RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales , Carcinoma de Células Escamosas , Transición Epitelial-Mesenquimal , Netrina-1 , Neoplasias Cutáneas , Animales , Humanos , Ratones , Células A549 , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores de Netrina/antagonistas & inhibidores , Receptores de Netrina/deficiencia , Receptores de Netrina/genética , Netrina-1/antagonistas & inhibidores , Netrina-1/deficiencia , Netrina-1/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Modelos Animales de Enfermedad , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Metástasis de la Neoplasia/tratamiento farmacológico , Análisis de Expresión Génica de una Sola Célula , RNA-Seq , Molécula de Adhesión Celular Epitelial/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
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.
Asunto(s)
Neoplasias Endometriales , Transición Epitelial-Mesenquimal , Netrina-1 , Animales , Femenino , Humanos , Ratones , Biopsia , Carboplatino/administración & dosificación , Carboplatino/farmacología , Carboplatino/uso terapéutico , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/genética , Neoplasias Endometriales/inmunología , Neoplasias Endometriales/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Perfilación de la Expresión Génica , Netrina-1/antagonistas & inhibidores , Paclitaxel/administración & dosificación , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , RNA-Seq , Análisis de Expresión Génica de una Sola Célula , Microambiente Tumoral/efectos de los fármacosRESUMEN
Netrin-1 is an evolutionarily conserved, secreted extracellular matrix protein involved in axon guidance at the central nervous system midline. Netrin-1 is expressed by cells localized at the central nervous system midline, such as those of the floor plate in vertebrate embryos. Growth cone turning assays and three-dimensional gel diffusion assays have shown that netrin-1 can attract commissural axons. Loss-of-function experiments further demonstrated that commissural axon extension to the midline is severely impaired in the absence of netrin-1 (refs 3, 7, 8, 9). Together, these data have long supported a model in which commissural axons are attracted by a netrin-1 gradient diffusing from the midline. Here we selectively ablate netrin-1 expression in floor-plate cells using a Ntn1 conditional knockout mouse line. We find that hindbrain and spinal cord commissural axons develop normally in the absence of floor-plate-derived netrin-1. Furthermore, we show that netrin-1 is highly expressed by cells in the ventricular zone, which can release netrin-1 at the pial surface where it binds to commissural axons. Notably, Ntn1 deletion from the ventricular zone phenocopies commissural axon guidance defects previously described in Ntn1-knockout mice. These results show that the classical view that attraction of commissural axons is mediated by a gradient of floor-plate-derived netrin-1 is inaccurate and that netrin-1 primarily acts locally by promoting growth cone adhesion.
Asunto(s)
Orientación del Axón , Conos de Crecimiento/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Médula Espinal/citología , Proteínas Supresoras de Tumor/metabolismo , Animales , Adhesión Celular , Femenino , Masculino , Ratones , Ratones Noqueados , Factores de Crecimiento Nervioso/deficiencia , Netrina-1 , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Proteínas Supresoras de Tumor/deficienciaRESUMEN
The UNC5H dependence receptors promote apoptosis in the absence of their ligand, netrin-1, and this is important for neuronal and vascular development and for limitation of cancer progression. UNC5H2 (also called UNC5B) triggers cell death through the activation of the serine-threonine protein kinase DAPk. While performing a siRNA screen to identify genes implicated in UNC5H-induced apoptosis, we identified the structural subunit PR65ß of the holoenzyme protein phosphatase 2A (PP2A). We show that UNC5H2/B recruits a protein complex that includes PR65ß and DAPk and retains PP2A activity. PP2A activity is required for UNC5H2/B-induced apoptosis, since it activates DAPk by triggering its dephosphorylation. Moreover, netrin-1 binding to UNC5H2/B prevents this effect through interaction of the PP2A inhibitor CIP2A to UNC5H2/B. Thus we show here that, in the absence of netrin-1, recruitment of PP2A to UNC5H2/B allows the activation of DAPk via a PP2A-mediated dephosphorylation and that this mechanism is involved in angiogenesis regulation.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteína Fosfatasa 2/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular , Humanos , Receptores de Netrina , Fosforilación , Células Tumorales CultivadasRESUMEN
The role of deleted in colorectal carcinoma (DCC) as a tumour suppressor has been a matter of debate for the past 15 years. DCC gene expression is lost or markedly reduced in the majority of advanced colorectal cancers and, by functioning as a dependence receptor, DCC has been shown to induce apoptosis unless engaged by its ligand, netrin-1 (ref. 2). However, so far no animal model has supported the view that the DCC loss-of-function is causally implicated as predisposing to aggressive cancer development. To investigate the role of DCC-induced apoptosis in the control of tumour progression, here we created a mouse model in which the pro-apoptotic activity of DCC is genetically silenced. Although the loss of DCC-induced apoptosis in this mouse model is not associated with a major disorganization of the intestines, it leads to spontaneous intestinal neoplasia at a relatively low frequency. Loss of DCC-induced apoptosis is also associated with an increase in the number and aggressiveness of intestinal tumours in a predisposing APC mutant context, resulting in the development of highly invasive adenocarcinomas. These results demonstrate that DCC functions as a tumour suppressor via its ability to trigger tumour cell apoptosis.
Asunto(s)
Progresión de la Enfermedad , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Receptores de Superficie Celular/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Apoptosis/genética , Caspasas/metabolismo , Células Cultivadas , Receptor DCC , Modelos Animales de Enfermedad , Fibroblastos , Silenciador del Gen , Genes APC , Células HEK293 , Humanos , Neoplasias Intestinales/metabolismo , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Factores de Crecimiento Nervioso/deficiencia , Factores de Crecimiento Nervioso/genética , Netrina-1 , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
The Hedgehog signaling is a determinant pathway for tumor progression. However, while inhibition of the Hedgehog canonical pathway-Patched-Smoothened-Gli-has proved efficient in human tumors with activating mutations in this pathway, recent clinical data have failed to show any benefit in other cancers, even though Sonic Hedgehog (SHH) expression is detected in these cancers. Cell-adhesion molecule-related/down-regulated by Oncogenes (CDON), a positive regulator of skeletal muscle development, was recently identified as a receptor for SHH. We show here that CDON behaves as a SHH dependence receptor: it actively triggers apoptosis in the absence of SHH. The pro-apoptotic activity of unbound CDON requires a proteolytic cleavage in its intracellular domain, allowing the recruitment and activation of caspase-9. We show that by inducing apoptosis in settings of SHH limitation, CDON expression constrains tumor progression, and as such, decreased CDON expression observed in a large fraction of human colorectal cancer is associated in mice with intestinal tumor progression. Reciprocally, we propose that the SHH expression, detected in human cancers and previously considered as a mechanism for activation of the canonical pathway in an autocrine or paracrine manner, actually provides a selective tumor growth advantage by blocking CDON-induced apoptosis. In support of this notion, we present the preclinical demonstration that interference with the SHH-CDON interaction triggers a CDON-dependent apoptosis in vitro and tumor growth inhibition in vivo. The latter observation qualifies CDON as a relevant alternative target for anticancer therapy in SHH-expressing tumors.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Moléculas de Adhesión Celular/genética , Línea Celular , Línea Celular Tumoral , Femenino , Proteínas Hedgehog/genética , Humanos , Masculino , Ratones , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/genéticaRESUMEN
Extracellular vesicles (EVs) are secreted, cell-derived, membrane-bound compartments implicated in various diseases for their ability to influence distant targets and as carriers of biomarkers. Here, we present a protocol for separating EVs from mammalian pancreatic cancer cells and their characterization using western blot and electron microscopy. We then demonstrate how they are utilized to affect tumor development in a murine model of metastatic pancreatic cancer including a method to quantify hepatic tumor burden in histologic samples. For complete details on the use and execution of this protocol, please refer to Dudgeon et al.1.
RESUMEN
The biology of metastatic pancreatic ductal adenocarcinoma (PDAC) is distinct from that of the primary tumor due to changes in cell plasticity governed by a distinct transcriptome. Therapeutic strategies that target this distinct biology are needed. We detect an upregulation of the neuronal axon guidance molecule Netrin-1 in PDAC liver metastases that signals through its dependence receptor (DR), uncoordinated-5b (Unc5b), to facilitate metastasis in vitro and in vivo. The mechanism of Netrin-1 induction involves a feedforward loop whereby Netrin-1 on the surface of PDAC-secreted extracellular vesicles prepares the metastatic niche by inducing hepatic stellate cell activation and retinoic acid secretion that in turn upregulates Netrin-1 in disseminated tumor cells via RAR/RXR and Elf3 signaling. While this mechanism promotes PDAC liver metastasis, it also identifies a therapeutic vulnerability, as it can be targeted using anti-Netrin-1 therapy to inhibit metastasis using the Unc5b DR cell death mechanism.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Hepáticas , Neoplasias Pancreáticas , Humanos , Netrina-1 , Retinoides , Células Estrelladas Hepáticas/metabolismo , Línea Celular Tumoral , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Neoplasias Hepáticas/metabolismo , Receptores de Netrina , Proteínas de Unión al ADN , Factores de Transcripción , Proteínas Proto-Oncogénicas c-etsRESUMEN
Drug resistance and cancer relapse represent significant therapeutic challenges after chemotherapy or immunotherapy, and a major limiting factor for long-term cancer survival. Netrin-1 was initially identified as a neuronal navigation cue but has more recently emerged as an interesting target for cancer therapy, which is currently clinically investigated. We show here that netrin-1 is an independent prognostic marker for clinical progression of breast and ovary cancers. Cancer stem cells (CSCs)/Tumor initiating cells (TICs) are hypothesized to be involved in clinical progression, tumor relapse and resistance. We found a significant correlation between netrin-1 expression and cancer stem cell (CSC) markers levels. We also show in different mice models of resistance to chemotherapies that netrin-1 interference using a therapeutic netrin-1 blocking antibody alleviates resistance to chemotherapy and triggers an efficient delay in tumor relapse and this effect is associated with CSCs loss. We also demonstrate that netrin-1 interference limits tumor resistance to immune checkpoint inhibitor and provide evidence linking this enhanced anti-tumor efficacy to a decreased recruitment of a subtype of myeloid-derived suppressor cells (MDSCs) called polymorphonuclear (PMN)-MDSCs. We have functionally demonstrated that these immune cells promote CSCs features and, consequently, resistance to anti-cancer treatments. Together, these data support the view of both a direct and indirect contribution of netrin-1 to cancer stemness and we propose that this may lead to therapeutic opportunities by combining conventional chemotherapies and immunotherapies with netrin-1 interfering drugs.
RESUMEN
BACKGROUND & AIMS: Expression of the netrin-1 dependence receptor UNC5C is reduced in many colorectal tumors; mice with the UNC5C mutations have increased progression of intestinal tumors. We investigated whether specific variants in UNC5C increase risk of colorectal cancer (CRC). METHODS: We analyzed the sequence of UNC5C in blood samples from 1801 patients with CRC and 4152 controls from 3 cohorts (France, United States, and Finland). Almost all cases from France and the United States had familial CRC; of the Finnish cases, 92 of 984 were familial. We analyzed whether CRC segregates with the UNC5C variant A628K in 3 families with histories of CRC. We also performed haplotype analysis to determine the origin of this variant. RESULTS: Of 817 patients with familial CRC, 14 had 1 of 4 different, unreported missense variants in UNC5C. The variants p.Asp353Asn (encodes D353N), p.Arg603Cys (encodes R603C), and p.Gln630Glu (encodes Q630E) did not occur significantly more often in cases than controls. The variant p.Ala628Lys (A628K) was detected in 3 families in the French cohort (odds ratio, 8.8; Wald's 95% confidence interval, 1.47-52.93; P = .03) and in 2 families in the US cohort (odds ratio, 1.9; P = .6) but was not detected in the Finnish cohort; UNC5C A628K segregated with CRC in families. Three families with A628K had a 109-kilobase identical haplotype that spanned most of UNC5C, indicating recent origin of this variant in white subjects (14 generations; 95% confidence interval, 6-36 generations). Transfection of HEK293T cells with UNC5C-A628K significantly reduced apoptosis compared with wild-type UNC5C, measured in an assay of active caspase-3. CONCLUSIONS: Inherited mutations in UNC5C prevent apoptosis and increase risk of CRC.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Apoptosis/genética , Mutación Missense/genética , Receptores de Superficie Celular/genética , Poliposis Adenomatosa del Colon/sangre , Estudios de Casos y Controles , Estudios de Cohortes , Análisis Mutacional de ADN , Predisposición Genética a la Enfermedad , Haplotipos/genética , Humanos , Immunoblotting , Inmunohistoquímica , Receptores de Netrina , Linaje , Reacción en Cadena de la Polimerasa , Receptores de Superficie Celular/sangre , Factores de RiesgoRESUMEN
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.
Asunto(s)
Neoplasias , Netrina-1 , Movimiento Celular , Proliferación Celular , Humanos , Receptores de Netrina/metabolismo , Netrina-1/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The discovery of bacterial-derived Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has revolutionized genome engineering and gene therapy due to its wide range of applications. One of the major challenging issues in CRISPR/Cas system is the lack of an efficient, safe, and clinically suitable delivery of the system's components into target cells. Here, we describe the development of polyethylenimine coated-bovine serum albumin nanoparticles (BSA-PEI NPs) for efficient delivery of CRISPR/Cas9 system in both DNA (px458 plasmid) and ribonucleoprotein (RNP) forms into MDA-MB-231 human breast cancer cell line. Our data showed that synthesized BSA-PEI (BP) NPs delivered plasmid px458 at concentrations of 0.15, 0.25, and 0.35 µg/µl with efficiencies of approximately 29.7, 54.8, and 84.1% into MDA-MB-231 cells, respectively. Our study demonstrated that Cas9/sgRNA RNP complex efficiently (~ 92.6%) delivered by BSA-PEI NPs into the same cells. Analysis of toxicity and biocompatibility of synthesized NPs on human red blood cells, MDA-MB-231 cells, and mice showed that the selected concentration (28 µg/µl) of BSA-PEI NPs for transfection had no remarkable toxicity effects. Thus, obtained results suggest BSA-PEI NPs as one of the most promising carrier for delivering CRISPR/Cas9 to target cells.
Asunto(s)
Sistemas CRISPR-Cas , Nanopartículas , Animales , Proteína 9 Asociada a CRISPR/genética , Humanos , Ratones , Polietileneimina , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Albúmina Sérica BovinaRESUMEN
Netrin-1, an axon navigation cue was proposed to play a crucial role during colorectal tumorigenesis by regulating apoptosis. The netrin-1 receptors DCC and UNC5H were shown to belong to the family of dependence receptors that share the ability to induce apoptosis in the absence of their ligands. Such a trait confers on these receptors a tumor suppressor activity. Expression of one of these dependence receptors at the surface of a tumor cell is indeed speculated to render this cell dependent on ligand availability for its survival, hence inhibiting uncontrolled cell proliferation or metastasis. Consequently, it is a selective advantage for a tumor cell to lose this dependence receptor activity, as previously described with losses of DCC and UNC5H expression in human cancers. However, the model predicts that a similar advantage may be obtained by gaining autocrine expression of the ligand. We describe here that, unlike human nonmetastatic breast tumors, a large fraction of metastatic breast cancers overexpress netrin-1. Moreover, we show that netrin-1-expressing mammary metastatic tumor cell lines undergo apoptosis when netrin-1 expression is experimentally decreased or when decoy soluble receptor ectodomains are added. Such treatments prevent metastasis formation both in a syngenic mouse model of lung colonization of a mammary cancer cell line and in a model of spontaneous lung metastasis of xenografted human breast tumor. Thus, netrin-1 expression observed in a large fraction of human metastatic breast tumors confers a selective advantage for tumor cell survival and potentially represents a promising target for alternative anticancer therapeutic strategies.
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Neoplasias de la Mama/patología , Factores de Crecimiento Nervioso/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Comunicación Autocrina , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/secundario , Ratones , Metástasis de la Neoplasia , Trasplante de Neoplasias , Factores de Crecimiento Nervioso/genética , Netrina-1 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Dependence receptors are known to promote survival and positive signaling such as proliferation, migration, and differentiation when activated, but to actively trigger apoptosis when unbound to their ligand. Their abnormal regulation was shown to be an important feature of tumorigenesis, allowing cancer cells to escape apoptosis triggered by these receptors while promoting in parallel major aspects of tumorigenesis such as proliferation, angiogenesis, invasiveness, and chemoresistance. This involvement in multiple cancer hallmarks has raised interest in dependence receptors as targets for cancer therapy. Although additional studies remain necessary to fully understand the complexity of signaling pathways activated by these receptors and to target them efficiently, it is now clear that dependence receptors represent very exciting targets for future cancer treatment. This manuscript reviews current knowledge on the contribution of dependence receptors to cancer and highlights the potential for therapies that activate pro-apoptotic functions of these proteins.
Asunto(s)
Neoplasias , Transducción de Señal , Apoptosis , Diferenciación Celular , Transformación Celular Neoplásica , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
The expression of the protein DCC (deleted in colorectal cancer) is lost or markedly reduced in numerous cancers and in the majority of colorectal cancers due to loss of heterozygosity in chromosome 18q, and has therefore been proposed to be a tumour suppressor. However, the rarity of mutations found in DCC, the lack of cancer predisposition of DCC mutant mice, and the presence of other tumour suppressor genes in 18q have raised doubts about the function of DCC as a tumour suppressor. Unlike classical tumour suppressors, DCC has been shown to induce apoptosis conditionally: by functioning as a dependence receptor, DCC induces apoptosis unless DCC is engaged by its ligand, netrin-1 (ref. 3). Here we show that inhibition of cell death by enforced expression of netrin-1 in mouse gastrointestinal tract leads to the spontaneous formation of hyperplastic and neoplastic lesions. Moreover, in the adenomatous polyposis coli mutant background associated with adenoma formation, enforced expression of netrin-1 engenders aggressive adenocarcinomatous malignancies. These data demonstrate that netrin-1 can promote intestinal tumour development, probably by regulating cell survival. Thus, a netrin-1 receptor or receptors function as conditional tumour suppressors.
Asunto(s)
Apoptosis , Transformación Celular Neoplásica , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Factores de Crecimiento Nervioso/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/metabolismo , Poliposis Adenomatosa del Colon/patología , Animales , Supervivencia Celular , Pollos , Neoplasias Colorrectales/genética , Progresión de la Enfermedad , Femenino , Mucosa Intestinal/metabolismo , Intestinos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Factores de Crecimiento Nervioso/genética , Netrina-1 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
BACKGROUND & AIMS: Netrin-1 was recently proposed to play a crucial role during colorectal tumorigenesis by regulating apoptosis. Because netrin-1 receptors belong to the family of dependence receptors, a selective advantage for a tumor is either to lose netrin-1 receptors or to gain autocrine expression of netrin-1. We have investigated whether netrin-1 is up-regulated in colorectal cancer and have searched for a link between NF-kappaB activation and netrin-1 up-regulation. METHODS: The level of netrin-1, netrin-1 receptors, ie, DCC, UNC5H1, UNC5H2, UNC5H3, and the proinflammatory markers cyclooxygenase-2 and inhibitor of nuclear factor-kappaB (IkappaB) alpha were analyzed in a panel of 59 primary sporadic colorectal carcinomas. Netrin-1 expression was investigated in tumor cells and in mouse colonic crypts in response to NF-kappaB activation but also in a mouse model of inflammation-induced colorectal cancer. Binding of NF-kappaB to netrin-1 promoter and effect of NF-kappaB activation to the proapoptotic activity of UNC5H2 were also analyzed. RESULTS: We show that colorectal tumors with a gain of netrin-1 are tumors that display increased activation of the NF-kappaB pathway. Moreover, netrin-1 up-regulation, which is associated with tumor formation in mice, is observed in mouse colonic crypts in response to NF-kappaB activation but also in a mouse model of inflammation-induced colorectal cancer. We demonstrate that the netrin-1 gene is a direct transcriptional target of NF-kappaB. We show that NF-kappaB-induced netrin-1 expression inhibits proapoptotic activity of the netrin-1 receptors. CONCLUSIONS: We propose that NF-kappaB activation that occurs in response to inflammation confers a selective advantage for tumor development through NF-kappaB-mediated netrin-1 up-regulation.
Asunto(s)
Neoplasias Colorrectales/fisiopatología , Regulación Neoplásica de la Expresión Génica , FN-kappa B/metabolismo , Factores de Crecimiento Nervioso/genética , Receptores de Superficie Celular/metabolismo , Proteínas Supresoras de Tumor/genética , Animales , Apoptosis/fisiología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Receptor DCC , Modelos Animales de Enfermedad , Células HCT116 , Humanos , Ratones , Ratones Endogámicos BALB C , Factores de Crecimiento Nervioso/metabolismo , Receptores de Netrina , Netrina-1 , Receptores de Superficie Celular/genética , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
Netrin-1 is upregulated in a large fraction of human neoplasms. In multiple animal models, interference with netrin-1 is associated with inhibition of tumor growth and metastasis. Although netrin-1 upregulation was initially described in cancer cells, we report here that in the human colorectal cancer database, the expression of netrin-1 and its receptor UNC5B correlates with a cancer-associated fibroblasts (CAF) signature. Both colon and lung CAF secreted netrin-1 when cocultured with respective cancer cells, and netrin-1 upregulation in CAF was associated with increased cancer cell stemness. Pharmacologic inhibition of netrin-1 with a netrin-1-mAb (Net1-mAb) abrogated the CAF-mediated increase of cancer stemness both in coculture experiments and in mice. Net-1-mAb inhibited intercellular signaling between CAF and cancer cells by modulating CAF-mediated expression of cytokines such as IL6. Together these data demonstrate that netrin-1 is upregulated not only in cancer cells but also in cancer-associated stromal cells. In addition to its direct activity on cancer cells, inhibition of netrin-1 may reduce proneoplastic CAF-cancer cell cross-talk, thus inhibiting cancer plasticity. SIGNIFICANCE: Netrin-1, a navigation cue during embryonic development, is upregulated in cancer-associated fibroblasts and regulates cancer cell stemness.
Asunto(s)
Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias del Colon/patología , Neoplasias Pulmonares/patología , Netrina-1/biosíntesis , Células A549 , Animales , Fibroblastos Asociados al Cáncer/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Plasticidad de la Célula/fisiología , Neoplasias del Colon/metabolismo , Femenino , Células HCT116 , Xenoinjertos , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Ratones SCID , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptores de Netrina/biosíntesis , Regulación hacia ArribaRESUMEN
BACKGROUND & AIMS: The UNC5H netrin-1 receptors (UNC5H1-3 also called UNC5A-C) belong to the functional dependence receptors family, which share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer a tumor-suppressor activity. Indeed, cells harboring these receptors are thought to be dependent on ligand availability for their survival, thereby inhibiting uncontrolled tumor cell proliferation. We investigate here whether UNC5C acts as a tumor suppressor in colorectal malignancies. METHODS: The level of UNC5C was analyzed in a panel of 86 primary sporadic colorectal carcinomas. Loss of heterozygosity in the UNC5C locus and epigenetic alterations in the UNC5C promoter were also analyzed. Intestinal tumor progression was monitored in mice bearing both UNC5C and APC1638N mutations, and apoptosis was measured in intestinal tumors developed in UNC5C/APC1638N mutant mice. RESULTS: We show here that UNC5C expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, in mice, inactivation of UNC5C is associated with increased intestinal tumor progression and a decrease in tumor cell apoptosis. CONCLUSIONS: The loss of UNC5C expression observed in human colorectal cancer is a selective advantage for tumor progression, in agreement with the dependence receptor hypothesis. Thus, the UNC5C dependence receptor is a tumor suppressor that regulates sporadic colorectal cancer.