Academic literature on the topic 'Netrin-1 (NTN1)'

Rationale: Therapeutic efforts to decrease atherosclerotic cardiovascular disease risk have focused largely on reducing atherogenic lipoproteins, yet lipid-lowering therapies alone are insufficient to fully regress plaque burden. We postulate that arterial repair requires resolution of a maladaptive immune response and that targeting factors that hinder inflammation resolution will facilitate plaque regression. Objective: The guidance molecule Ntn1 (netrin-1) is secreted by macrophages in atherosclerotic plaques, where it sustains inflammation by enhancing macrophage survival and blocking macrophage emigration. We tested whether silencing Ntn1 in advanced atherosclerosis could resolve arterial inflammation and regress plaques. Methods and Results: To temporally silence Ntn1 in myeloid cells, we generated genetically modified mice in which Ntn1 could be selectively deleted in monocytes and macrophages using a tamoxifen-induced CX3CR1-driven cre recombinase ( Ntn1 fl/fl Cx3cr1 creERT2+ ) and littermate control mice ( Ntn1 fl/fl Cx3cr1 WT ). Mice were fed Western diet in the setting of hepatic PCSK9 (proprotein convertase subtilisin/kexin type 9) overexpression to render them atherosclerotic and then treated with tamoxifen to initiate deletion of myeloid Ntn1 (Mø ΔNtn1 ) or not in controls (Mø WT ). Morphometric analyses performed 4 weeks later showed that myeloid Ntn1 silencing reduced plaque burden in the aorta (−50%) and plaque complexity in the aortic root. Monocyte-macrophage tracing experiments revealed lower monocyte recruitment, macrophage retention, and proliferation in Mø ΔNtn1 compared with Mø WT plaques, indicating a restructuring of monocyte-macrophage dynamics in the artery wall upon Ntn1 silencing. Single-cell RNA sequencing of aortic immune cells before and after Ntn1 silencing revealed upregulation of gene pathways involved in macrophage phagocytosis and migration, including the Ccr7 chemokine receptor signaling pathway required for macrophage emigration from plaques and atherosclerosis regression. Additionally, plaques from Mø ΔNtn1 mice showed hallmarks of inflammation resolution, including higher levels of proresolving macrophages, IL (interleukin)-10, and efferocytosis, as compared to plaques from Mø WT mice. Conclusion: Our data show that targeting Ntn1 in advanced atherosclerosis ameliorates atherosclerotic inflammation and promotes plaque regression.

Abstract A hallmark of pancreatic ductal adenocarcinoma (PDAC) is its proclivity for metastasis as evidenced by the fact that 85% are stage IV at diagnosis. This highlights the need to better understand the biology of metastatic PDAC and identify novel therapies for this patient population. Axon guidance genes have been shown to be involved in PDAC progression, but their role is unclear. We have investigated the role of the axon guidance molecule Netrin-1 and its receptors Unc5b and DCC in PDAC. We found that in both murine and human samples that NTN1 expression is increased in metastatic PDAC and the quasi-mesenchymal subtype. Murine and TCGA data indicate that Unc5b is the dominant NTN1 receptor and genetic knock-down (KD) or knock-out (KO) of either Netrin-1 or Unc5b decreases migration, invasion, and cell survival in vitro and hepatic metastatic growth in vivo. The mechanism of Netrin-1 upregulation in metastatic PDAC is unknown. We found that hepatic stellate cell (HSC) secreted retinoic acid upregulates NTN1 through both an RXR/RAR and Elf mediated mechanism. To determine if NTN1 is involved in the process of HSC activation we found that recombinant NTN1 added to HSCs in vitro induced activation. We examined the livers of mice harboring orthotopic PDAC tumors using murine pancreatic cancer lines that were either NTN1 wild type (WT) or KO, and found that the NTN-expressing lines increased HSC activation providing evidence that NTN1 is important for long distance intercellular communication between primary pancreatic tumors and the pre-metastatic liver. We detected NTN1 within extracellular vesicles, and mice pre-conditioned with EVs from NTN1 KO cells demonstrated a decreased metastatic burden as compared mice preconditioned with NTN1 WT cells. Treatment of several murine PDAC models (autochthonous and metastatic) with a monoclonal antibody to NTN1 led to decreased metastases and increased survival. These studies reveal that NTN1 is upregulated in metastatic PDAC mediated by a novel mechanism that involves EVs, HSC activation and RXR/RAR signaling. These studies provide pre-clinical evidence to support a human clinical trial of anti-NTN1 therapy in PDAC. Citation Format: Crissy Dudgeon, Anthony Casabianca, Chris Harris, Igor Astsaturov, Charline Ogier, Xiaoyang Su, Jason Pitarresi, Wade Narrow, Fady Soliman, Tracy Withers, Patrick Mehlen, Darren Carpizo. Retinoic acid produced by hepatic stellate cells facilitates Netrin-1 mediated pancreatic cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B020.

Abstract Background: Nerves are a major component of the tumor microenvironment, with active crosstalk between cancer cells and nerves that may contribute to cancer progression. Netrin1 (Ntn1) is an axon guidance molecule that is important during early neural development. Recent studies suggest that Ntn1 may also play a role in tumorigenesis but its role in pancreatic cancer is not well understood. We hypothesized that Ntn1 plays a pro-tumorigenic role in pancreatic ductal adenocarcinoma (PDAC) progression and metastasis in part through modulation of nerves in the microenvironment. Methods: Expression of Ntn1 and its receptor Neogenin1(Neo1) were investigated in mouse models of spontaneous PDAC (LSL-Kras+/G12D/Pdx1-Cre; KC, LSL-Kras+/G12D/LSL-Trp53+/R172H/Pdx1-Cre; KPC). The influence of Ntn1 on pancreatic cancer progression and innervation was evaluated by KC mice with or without conditional knockout of Ntn1 (LSL-Kras+/G12D/Pdx1-Cre/Netrin1fl/fl; KCN). Murine PDAC cell lines and organoids were used to examine the functional role of Ntn1 and Neo1 through genetic and pharmacological modulations. The capacity of these cell lines to generate metastasis and the effect on neurite outgrowth was studied in a co-culture system with Dorsal Root Ganglia (DRG). Results: Expression of Ntn1 was not observed in normal acinar cells or ductal cells, but was found in both PanIN lesions and PDAC. Neo1 expression was similar to Ntn1 and there was a positive correlation between the expression of Ntn1 and Neo1. A lower rate of PanIN progression was observed in KCN mice compared to KC mice. In the metastatic model, overexpression of Ntn1 increased tumor burden and decreased survival while injections of a neutralizing Ntn1 antibody or genetic knockdown of Neo1 improved both endpoints. Ntn1 and Neo1 were upregulated in pancreatic organoids upon KRAS activation. Recombinant Ntn1 (rNTN1) promoted organoid forming capacity and upregulate Sox2 and Sox9 gene expression, which was inhibited by genetic knockdown of Neo1 or an anti-Neo1 antibody, supporting a direct autocrine role in modulating stemness. Nerve fibers were significantly increased at the site of PDAC liver metastasis, with Ntn1 showing the highest expression level among neurotrophins, as it was significantly increased in metastatic PDAC cells. Consistent with this observation, we observed less innervation in KCN mice, which correlated with reduced PanIN progression. Co-culture of DRGs with PDAC cells that overexpressed Ntn1 resulted in much greater axonal elongation consistent with a role of Ntn1 in promoting innervation. Conclusion: The Netrin1/Neogenin1 interaction is an important regulator of PDAC progression, acting both directly on PDAC cells to promote stemness, and indirectly via modulating tumor innervation. Blockade of Ntn1/Neo1 interaction reduces the capacity of PDAC to metastasize to the liver and improves survival. Overall, targeting the Ntn1/Neo1 interaction may represent a potential new therapeutic approach for the treatment of pancreatic cancer. Citation Format: Yosuke Ochiai, Sunagawa Masaki, Ermanno Malagola, Hiroki Kobayashi, Feijing Wu, Ruth A. White, Leah B. Zamechek, Timothy C. Wang. Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B100.

Intramuscular fat (IMF) content is an important economic trait for pork quality. Our previous results regarding the genome-wide association between IMF content and copy number variations (CNVs) indicated that the CNV within Netrin-1(NTN1-CNV) was significantly associated with IMF. In order to validate the effect of NTN1-CNV, we detected the Netrin-1 (NTN1) gene dose and protein expression content in the longissimus dorsi of different IMF content pigs using Western blotting and investigated the expression of NTN1 RNA in different tissues using real-time quantitative polymerase chain reaction (qPCR). The knock-down of the NTN1 gene in C2C12 and 3T3-L1 cells and over-expression in C2C12 cells during the proliferation and differentiation stage were also investigated to explore the possible pathway of action of NTN1. The results showed that in individuals with IMF content differences, the gene dose of NTN1 and the expression of NTN1 protein were also significantly different, which indicated that NTN1-CNV may directly affect IMF by its coding protein. NTN1 had the highest expression in pig longissimus dorsi and backfat tissues, which indicates that NTN1 may play an important role in muscle and fat tissues. The in vitro validation assay indicated that NTN1 silencing could promote the proliferation and inhibit the differentiation of C2C12 cells, with no effect on 3T3-L1 cells. Additionally, NTN1 over-expression could inhibit the proliferation and promote the differentiation of C2C12 cells. Combined with previous research, we conclude that NTN1-CNV may affect IMF by its gene dose, and the expression of NTN1 may affect the proliferation and differentiation of muscle cells by the AMP-activated protein kinase (AMPK) pathway and finally influence the IMF.

Netrin 1 (Ntn1) is a cell migration protein with an anti-inflammatory effect, which may play a key role in the pathological development of type 2 diabetes (T2D). In this study, we evaluate the relationships between the serum concentrations of Ntn1, glucose, and high-sensitivity C-reactive Protein (hsCRP). We carried out a cross-sectional study including 90 individuals divided into three groups (n = 30): healthy subjects, individuals with obesity without glucose alterations, and individuals with newly diagnosed T2D. Serum concentrations of Ntn1 and hs-CRP were determined by enzyme-linked immunosorbent assay (ELISA). The serum concentration of Ntn1 was higher in individuals with newly diagnosed T2D (0.33 ± 0.22 ng/mL), in comparison to healthy subjects and individuals with obesity (0.13 ± 0.06 and 0.15 ± 0.07 ng/mL, respectively). In addition, we observed a positive association between the levels of Ntn1 and hsCRP (rho = 0.443; p < 0.001) as well as with serum glucose (rho = −0.110; p = 0.05). The serum concentration of Ntn1 was higher in individuals with T2D, in comparison with the other groups in this study, and presented a positive correlation with hsCRP. Therefore, Ntn1 can be considered a promising risk biomarker and a potential therapeutic target for T2D.

AbstractPrevious genome-wide association study of nonsyndromic cleft lip with or without cleft palate (NSCL/P) identified a susceptible variant (rs4791774). We hypothesized that the functional single nucleotide polymorphism (SNP) may be in linkage disequilibrium with this lead SNP. The potential functional SNP (rs4791331) was identified by bioinformatic analysis. A case–control study with 891 orofacial cleft cases and 830 controls was designed to investigate its association with orofacial cleft. The allele-specific DNA-protein binding preference was predicted by JASPAR database. Cell proliferation, cycle and apoptosis, luciferase activity and netrin-1 (NTN1) expression were examined after transfection with the rs4791331 C/T vector in HEK-293 and HEPM cell lines. Forty-six lip tissues of NSCL/P patients were collected to detect NTN1 expression. ntn1a knockout zebrafish models were generated by CRISPR/Cas9 and observed with micro-CT. In the case–control study, the rs4791331-T allele was associated with an increased risk of nonsyndromic orofacial cleft (OR = 1.41, 95% CI = 1.19–1.68), as well as the subgroups cleft lip only (OR = 1.46, 95% CI = 1.14–1.87) and cleft lip and palate (OR = 1.58, 95% CI = 1.27–1.96). The T allele of rs4791331 exhibited anti-apoptotic effects and promoted cell cycle progression at the G1/S transition. Decreased enhancer activity and reduced NTN1 expression following transfection of the T allele were observed. Carriers of the CT/TT genotypes showed significantly lower expression of NTN1 than CC carriers. The ntn1a−/− zebrafish showed relatively wider intermaxillary fissures. These results indicate that rs4791331 (C > T) disrupted motif binding and led to abnormal expression of NTN1, which may be involved in the development of NSCL/P.

Resolution of inflammation is now recognized as a biosynthetically active process involving pro-resolving mediators. Here, we show in zymosan-initiated peritoneal inflammation that the vagus nerve regulates local expression of netrin-1, an axonal guidance molecule that activates resolution, and that vagotomy reduced local pro-resolving mediators, thereby delaying resolution. In netrin-1+/− mice, resolvin D1 (RvD1) was less effective in reducing neutrophil influx promoting resolution of peritonitis compared with Ntn1+/+. Netrin-1 shortened the resolution interval, decreasing exudate neutrophils, reducing proinflammatory mediators, and stimulating the production of resolvins, protectins, and lipoxins. Human monocytes incubated with netrin-1 produced proresolving mediators, including resolvins and lipoxins. Netrin-1 and RvD1 displayed bidirectional activation in that they stimulated each other’s expression and enhanced efferocytosis. These results indicate that the vagus nerve regulates both netrin-1 and pro-resolving lipid mediators, which act in a bidirectional fashion to stimulate resolution, and provide evidence for a novel mechanism for local neuronal control of resolution.

Netrin (NTN)-1 exhibits pro- and anti-inflammatory roles in different settings, playing important roles in the obesity-associated low-grade chronic inflammation. We aimed to determine the impact of NTN-1 on obesity and obesity-associated type 2 diabetes, as well as its role in visceral adipose tissue (VAT) inflammation. A total of 91 subjects were enrolled in this case-control study. Circulating levels of NTN-1 and its receptor neogenin (NEO)-1 were determined before and after weight loss achieved by caloric restriction and bariatric surgery. mRNA levels of NTN1 and NEO1 were assessed in human VAT, liver, and peripheral blood mononuclear cells. In vitro studies in human visceral adipocytes and human monocytic leukemia cells (THP-1)-derived macrophages were performed to analyze the impact of inflammation-related mediators on the gene expression levels of NTN1 and its receptor NEO1 as well as the effect of NTN-1 on inflammation. Increased (p < 0.001) circulating concentrations of NTN-1 in obesity decreased (p < 0.05) after diet-induced weight loss being also associated with a reduction in glucose (p < 0.01) and insulin levels (p < 0.05). Gene expression levels of NTN1 and NEO1 were upregulated (p < 0.05) in the VAT from patients with obesity with the highest expression in the stromovascular fraction cells compared with mature adipocytes (p < 0.01). NTN1 expression levels were enhanced (p < 0.01) under hypoxia and by inflammatory factors in both adipocytes and macrophages. Adipocyte-conditioned media strongly upregulated (p < 0.001) the mRNA levels of NTN1 in macrophages. The treatment of adipocytes with NTN-1 promoted the upregulation (p < 0.05) of pro-inflammatory and chemotactic molecules as well as its receptor NEO1. Collectively, these findings suggest that NTN-1 regulates VAT chronic inflammation and insulin resistance in obesity.

Netrin (NTN)-1, an extracellular matrix protein with a crucial role in inflammation, is dysregulated during obesity (OB) and influences colon cancer (CC) progression. To decipher the mechanisms underlying CC development during obesity, we examined the expression of NTN1 and its receptors in the visceral adipose tissue (VAT) of 74 (25 normal weight (NW)) (16 with CC) and 49 patients with OB (12 with CC). We also evaluated the effect of caloric restriction (CR) on the gene expression levels of Ntn1 and its receptors in the colon from a rat model fed a normal diet. The impact of adipocyte-conditioned media (ACM) from patients with OB and NTN-1 was assessed on the expression levels of neogenin 1(NEO1), deleted in colorectal carcinomas (DCC) and uncoordinated-5 homolog B (UNC5B) in Caco-2 and HT-29 human colorectal cell lines, as well as on Caco-2 cell migration. Increased NTN1 and NEO1 mRNA levels in VAT were due to OB (p < 0.05) and CC (p < 0.001). In addition, an upregulation in the expression levels of DCC and UNC5B in patients with CC (p < 0.01 and p < 0.05, respectively) was observed. Decreased (p < 0.01) Ntn1 levels in the colon from rats submitted to CR were found. In vitro experiments showed that ACM increased DCC (p < 0.05) and NEO1 (p < 0.01) mRNA levels in HT-29 and Caco-2 cell lines, respectively, while UNC5B decreased (p < 0.01) in HT-29. The treatment with NTN-1 increased (p < 0.05) NEO1 mRNA levels in HT-29 cells and DCC (p < 0.05) in both cell lines. Finally, we revealed a potent migratory effect of ACM and NTN-1 on Caco-2 cells. Collectively, these findings point to increased NTN-1 during OB and CC fuelling cancer progression and exerting a strong migratory effect on colon cancer cells.

457 Background: DCC (deleted in colon cancer; 18q21.3) is frequently lost in colorectal cancers (CRC), but few mutations in DCC have been discovered, even in tumors with 18q loss of heterozygosity. DCC has been shown to be a dependence receptor, with differential signaling depending on the presence (proliferative) or absence (pro-apoptotic) of the netrin-1 ligand (NTN1). DCC-mutated CRC tend to present at advanced stage and have a poor prognosis. RET, another dependence receptor, is a possible tumor suppressor gene in CRC and associates with CRC progression. Given the apparent role of DCC and RET in CRC progression, we carried out a genetic association study to determine if specific genetic variants in these pathways associate with advanced vs. early CRC. Methods: Imputed HapMap genome-wide association study (GWAS) from the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), a collection of 19 international case-control and cohort studies, was used to identify single nucleotide polymorphisms (SNPs) in DCC, NTN1, RET and interacting genes within 5kb upstream to 500mb downstream of each of the 54 genes. With the resultant 10,102 SNPs, we performed a stage-stratified analysis, comparing advanced (AJCC stage III-IV, n = 3500) to early CRC (I-II, n = 5300). An inverse-variance weighted fixed effect meta-analysis was performed with significance set at p=0.05/10102 SNPs=5x10-6 for multiple test correction. Results: Of the examined SNPs within DCC, the lowest p-value comparing advanced vs. early CRC was 3.6x10-3. SNPs in DOCK1 (dedicator of cytokinesis 1), which complexes with DCC and netrin-1, were associated with advanced CRC at p=1.11x10-3. SNPs in NTN1 and RET reached significance only at p = 1.73x10-2 and 1.53x10-2, respectively. No SNPs reached the pre-determined level of statistical significance. Conclusions: Our current analysis does not provide clear evidence for candidate SNPs associated with advanced CRC. Further approaches include expanding the analysis to include 1,000 Genome Project and ExomeChip data (~30,000 added SNPs), comparison of cases and controls, and evaluating candidate SNP-SNP interactions to better evaluate pathway pathogenesis. We plan to present an updated analysis at the symposium.

L'inflammation hépatique chronique peut entraîner des maladies chroniques du foie (MCF), notamment l'hépatite, la cirrhose et le carcinome hépatocellulaire (CHC), qui est la tumeur maligne primaire du foie la plus fréquente et le troisième cancer le plus fréquent en termes de mortalité dans le monde. Indépendamment des facteurs étiologiques, toutes les MCFs partagent de nombreux mécanismes physiopathologiques communs : réponse aux protéines non pliées (UPR), inflammation chronique et fibrose. A l'aide d'un modèle animal et d'échantillons cliniques nous avons étudié un autre dénominateur commun des MCFs et du CHC : la molécule neurotrope de guidage axonal netrin-1 et ses récepteurs à dépendance UNC5A, UNC5b et UNC5C. La nétrine-1, connue de ces propriétés pro-oncogéniques dans d'autres tumeurs solides, est induite pendant l'inflammation hépatique, alors que le signal pro-apoptotique des récepteurs UNC5 est atténué avec un équilibre ligand/récepteur globalement augmenté dans la cirrhose et le CHC. L'inflammation chronique est médiée par de multiples acteurs du système immunitaire. L'implication du système nerveux autonome (SNA) dans l'inflammation hépatique et la progression du CLD reste mal comprise. En étudiant l'implication des signaux neuronaux pré-synaptiques et post-synaptiques dans la cirrhose et le CHC, nous avons observé le remaniement de l'équilibre entre les systèmes nerveux sympathique (adrénergique) et parasympathique (cholinergique) intra-hépatiques. Le modèle animal de CHC cirrhotique a montré l'établissement progressif d'une orientation cholinergique à travers les différents stades de la fibrose. Le signal cholinergique global du CHC était associé à un microenvironnement anti-inflammatoire et à une survie plus faible chez les patients. La progression observée du CLD vers le CHC in vivo était accompagnée de la surexpression de marqueurs neuronaux immatures dans le CHC. Dans l'ensemble, nous avons montré que le bras parasympathique du SNA est impliqué dans la physiopathologie du CHC, suggérant l'utilisation de médicaments ciblant le SNA, dont beaucoup sont cliniquement sûrs et bien caractérisés, dans les études sur le CHC. En tentant d'établir le lien entre la netrine-1 ayant des propriétés anti-apoptotique et chimiotactique et SNA intrahépatique, nous avons identifié la corrélation positive entre le système netrine-1/UNC5s et le signal cholinergique dans le CHC. Le ciblage de la netrine-1 par l'anticorps monoclonal NP137, actuellement étudié dans les essais cliniques dans le traitement des tumeurs solides avancées, a montré un remaniement du SNA, confirmant la sensibilité du SNA à l'axe netrine-1/UNC5 dans le contexte hépatique pathologique. Les anomalies moléculaires les plus courantes dans le CHC au niveau du promoteur du gène TERT et au niveau du gène CTNNB1, ont montré une association avec le système netrin-1/UNC5, alors que les mutations dans T53, le régulateur bien connu de NTN1 et UNC5s n'ont pas montré d'implication dans le remodelage de l'axe netrin-1/UNC5 dans les échantillons cliniques de CHC, également insensibles au statut fonctionnel de la protéine p53. Globalement, les échantillons cliniques hébergeant une mutation dan le gène CTNNB1 sont corrélés à la polarité adrénergique du CHC, tandis que les mutations dans le gène TP53 apparaissent comme positivement associées à la polarité cholinergique du CHC. Dans l'ensemble, les résultats de ma thèse suggèrent le rôle pro-cancérogène de la nétrine-1 et l’implication de la neuroregulation associée via l’orientation de SNA dans le CLD et le CHC
Chronic liver inflammation can lead to chronic liver diseases (CLD), including hepatitis, cirrhosis and hepatocellular carcinoma (HCC), which is the most common malignant primary liver tumor and is the 3rd most common cancer in terms of mortality worldwide. Regardless of the etiological factor all CLDs share numerous common patho-physiogycal mechanisms: unfolded protein response (UPR), chronic inflammation and fibrosis. Using animal model and clinical samples we studied another common denominator of CLDs and HCC, which is the system of axon guidance cue netrin-1 and its dependence receptors UNC5s. Netrin-1, known as pro-oncogenic in other solid tumors, is induced during hepatic inflammation, and the pro-apoptotic signal of UNC5 receptors is attenuated with overall increased ligand/receptor balance in cirrhosis and HCC. Chronic inflammation is mediated by multiple actors of the immune system. The implication of autonomic nervous system (ANS) in hepatic inflammation and CLD progression remains poorly understood. Looking at the involvement of pre-synaptic and post-synaptic neuronal signals in the cirrhosis and HCC, we observed the reshaping of the balance between intrahepatic sympathetic (adrenergic) and parasympathetic (cholinergic) nervous systems. In vivo model of cirrhotic HCC showed the progressive establishment of cholinergic orientation throughout the different stages of fibrosis. The overall cholinergic signal of HCC was associated with anti-inflammatory microenvironment and poorer survival in patients. The observed CLD-to-HCC progression in vivo was accompanied by the overexpression of immature neuronal markers in HCC. Altogether, we showed that the parasympathetic arm of the ANS is implicated in the patho-physiology of HCC, and encourage for the use of ANS-targeting drugs in HCC studies, many of which are clinically safe and well characterized. Trying to establish the connection between pro-survival and chemotactic netrin-1 and intrahepatic ANS, we found the positive association between netrin-1/UNC5s and cholinergic signal in HCC. Netrin-1 targeting by the monoclonal antibody NP137, currently studied in the clinical trials in the treatment of advanced solid tumors, showed remodeling of ANS orientation, confirming the sensitivity of ANS to netrin-1/UNC5 axis in the hepatic pathological context. The most common molecular anomalies in HCC in the TERT promoter and CTNNB1 gene, showed an association with netrin-1/UNC5 system, whereas mutations in T53, the well-known regulator of NTN1 and UNC5s expression did not show any implication in netrin-1/UNC5 axis reshuffling in clinical HCC samples, also unsensitive to functionality status of p53. In average, CTNNB1-mutated clinical samples correlated with adrenergic polarity of HCC, whereas TP53 mutations appear to be positively associated with cholinergic polarity of HCC. Taken together, my thesis results suggest the putative pro-cancerogenic role of the netrin-1 and the implication of the neuroregulation via ANS in the CLD and HCC development