Pharmacological targeting of netrin-1 inhibits EMT in cancer.

Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1-7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195 ), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line-which undergoes EMT following TGFß1 administration8,9-with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.

Netrin-1 blockade inhibits tumour growth and EMT features in endometrial cancer.

Netrin-1 is upregulated in cancers as a protumoural mechanism1. Here we describe netrin-1 upregulation in a majority of human endometrial carcinomas (ECs) and demonstrate that netrin-1 blockade, using an anti-netrin-1 antibody (NP137), is effective in reduction of tumour progression in an EC mouse model. We next examined the efficacy of NP137, as a first-in-class single agent, in a Phase I trial comprising 14 patients with advanced EC. As best response we observed 8 stable disease (8 out of 14, 57.1%) and 1 objective response as RECIST v.1.1 (partial response, 1 out of 14 (7.1%), 51.16% reduction in target lesions at 6 weeks and up to 54.65% reduction during the following 6 months). To evaluate the NP137 mechanism of action, mouse tumour gene profiling was performed, and we observed, in addition to cell death induction, that NP137 inhibited epithelial-to-mesenchymal transition (EMT). By performing bulk RNA sequencing (RNA-seq), spatial transcriptomics and single-cell RNA-seq on paired pre- and on-treatment biopsies from patients with EC from the NP137 trial, we noted a net reduction in tumour EMT. This was associated with changes in immune infiltrate and increased interactions between cancer cells and the tumour microenvironment. Given the importance of EMT in resistance to current standards of care2, we show in the EC mouse model that a combination of NP137 with carboplatin-paclitaxel outperformed carboplatin-paclitaxel alone. Our results identify netrin-1 blockade as a clinical strategy triggering both tumour debulking and EMT inhibition, thus potentially alleviating resistance to standard treatments.

Investigation of effect of paclitaxel on netrin 1 and factor 8 in Ehrlich solid tumors / Investigación del efecto del paclitaxel en netrina 1 y factor 8 en tumores sólidos de Ehrlich

SUMMARY: Cancer known as a malignant tumor, is a class of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. The Ehrlich tumor is a mammary adenocarcinoma of mice developed in solid and ascitic forms. This study was aimed to investigate the effects of paclitaxel on Netrin 1 and Factor 8 expression and also in tumor cell proliferation, apoptosis, angiogenesis, and development of tumor in Ehrlich solid tumors treated with paclitaxel. In this study, 26 adult Balb/C male mice were used. 6 of them were used as stock. Ehrlich ascites cells taken from animals in stock were injected subcutaneously from the neck area to all animals. The mice were randomly assigned to two groups of ten rats per group. Paclitaxel treatment group 10 mg/kg were administered to mice intraperitoneally (i.p.) 4,9, and 14th days. 15th day the animals were sacrificed and tumor tissues were taken. Paraffin-embedded solid tumor sections were stained Hematoxylin & Eosin, Masson's Trichrome. Also solid tumor sections were stained immunohistochemically with Netrin1 and Factor 8. Tunel method was applied to determine apoptosis. Paclitaxel applied as a therapeutic Ehrlich solid tumor reduced the volume of tumors in the treatment groups. At the end of the experiments, in the treatment groups' significantly reduced the Netrin 1 expression and microvessel density compared to the group control. Also paclitaxel in the treatment group increased the number of apoptotic cells. We suggest that decreasing the expression of Netrin 1 would be reduced vessel density and increased apoptosis.

RESUMEN: El cáncer, conocido como tumor maligno, es una clase de enfermedad que involucra un crecimiento celular anormal con potencial de invadir o diseminarse a otras partes del cuerpo. El tumor de Ehrlich es un adenocarcinoma mamario de ratones desarrollado en formas sólidas y ascíticas. Este estudio tuvo como objetivo investigar los efectos del paclitaxel en la expresión de Netrin 1 y Factor 8 y también en la proliferación de células tumorales, apoptosis, angiogénesis y desarrollo de tumores sólidos de Ehrlich tratados con paclitaxel. En esta investigación se utilizaron 26 ratones machos Balb / C adultos. Seis de ellos se utilizaron como stock. Se inyectaron por vía subcutánea células de ascitis de Ehrlich tomadas de animales en la zona del cuello. Los ratones se asignaron aleatoriamente a dos grupos de diez ratas por grupo. Se administraron 10 mg/kg del grupo de tratamiento con paclitaxel a ratones por vía intraperitoneal (i.p.) 4, 9 y 14 días. El día 15 se sacrificaron los animales y se extrajeron los tejidos tumorales. Las secciones de tumor sólido incluidas en parafina se tiñeron con hematoxilina y eosina y tricrómico de Masson. También se tiñeron inmunohisto-químicamente secciones de tumor sólido con Netrin1 y Factor 8. Se aplicó el método Tunel para determinar la apoptosis. El paclitaxel aplicado como tumor sólido terapéutico de Ehrlich redujo el volumen de tumores en los grupos de tratamiento. Al final de los experimentos, en los grupos de tratamiento se redujo significativamente la expresión de Netrin 1 y la densidad de microvasos en comparación con el grupo control. Además, el paclitaxel en el grupo tratamiento aumentó el número de células apoptóticas. Sugerimos que la disminución de la expresión de Netrin 1 reduciría la densidad de los vasos y aumentaría la apoptosis.

Netrin-1 reduces lung ischemia-reperfusion injury by increasing the proportion of regulatory T cells.

OBJECTIVE: Inflammation is the primary mechanism of lung ischemia-reperfusion injury (LIRI) and neurologic factors can regulate inflammatory immune responses. Netrin-1 is an axonal guidance molecule, but whether Netrin-1 plays a role in LIRI remains unclear. METHODS: A mouse model of LIRI was established. Immunohistochemistry was used to detect expression of Netrin-1 and to enumerate macrophages and T cells in lung tissue. The proportion of regulatory T cells (Tregs) was assessed by flow cytometry. Levels of apoptosis were assessed by terminal deoxynucleotidyl transferase dUTP nick end staining. RESULTS: Numbers of macrophages and T cells in the lung tissues of mice with LIRI were elevated, while expression of netrin-1 was significantly decreased. Flow cytometry showed that the proportion of Tregs in mice with LIRI was significantly decreased. The proportion of Tregs among lymphocytes was positively correlated with netrin-1 expression. In vitro experiments showed that netrin-1 promoted an increase in Treg proportion through the A2b receptor. Animal experiments showed that netrin-1 could inhibit apoptosis and reduce T cell and macrophage infiltration by increasing the proportion of Tregs, ultimately reducing LIRI. Treg depletion using an anti-CD25 monoclonal antibody blocked the effects of netrin-1. CONCLUSION: Netrin-1 reduced LIRI by increasing the proportion of Tregs.

Netrin-1 and Its Receptor DCC Are Causally Implicated in Melanoma Progression.

Deleted in colorectal cancer (DCC), the receptor for the multifunctional cue netrin-1, acts as a tumor suppressor in intestinal cancer and lung metastasis by triggering cancer cell death when netrin-1 is lowly expressed. Recent genomic data highlighted that DCC is the third most frequently mutated gene in melanoma; we therefore investigated whether DCC could act as a melanoma tumor suppressor. Reexpressing DCC in human melanoma cell lines promoted tumor cell death and tumor growth inhibition in xenograft mouse models. Genetic silencing of DCC prodeath activity in a BRAFV600E mouse model increased the proportion of mice with melanoma, further supporting that DCC is a melanoma tumor suppressor. Netrin-1 expression was elevated in melanoma compared with benign melanocytic lesions. Upregulation of netrin-1 in the skin cells of a BRAFV600E-mutated murine model reduced cancer cell death and promoted melanoma progression. Therapeutic antibody blockade of netrin-1 combined with dacarbazine increased overall survival in several mouse melanoma models. Together, these data support that interfering with netrin-1 could be a viable therapeutic approach in patients with netrin-1-expressing melanoma. SIGNIFICANCE: Netrin-1 and its receptor DCC regulate melanoma progression, suggesting therapeutic targeting of this signaling axis as a viable option for melanoma treatment.

Therapeutic Targeting of the Notch Pathway in Glioblastoma Multiforme.

BACKGROUND: Glioblastoma (GBM) is the most common and deadly form of brain tumor. After standard treatment of resection, radiotherapy, and chemotherapy, the 5-year survival is <5%. In recent years, research has uncovered several potential targets within the Notch signaling pathway, which may lead to improved patient outcomes. METHODS: A literature search was performed for articles containing the terms "Glioblastoma" and "Receptors, Notch" between 2003 and July 2015. Of the 62 articles retrieved, 46 met our criteria and were included in our review. Nine articles were identified from other sources and were subsequently included, leaving 55 articles reviewed. RESULTS: Of the 55 articles reviewed, 47 used established human GBM cell lines. Seventeen articles used human GBM surgical samples. Forty-five of 48 articles that assessed Notch activity showed increased expression in GBM cell lines. Targeting the Notch pathway was carried out through Notch knockdown and overexpression and targeting δ-like ligand, Jagged, γ-secretase, ADAM10, ADAM17, and Mastermindlike protein 1. Arsenic trioxide, microRNAs, and several other compounds were shown to have an effect on the Notch pathway in GBM. Notch activity in GBM was also shown to be associated with hypoxia and certain cancer-related molecular pathways such as PI3K/AKT/mTOR and ERK/MAPK. Most articles concluded that Notch activity amplifies malignant characteristics in GBM and targeting this pathway can bring about amelioration of these effects. CONCLUSIONS: Recent literature suggests targeting the Notch pathway has great potential for future therapies for GBM.

Ultrasound molecular imaging as a non-invasive companion diagnostic for netrin-1 interference therapy in breast cancer.

In ultrasound molecular imaging (USMI), ligand-functionalized microbubbles (MBs) are used to visualize vascular endothelial targets. Netrin-1 is upregulated in 60% of metastatic breast cancers and promotes tumor progression. A novel netrin-1 interference therapy requires the assessment of netrin-1 expression prior to treatment. In this study, we studied netrin-1 as a target for USMI and its potential as a companion diagnostic in breast cancer models. Methods: To verify netrin-1 expression and localization, an in vivo immuno-localization approach was applied, in which anti-netrin-1 antibody was injected into living mice 24 h before tumor collection, and revealed with secondary fluorescent antibody for immunofluorescence analysis. Netrin-1 interactions with the cell surface were studied by flow cytometry. Netrin-1-targeted MBs were prepared using MicroMarker Target-Ready (VisualSonics), and validated in in vitro binding assays in static conditions or in a flow chamber using purified netrin-1 protein or netrin-1-expressing cancer cells. In vivo USMI of netrin-1 was validated in nude mice bearing human netrin-1-positive SKBR7 tumors or weakly netrin-1-expressing MDA-MB-231 tumors using the Vevo 2100 small animal imaging device (VisualSonics). USMI feasibility was further tested in transgenic murine FVB/N Tg(MMTV/PyMT634Mul) (MMTV-PyMT) mammary tumors. Results: Netrin-1 co-localized with endothelial CD31 in netrin-1-positive breast tumors. Netrin-1 binding to the surface of endothelial HUVEC and cancer cells was partially mediated by heparan sulfate proteoglycans. MBs targeted with humanized monoclonal anti-netrin-1 antibody bound to netrin-1-expressing cancer cells in static and dynamic conditions. USMI signal was significantly increased with anti-netrin-1 MBs in human SKBR7 breast tumors and transgenic murine MMTV-PyMT mammary tumors compared to signals recorded with either isotype control MBs or after blocking of netrin-1 with humanized monoclonal anti-netrin-1 antibody. In weakly netrin-1-expressing human tumors and normal mammary glands, no difference in imaging signal was observed with anti-netrin-1- and isotype control MBs. Ex vivo analysis confirmed netrin-1 expression in MMTV-PyMT tumors. Conclusions: These results show that USMI allowed reliable detection of netrin-1 on the endothelium of netrin-1-positive human and murine tumors. Significant differences in USMI signal for netrin-1 reflected the significant differences in netrin-1 mRNA & protein expression observed between different breast tumor models. The imaging approach was non-invasive and safe, and provided the netrin-1 expression status in near real-time. Thus, USMI of netrin-1 has the potential to become a companion diagnostic for the stratification of patients for netrin-1 interference therapy in future clinical trials.

Resveratrol ameliorates depressive disorder through the NETRIN1-mediated extracellular signal-regulated kinase/cAMP signal transduction pathway.

Depressive disorder is a mental health disorder caused by the dysfunction of nerve regeneration, neuroendocrine and neurobiochemistry, which frequently results in cognitive impairments and disorder. Evidence has shown that resveratrol offers benefits for the treatment of depressive disorder. In the present study, the therapeutic effects of resveratrol were investigated and the potential mechanisms mediated by resveratrol were analyzed in hippocampal neuron cells. The anti­oxidative stress and anti­inflammatory properties of resveratrol were also examined in vitro and in vivo. The results revealed that resveratrol administration inhibited the inflammation in hippocampal neuron cells induced by ouabain. Oxidative stress in the hippocampal neuron cells was ameliorated by resveratrol treatment in vitro and in vivo. In addition, the apoptosis of hippocampal neuron cells was inhibited by the upregulation of anti­apoptotic genes, including P53, B­cell lymphoma­2 (Bcl­2) and Bcl­2­associated death promoter, and the downregulation of the cleaved caspase­3 and caspase­9. The analysis of the mechanism revealed that that resveratrol treatment suppressed the apoptosis of hippocampal neuron cells through the NETRIN1­mediated extracellular signal­regulated kinase/cAMP signal transduction pathway. The results of the in vivo assay showed that resveratrol treatment led to improvements in cognitive competence, learning memory ability and anxiety in a mouse model of depressive disorder induced by ouabain. In conclusion, these results indicated that resveratrol treatment had protective effects against oxidative stress and neuroinflammatory pathogenesis through the NETRIN1­mediated extracellular signal­regulated kinase/cAMP signal transduction pathway, suggesting that resveratrol treatment may be a potential antidepressant agent for the treatment of depressive disorder.

Netrin-1 promotes glioma growth by activating NF-κB via UNC5A.

Gliomas, a common type of brain tumor, are characterized by aggressive infiltration, making it difficultly to cure by surgery. Netrin-1, an extracellular guidance cue critical for neuronal axon path-finding, has been reported to play an important role in cell invasion and migration in several types of cancers. However, the role of netrin-1 in glioma remains largely unknown. Here, we provide evidence suggested that Netrin-1 has a critical role in glioma growth. We found that netrin-1 was significantly increased in glioma samples and positively correlated with cell proliferation, tumor grade and malignancy. Netrin-1 knockdown reduced cell proliferation and attenuated tumor growth in a xenograft mouse model. Further studies found that netrin-1 induced NF-κB p65ser536 phosphorylation and c-Myc expression in vitro and in vivo. Interestingly, activation of NF-κB by netrin-1 was dependent on UNC5A receptor, because suppression of UNC5A significantly inhibited NF-κB p65ser536 phosphorylation, c-Myc up-regulation and reduced cell proliferation. Taken together, these results suggested netrin-1 promotes glioma cell proliferation by activating NF-κB signaling via UNC5A, netrin-1 may be a potential therapeutic target for the treatment of glioma.

Netrin-1 restores cell injury and impaired angiogenesis in vascular endothelial cells upon high glucose by PI3K/AKT-eNOS.

Diabetic foot ulceration (DFU) represents a common vascular complication of diabetes mellitus (DM) with high morbidity and disability resulting from amputation. Netrin-1 level was decreased in type 2 DM patients and has been identified as a protective regulator against diabetes-triggered myocardial infarction and nephropathy. Unfortunately, its role and molecular mechanism in DFU remain poorly elucidated. Here, netrin-1 levels were reduced in DM and DFU patients relative to healthy controls, with netrin-1 levels being the lowest in DFU patients. Moreover, exposure to high glucose (HG) also suppressed netrin-1 expression in human umbilical vein endothelial cells (HUVECs). Elevated netrin-1 expression by infection with Ad-netrin-1 adenovirus vector protected against HUVEC injury in response to HG by ameliorating the inhibitory effects on cell viability, lactate dehydrogenase (LDH) and malondialdehyde (MDA) levels, cell apoptotic rate and caspase-3 activity. Importantly, HG-impaired angiogenesis was improved after netrin-1 overexpression by elevating cell migration, capillary-like tube formation and VEGF production. Mechanism assay substantiated that netrin-1 elevation increased the phosphorylation levels of AKT and eNOS, and NO production, which was notably suppressed by HG, indicating that netrin-1 overexpression restored HG-triggered impairment of the PI3K/AKT-eNOS pathway. More intriguingly, preconditioning with LY294002 (PI3K/AKT antagonist) or NG-monomethyl-l-arginine (eNOS inhibitor) antagonized netrin-1-induced activation of the PI3K/AKT-eNOS pathway. Concomitantly, treatment with these antagonists also attenuated the protective role of netrin-1 in endothelial dysfunction upon HG stimulation. These results suggest that elevation of netrin-1 may restore HG-triggered impairment of HUVEC and angiogenesis by activating the PI3K/AKT-eNOS pathway, indicating a potential agent for wound healing in DFU patients.