activity. SIGNIFICANCE: Netrins are widely perceived as procancer proteins; however, we uncover an anticancer function for Netrin-1 and its receptor UNC5B.

Identification of UNC5B as a novel aggressive biomarker for osteosarcoma based on basement membrane genes.

BACKGROUND: The prognosis of patients with metastatic osteosarcoma is poor, and the variation of basement membrane genes (BMGs) is associated with cancer metastasis. However, the role of BMGs in osteosarcoma has been poorly studied. METHODS: BMGs were collected and differentially expressed BMGs (DE-BMGs) were found through difference analysis. DE-BMGs were further screened by univariate Cox regression and Lasso regression analyses, and six key BMGs were identified and defined as basement membrane genes signatures (BMGS). Then, BMGS was used to construct the osteosarcoma BMGS risk score system, and the osteosarcoma patients were divided into high- and low-risk groups based on the median risk score. Single-sample gene set enrichment analysis (ssGSEA) and ESTIMATE scores were used to investigate the differences in immune infiltration between the two scoring groups. Additionally, we investigated whether UNC5B affects various features in tumors by bioinformatic analysis and whether UNC5B was involved in multiple biological functions of osteosarcoma cells by wound healing assay, transwell assay, and western blot. RESULTS: The osteosarcoma BMGS risk score reliably predicts the risk of metastasis, patient prognosis, and immunity. UNC5B expression was elevated in osteosarcoma, and correlated with various characteristics such as immune infiltration, prognosis, and drug sensitivity. In vitro assays showed that UNC5B knockdown reduced osteosarcoma cells' capacity for migration and invasion, and EMT process. CONCLUSION: A novel BMGS risk score system that can effectively predict the prognosis of osteosarcoma was developed and validated. The UNC5B gene in this system is one of the key aggressive biomarkers of osteosarcoma.

Netrin signaling mediates survival of dormant epithelial ovarian cancer cells.

Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, -3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and -3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis.

High-grade serous ovarian cancer (or HGSOC for short) is the fifth leading cause of cancer-related deaths in women. It is generally diagnosed at an advanced stage of disease when the cancer has already spread to other parts of the body. Surgical removal of tumors and subsequent treatment with chemotherapy often reduces the signs and symptoms of the disease for a time but some cancer cells tend to survive so that patients eventually relapse. The HGSOC cells typically spread from the ovaries by moving through the liquid surrounding organs in the abdomen. The cells clump together and enter an inactive state known as dormancy that allows them to survive chemotherapy and low-nutrient conditions. Understanding how to develop new drug therapies that target dormant cancer cells is thought to be an important step in prolonging the life of HGSOC patients. Cancer cells are hardwired to multiply and grow, so Perampalam et al. reasoned that becoming dormant poses challenges for HGSOC cells, which may create unique vulnerabilities not shared by proliferating cancer cells. To find out more, the researchers used HGSOC cells that had been isolated from patients and grown in the laboratory. The team used a gene editing technique to screen HGSOC cells for genes required by the cells to survive when they are dormant. The experiments found that genes involved in a cell signaling pathway, known as Netrin signaling, were critical for the cells to survive. Previous studies have shown that Netrin signaling helps the nervous system form in embryos and inhibits a program of controlled cell death in some cancers. Perampalam et al. discovered that Netrins were present in the environment immediately surrounding dormant HGSOC cells. Human HGSOC patients with higher levels of Netrin gene expression had poorer prognoses than patients with lower levels of Netrin gene expression. Further experiments demonstrated that Netrins help dormant HGSOC cells to spread around the body. These findings suggest that Netrin signalling may provide useful targets for future drug therapies against dormant cells in some ovarian cancers. This could include repurposing drugs already in development or creating new inhibitors of this pathway.

Long noncoding RNA UNC5B-AS1 suppresses cell proliferation by sponging miR-24-3p in glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary CNS tumor, characterized by high mortality and heterogeneity. However, the related lncRNA signatures and their target microRNA (miRNA) for GBM are still mostly unknown. Therefore, it is critical that we discover lncRNA markers in GBM and their biological activities. MATERIALS AND METHODS: GBM-related RNA-seq data were obtained from the Cancer Genome Atlas (TCGA) database. The "edger" R package was used for differently expressed lncRNAs (DELs) identification. Then, we forecasted prospective miRNAs that might bind to lncRNAs by Cytoscape software. Survival analysis of those miRNAs was examined by the starBase database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the miRNAs' target genes was conducted by the Gene Set Enrichment Analysis (GSEA) database and R software. Moreover, the proliferative ability of unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1) cells was evaluated by Cell Counting Kit-8 (CCK-8) analysis. Mechanistically, the regulatory interaction between UNC5B-AS1 and miRNA in GBM biological processes was studied using CCK-8 analysis. RESULTS: Our results indicated that overexpression of UNC5B-AS1 has been shown to suppress GBM cell growth. Mechanistically, miR-24-3p in GBM was able to alleviate the anti-oncogenic effects of UNC5B-AS1 on cell proliferation. CONCLUSION: The discovery of the novel UNC5B-AS1-miR-24-3p network suggests possible lncRNA and miRNA roles in the development of GBM, which may have significant ramifications for the analysis of clinical prognosis and the development of GBM medications.

Fibronectin leucine-rich transmembrane protein 2 drives monocyte differentiation into macrophages via the UNC5B-Akt/mTOR axis.

Upon migrating into the tissues, hematopoietic stem cell (HSC)-derived monocytes differentiate into macrophages, playing a crucial role in determining innate immune responses towards external pathogens and internal stimuli. However, the regulatory mechanisms underlying monocyte-to-macrophage differentiation remain largely unexplored. Here we divulge a previously uncharacterized but essential role for an axon guidance molecule, fibronectin leucine-rich transmembrane protein 2 (FLRT2), in monocyte-to-macrophage maturation. FLRT2 is almost undetectable in human monocytic cell lines, human peripheral blood mononuclear cells (PBMCs), and mouse primary monocytes but significantly increases in fully differentiated macrophages. Myeloid-specific deletion of FLRT2 (Flrt2ΔMyel ) contributes to decreased peritoneal monocyte-to-macrophage generation in mice in vivo, accompanied by impaired macrophage functions. Gain- and loss-of-function studies support the promoting effect of FLRT2 on THP-1 cell and human PBMC differentiation into macrophages. Mechanistically, FLRT2 directly interacts with Unc-5 netrin receptor B (UNC5B) via its extracellular domain (ECD) and activates Akt/mTOR signaling. In vivo administration of mTOR agonist MYH1485 reverses the impaired phenotypes observed in Flrt2ΔMyel mice. Together, these results identify FLRT2 as a novel pivotal endogenous regulator of monocyte differentiation into macrophages. Targeting the FLRT2/UNC5B-Akt/mTOR axis may provide potential therapeutic strategies directly relevant to human diseases associated with aberrant monocyte/macrophage differentiation.

EPB41L4A-AS1 and UNC5B-AS1 have diagnostic and prognostic significance in osteosarcoma.

BACKGROUND: Deregulation of lncRNAs has been observed in human osteosarcoma. This study explored the diagnostic and prognostic significance of EPB41L4A-AS1 and UNC5B-AS1 in osteosarcoma. METHODS: Relative levels of EPB41L4A-AS1 and UNC5B-AS1 were detected in osteosarcoma tissue samples and cells. The ability to distinguish osteosarcoma from health was assessed by receiver operating characteristic (ROC) curve construction. Kaplan-Meier (K-M) and Cox proportional-hazards analyses were performed for prognosis factors. The bioinformatics approach was used to identify targeting miRNA for EPB41L4A-AS1 and UNC5B-AS1. Kaplan-Meier survival curves and Whitney Mann U tests were conducted for validating the statistical significance. In cell culture experiments, the influence of EPB41L4A-AS1 and UNC5B-AS1 on proliferation, migration, and invasion of the osteosarcoma cell line was examined by CCK-8 and Transwell assays. RESULTS: Levels of EPB41L4A-AS1 and UNC5B-AS1 were upregulated in osteosarcoma patients and cells compared with the healthy participants and normal cell lines. EPB41L4A-AS1 and UNC5B-AS1 have a potent ability to distinguish the patients with osteosarcoma from the health. EPB41L4A-AS1 and UNC5B-AS1 levels correlated with SSS stage. Patients with high levels of EPB41L4A-AS1 and UNC5B-AS1 had significantly shorter survival times. EPB41L4A-AS1 and UNC5B-AS1 were independent prognostic indexes for overall survival. miR-1306-5p was a common target for EPB41L4A-AS1 and UNC5B-AS1. A propulsive impact on cell proliferation, migration, and invasion by EPB41L4A-AS1 and UNC5B-AS1 was observed, but can be rescued by miR-1306-5p. CONCLUSIONS: It was concluded that upregulations of EPB41L4A-AS1 and UNC5B-AS1 expression were diagnostic and prognostic biomarkers for human osteosarcoma. EPB41L4A-AS1 and UNC5B-AS1 contribute to the biological behavior of osteosarcoma via miR-1306-5p.

A Systematic Analysis of the Role of Unc-5 Netrin Receptor A (UNC5A) in Human Cancers.

Unc-5 netrin receptor A (UNC5A), a netrin family receptor, plays a key role in neuronal development and subsequent differentiation. Recently, studies have found that UNC5A plays an important role in multiple cancers, such as bladder cancer, non-small cell lung carcinoma, and colon cancer but its pan-cancer function is largely unknown. Herein, the R software and multiple databases or online websites (The Cancer Genome Atlas (TCGA), The Genotype-Tissue Expression (GTEx), The Tumor Immune Estimation Resource (TIMER), The Gene Set Cancer Analysis (GSCA), Gene Expression Profiling Interactive Analysis (GEPIA), and cBioPortal etc.) were utilized to examine the role of UNC5A in pan-cancer. UNC5A was found to be highly expressed across multiple human cancer tissues and cells, was linked to clinical outcomes of patients, and was a potential pan-cancer biomarker. The mutational landscape of UNC5A exhibited that patients with UNC5A mutations had poorer progress free survival (PFS) in head and neck squamous cell carcinoma (HNSC) and prostate adenocarcinoma (PRAD). Furthermore, UNC5A expression was associated with tumor mutation burden (TMB), neoantigen, tumor microenvironment (TME), tumor microsatellite instability (MSI), immunomodulators, immune infiltration, DNA methylation, immune checkpoint (ICP) genes, and drug responses. Our results suggest the potential of UNC5A as a pan-cancer biomarker and an efficient immunotherapy target, which may also guide drug selection for some specific cancer types in clinical practice.

GPC3-Unc5 receptor complex structure and role in cell migration.

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.

Netrin-1 induces the anti-apoptotic and pro-survival effects of B-ALL cells through the Unc5b-MAPK axis.

BACKGROUND: B-cell acute lymphoblastic leukemia (B-ALL) comprises over 85% of all acute lymphoblastic leukemia (ALL) cases and is the most common childhood malignancy. Although the 5 year overall survival of patients with B-ALL exceeds 90%, patients with relapsed or refractory B-ALL may suffer from poor prognosis and adverse events. The axon guidance factor netrin-1 has been reported to be involved in the tumorigenesis of many types of cancers. However, the impact of netrin-1 on B-ALL remains unknown. METHODS: The expression level of netrin-1 in peripheral blood samples of children with B-ALL and children without neoplasia was measured by enzyme-linked immunosorbent assay (ELISA) kits. Then, CCK-8 cell proliferation assays and flow cytometric analysis were performed to detect the viability and apoptosis of B-ALL cells (Reh and Sup B15) treated with exogenous recombinant netrin-1 at concentrations of 0, 25, 50, and 100 ng/ml. Furthermore, co-immunoprecipitation(co-IP) was performed to detect the receptor of netrin-1. UNC5B expression interference was induced in B-ALL cells with recombinant lentivirus, and then CCK-8 assays, flow cytometry assays and western blotting assays were performed to verify that netrin-1 might act on B-ALL cells via the receptor Unc5b. Finally, western blotting and kinase inhibitor treatment were applied to detect the downstream signaling pathway. RESULTS: Netrin-1 expression was increased in B-ALL, and netrin-1 expression was upregulated in patients with high- and intermediate-risk stratification group of patients. Then, we found that netrin-1 induced an anti-apoptotic effect in B-ALL cells, implying that netrin-1 plays an oncogenic role in B-ALL. co-IP results showed that netrin-1 interacted with the receptor Unc5b in B-ALL cells. Interference with UNC5B was performed in B-ALL cells and abolished the antiapoptotic effects of netrin-1. Further western blotting was applied to detect the phosphorylation levels of key molecules in common signaling transduction pathways in B-ALL cells treated with recombinant netrin-1, and the FAK-MAPK signaling pathway was found to be activated. The anti-apoptotic effect of netrin-1 and FAK-MAPK phosphorylation was abrogated by UNC5B interference. FAK inhibitor treatment and ERK inhibitor treatment were applied and verified that the FAK-MAPK pathway may be downstream of Unc5b. CONCLUSION: Taken together, our findings suggested that netrin-1 induced the anti-apoptotic effect of B-ALL cells through activation of the FAK-MAPK signaling pathway by binding to the receptor Unc5b. Video Abstract.

Somatotopy of Mouse Spinothalamic Innervation and the Localization of a Noxious Stimulus Requires Deleted in Colorectal Carcinoma Expression by Phox2a Neurons.

Anterolateral system (AS) neurons transmit pain signals from the spinal cord to the brain. Their morphology, anatomy, and physiological properties have been extensively characterized and suggest that specific AS neurons and their brain targets are concerned with the discriminatory aspects of noxious stimuli, such as their location or intensity, and their motivational/emotive dimension. Among the recently unraveled molecular markers of AS neurons is the developmentally expressed transcription factor Phox2a, providing us with the opportunity to selectively disrupt the embryonic wiring of AS neurons to gain insights into the logic of their adult function. As mice with a spinal-cord-specific loss of the netrin-1 receptor deleted in colorectal carcinoma (DCC) have increased AS neuron innervation of ipsilateral brain targets and defective noxious stimulus localization or topognosis, we generated mice of either sex carrying a deletion of Dcc in Phox2a neurons. Such DccPhox2a mice displayed impaired topognosis along the rostrocaudal axis but with little effect on left-right discrimination and normal aversive responses. Anatomical tracing experiments in DccPhox2a mice revealed defective targeting of cervical and lumbar AS axons within the thalamus. Furthermore, genetic labeling of AS axons revealed their expression of DCC on their arrival in the brain, at a time when many of their target neurons are being born and express Ntn1 Our experiments suggest a postcommissural crossing function for netrin-1:DCC signaling during the formation of somatotopically ordered maps and are consistent with a discriminatory function of some of the Phox2a AS neurons.SIGNIFICANCE STATEMENT How nociceptive (pain) signals are relayed from the body to the brain remains an important question relevant to our understanding of the basic physiology of pain perception. Previous studies have demonstrated that the AS is a main effector of this function. It is composed of AS neurons located in the spinal cord that receive signals from nociceptive sensory neurons that detect noxious stimuli. In this study, we generate a genetic miswiring of mouse AS neurons that results in a decreased ability to perceive the location of a painful stimulus. The precise nature of this defect sheds light on the function of different kinds of AS neurons and how pain information may be organized.

A novel signature to guide osteosarcoma prognosis and immune microenvironment: Cuproptosis-related lncRNA.

Objective: Osteosarcoma (OS) is a common bone malignancy with poor prognosis. We aimed to investigate the relationship between cuproptosis-related lncRNAs (CRLncs) and the survival outcomes of patients with OS. Methods: Transcriptome and clinical data of 86 patients with OS were downloaded from The Cancer Genome Atlas (TCGA). The GSE16088 dataset was downloaded from the Gene Expression Omnibus (GEO) database. The 10 cuproptosis-related genes (CRGs) were obtained from a recently published article on cuproptosis in Science. Combined analysis of OS transcriptome data and the GSE16088 dataset identified differentially expressed CRGs related to OS. Next, pathway enrichment analysis was performed. Co-expression analysis obtained CRLncs related to OS. Univariate COX regression analysis and least absolute shrinkage and selection operator (LASSO) regression analysis were used to construct the risk prognostic model of CRLncs. The samples were divided evenly into training and test groups to verify the accuracy of the model. Risk curve, survival, receiver operating characteristic (ROC) curve, and independent prognostic analyses were performed. Next, principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE) analysis were performed. Single-sample gene set enrichment analysis (ssGSEA) was used to explore the correlation between the risk prognostic models and OS immune microenvironment. Drug sensitivity analysis identified drugs with potential efficacy in OS. Real-time quantitative PCR, Western blotting, and immunohistochemistry analyses verified the expression of CRGs in OS. Real-time quantitative PCR was used to verify the expression of CRLncs in OS. Results: Six CRLncs that can guide OS prognosis and immune microenvironment were obtained, including three high-risk CRLncs (AL645608.6, AL591767.1, and UNC5B-AS1) and three low-risk CRLncs (CARD8-AS1, AC098487.1, and AC005041.3). Immune cells such as B cells, macrophages, T-helper type 2 (Th2) cells, regulatory T cells (Treg), and immune functions such as APC co-inhibition, checkpoint, and T-cell co-inhibition were significantly downregulated in high-risk groups. In addition, we obtained four drugs with potential efficacy for OS: AUY922, bortezomib, lenalidomide, and Z.LLNle.CHO. The expression of LIPT1, DLAT, and FDX1 at both mRNA and protein levels was significantly elevated in OS cell lines compared with normal osteoblast hFOB1.19. The mRNA expression level of AL591767.1 was decreased in OS, and that of AL645608.6, CARD8-AS1, AC005041.3, AC098487.1, and UNC5B-AS1 was upregulated in OS. Conclusion: CRLncs that can guide OS prognosis and the immune microenvironment and drugs that may have a potential curative effect on OS obtained in this study provide a theoretical basis for OS survival research and clinical decision-making.

PTBP1 knockdown promotes neural differentiation of glioblastoma cells through UNC5B receptor.

Rationale: Cell reprogramming technology is utilized to prevent cancer progression by transforming cells into terminally differentiated, non-proliferating states. Polypyrimidine tract binding protein 1 (PTBP1) is an RNA binding protein required for the growth of neurons and may directly transform multiple normal human cells into functioning neurons in vitro and in vivo when expressed at low levels. As a result, we identified it as a key to inhibiting cancer cell proliferation by boosting glioblastoma cell neural differentiation. Methods: Immunocytofluorescence (ICF) targeting TUJ1, MAP2, KI67, and EdU were utilized to evaluate glioblastoma cell reprogramming under PTBP1 knockdown or other conditions. PTBP1 and other target genes were detected using Western blotting and qRT-PCR. Activating protein phosphatase 2A (PP2A) and RhoA were detected using specific kits. CCK8 assays were employed to detect cell viability. Bioluminescence, immunohistofluorescence (IHF), and Kaplan-Meier survival analyses were utilized to demonstrate the in vivo reprogramming efficiency of PTBP1 knockdown in U87 murine glioblastoma model. In this study, RNA-seq technology was used to examine the intrinsic pathway. Results: The expression of TUJ1 and MAP2 neural markers, as well as the absence of KI67 and EdU proliferative markers in U251, U87, and KNS89 cells, indicated that glioblastoma cell reprogramming was successful. In vivo, U87 growth generated xenografts was substantially shrank due to PTBP1 knockdown induced neural differentiation, and these tumor-bearing mice had a prolonged survival time. Following RNA-seq, ten potential downstream genes were eliminated. Lentiviral interference and inhibitors blocking tests demonstrated that UNC5B receptor and its downstream signaling were essential in the neural differentiation process mediated by PTBP1 knockdown in glioblastoma cells. Conclusions: Our results indicate that PTBP1 knockdown promotes neural differentiation of glioblastoma cells via UNC5B receptor, consequently suppressing cancer cell proliferation in vitro and in vivo, providing a promising and feasible approach for glioblastoma treatment.

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.

[Netrin-1, a novel antitumoral target]. / La nétrine-1, une nouvelle cible antitumorale.

Netrin-1, a secreted molecule that was first described for its role in guidance during embryogenesis, was then brought to light for its overexpression in a large number of aggressive cancers. Netrin-1 is a ligand of "dependence receptors". In adults, the interaction between Netrine-1 and these receptors triggers the survival, proliferation, and migration of different cell types. This will confer better survival properties to tumor cells, making them more prone to form aggressive tumors. A recently developed novel therapy aims at inhibiting the binding of Netrin-1 to these receptors in order to trigger cell death by apoptosis. This article presents a review of the functional characteristics of the Netrin-1 molecule, and the potential effects of a novel targeted therapy against Netrin-1 that could lead to very promising results in combination with conventional anti-cancer treatments.

Title: La nétrine-1, une nouvelle cible antitumorale. Abstract: La nétrine-1, une molécule sécrétée mise en évidence pour son rôle de guidage au cours de l'embryogenèse, a été également décrite pour être surexprimée dans de nombreux cancers agressifs. Elle est le ligand de récepteurs dits « à dépendance ¼, à l'origine, chez l'adulte, de la survie, de la prolifération et de la migration de différents types cellulaires, ce qui confère aux cellules cancéreuses des propriétés avantageuses leur permettant de se développer sous forme de tumeurs agressives. Une stratégie thérapeutique consiste à inhiber l'interaction de la nétrine-1 avec son récepteur, ce qui déclenche la mort des cellules par apoptose. Cet article présente une revue des caractéristiques fonctionnelles de cette molécule et les effets potentiels d'une nouvelle thérapie ciblée sur la nétrine-1, dont la combinaison avec les traitements conventionnels pourrait être des plus prometteurs.

The netrin-1 receptor DCC promotes the survival of a subpopulation of midbrain dopaminergic neurons: Relevance for ageing and Parkinson's disease.

Mechanisms that determine the survival of midbrain dopaminergic (mDA) neurons in the adult central nervous system (CNS) are not fully understood. Netrins are a family of secreted proteins that are essential for normal neural development. In the mature CNS, mDA neurons express particularly high levels of netrin-1 and its receptor Deleted in Colorectal Cancer (DCC). Recent findings indicate that overexpressing netrin-1 protects mDA neurons in animal models of Parkinson's disease (PD), with a proposed pro-apoptotic dependence function for DCC that triggers cell death in the absence of a ligand. Here, we sought to determine if DCC expression influences mDA neuron survival in young adult and ageing mice. To circumvent the perinatal lethality of DCC null mice, we selectively deleted DCC from mDA neurons utilizing DATcre /loxP gene-targeting and examined neuronal survival in adult and aged animals. Reduced numbers of mDA neurons were detected in the substantia nigra pars compacta (SNc) of young adult DATcre /DCCfl/fl mice, with further reduction in aged DATcre /DCCfl/fl animals. In contrast to young adults, aged mice also exhibited a gene dosage effect, with fewer SNc mDA neurons in DCC heterozygotes (DATcre /DCCfl/wt ). Notably, loss of mDA neurons in the SN was not uniform. Neuronal loss in the SN was limited to ventral tier mDA neurons, while mDA neurons in the dorsal tier of the SN, which resist degeneration in PD, were spared from the effect of DCC deletion in both young and aged mice. In the ventral tegmental area (VTA), young adult mice with conditional deletion of DCC had normal mDA neuronal numbers, while significant loss occurred in aged DATcre /DCCfl/fl and DATcre /DCCfl/wt mice compared to age-matched wild-type mice. Our results indicate that expression of DCC is required for the survival of subpopulations of mDA neurons and may be relevant to the degenerative processes in PD.

UNC5B-AS1 promotes the proliferation, migration and EMT of hepatocellular carcinoma cells via regulating miR-4306/KDM2A axis.

Being one of the most prevalent malignancies, hepatocellular carcinoma (HCC) threatens the health of population all over the world. Numerous researches have confirmed that long noncoding RNAs (lncRNAs) play an important role in tumor progression. Nonetheless, the mechanisms of unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1) in HCC remain obscure. Thus, this study aims to investigate the regulatory role and mechanism of UNC5B-AS1 in HCC cells. In our research, UNC5B-AS1 was subjected to gene expression analysis by RT-qPCR. Biological functions of UNC5B-AS1 in HCC cells were measured by MTT, colony formation, EdU and transwell assays. The combination between UNC5B-AS1, lysine demethylase 2A (KDM2A) and miR-4306 was validated by mechanism assays. Result showed UNC5B-AS1 was upregulated in HCC tissues and cells, contributing to the development of cancer staging and survival rate of HCC patients. Moreover, UNC5B-AS1 deficiency inhibited the proliferation, migration and epithelial-mesenchymal transition (EMT) of HCC cells. Furthermore, UNC5B-AS1 could interact with miR-4306 in HCC cells. Similarly, KDM2A was proved as the target gene of miR-4306. Finally, miR-4306 downregulation or KDM2A overexpression reversed the prohibitive role of UNC5B-AS1 knockdown in HCC progression. In short, UNC5B-AS1 accelerates the proliferation, migration and EMT of HCC cells via the regulation of miR-4306/KDM2A axis.

Inhibition of the long non-coding RNA UNC5B-AS1/miR-4455/RSPO4 axis reduces cervical cancer growth in vitro and in vivo.

BACKGROUND: Long non-coding RNAs (lncRNAs) are significant regulatory factors for the initiation and development of numerous malignant tumors, including cervical cancer (CC). The expression of lncRNA unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1, also known as UASR1) is up-regulated in tissues of cervical squamous cell carcinoma and endocervical adenocarcinoma compared to in normal tissues based on the GEPIA database. In the present study, we explored the functions of UNC5B-AS1 and its underlying mechanism with respect to CC development. METHODS: A real-time quantitative polymerase chain reaction was applied for the detection of UNC5B-AS1 expression in CC cells. Cell counting kit-8, colony formation and transwell assays, as well as western blot and flow cytometry analyses, were employed to detect the biological effects of UNC5B-AS1 knockdown on malignant phenotypes of CC cells in vitro. In addition, the combination between microRNA-4455 (miR-4455) and UNC5B-AS1 or R-spondin 4 (RSPO4) was explored by RNA immunoprecipitation, luciferase reporter and RNA pulldown assays. A tumor xenograft nude mice model was established to explore the effect of UNC5B-AS1 depletion or miR-4455 overexpression on tumor growth. RESULTS: UNC5B-AS1 is up-regulated in CC tissues and cells. The knockdown of UNC5B-AS1 inhibits CC cell proliferation, migration and invasion and promotes CC cell apoptosis. Mechanistically, UNC5B-AS1 binds with miR-4455 to up-regulate RSPO4 expression. RSPO4 is targeted by miR-4455 and its expression is negatively regulated by miR-4455 expression. In vivo assays revealed that UNC5B-AS1 depletion or miR-4455 overexpression inhibits tumor growth by regulating RSPO4 expression. CONCLUSIONS: Inhibition of UNC5B-AS1/miR-4455/RSPO4 reduces CC growth both in vitro and in vivo, furnishing new insights into molecular studies on UNC5B-AS1 with respect to CC development.

Netrin-1 in Glioblastoma Neovascularization: The New Partner in Crime?

Glioblastoma (GBM) is the most aggressive and common primary tumor of the central nervous system. It is characterized by having an infiltrating growth and by the presence of an excessive and aberrant vasculature. Some of the mechanisms that promote this neovascularization are angiogenesis and the transdifferentiation of tumor cells into endothelial cells or pericytes. In all these processes, the release of extracellular microvesicles by tumor cells plays an important role. Tumor cell-derived extracellular microvesicles contain pro-angiogenic molecules such as VEGF, which promote the formation of blood vessels and the recruitment of pericytes that reinforce these structures. The present study summarizes and discusses recent data from different investigations suggesting that Netrin-1, a highly versatile protein recently postulated as a non-canonical angiogenic ligand, could participate in the promotion of neovascularization processes in GBM. The relevance of determining the angiogenic signaling pathways associated with the interaction of Netrin-1 with its receptors is posed. Furthermore, we speculate that this molecule could form part of the microvesicles that favor abnormal tumor vasculature. Based on the studies presented, this review proposes Netrin-1 as a novel biomarker for GBM progression and vascularization.

A ligand-insensitive UNC5B splicing isoform regulates angiogenesis by promoting apoptosis.

The Netrin-1 receptor UNC5B is an axon guidance regulator that is also expressed in endothelial cells (ECs), where it finely controls developmental and tumor angiogenesis. In the absence of Netrin-1, UNC5B induces apoptosis that is blocked upon Netrin-1 binding. Here, we identify an UNC5B splicing isoform (called UNC5B-Δ8) expressed exclusively by ECs and generated through exon skipping by NOVA2, an alternative splicing factor regulating vascular development. We show that UNC5B-Δ8 is a constitutively pro-apoptotic splicing isoform insensitive to Netrin-1 and required for specific blood vessel development in an apoptosis-dependent manner. Like NOVA2, UNC5B-Δ8 is aberrantly expressed in colon cancer vasculature where its expression correlates with tumor angiogenesis and poor patient outcome. Collectively, our data identify a mechanism controlling UNC5B's necessary apoptotic function in ECs and suggest that the NOVA2/UNC5B circuit represents a post-transcriptional pathway regulating angiogenesis.

UNC5B Promotes Vascular Endothelial Cell Senescence via the ROS-Mediated P53 Pathway.

Vascular endothelial cell senescence is involved in human aging and age-related vascular disorders. Guidance receptor UNC5B is implicated in oxidative stress and angiogenesis. Nonetheless, little is known about the role of UNC5B in endothelial cell senescence. Here, we cultured primary human umbilical vein endothelial cells to young and senescent phases. Subsequently, the expression of UNC5B was identified in replicative senescent cells, and then, its effect on endothelial cell senescence was confirmed by UNC5B-overexpressing lentiviral vectors and RNA interference. Overexpression of UNC5B in young endothelial cells significantly increased senescence-associated ß-galactosidase-positive cells, upregulated the mRNAs expression of the senescence-associated secretory phenotype genes, reduced total cell number, and inhibited the potential for cell proliferation. Furthermore, overexpression of UNC5B promoted the generation of intracellular reactive oxygen species (ROS) and activated the P53 pathway. Besides, overexpression of UNC5B disturbed endothelial function by inhibiting cell migration and tube formation. Nevertheless, silencing UNC5B generated conflicting outcomes. Blocking ROS production or inhibiting the function of P53 rescued endothelial cell senescence induced by UNC5B. These findings suggest that UNC5B promotes endothelial cell senescence, potentially by activating the ROS-P53 pathway. Therefore, inhibiting UNC5B might reduce endothelial cell senescence and hinder age-related vascular disorders.