ProNGF in breast cancer

Le NGF (nerve growth factor), membre prototypique des neurotrophines, a été largement décrit pour son rôle dans le système nerveux où il active la survie et la différenciation neuronale via son récepteur tyrosine kinase TrkA et le récepteur commun à toutes les neurotrophines p75NTR. Plus récemment, il a été décrit que le précurseur du NGF, le proNGF, peut être sécrété et induire la mort neuronale en se liant à p75NTR et à la sortiline. Plusieurs études ont montré l’implication des neurotrophines dans la pathologie tumorale et les travaux pionniers de notre laboratoire ont démontré plus particulièrement l’effet mitogénique et l’action anti-apoptotique du NGF dans le cancer du sein. Néanmoins, aucune donnée n’a été rapportée concernant le rôle du proNGF dans ce cancer, c’est pourquoi nous avons décidé de mener cette étude. Nous avons démontré que le proNGF est produit et sécrété par les cellules cancéreuses mammaires. Ainsi, des western-blots ont permis de mettre en évidence une bande de 25 kDa qui diminue après transfection de siRNA contre le proNGF. De plus, par immunocytochimie, nous avons observé que le proNGF est localisé dans des vésicules intracellulaires qui sont libérées suite à un traitement avec un inducteur de sécrétion comme la ionomycine. En outre, le proNGF est détecté par spectrométrie de masse dans le milieu conditionné des cellules cancéreuses. Par ailleurs, en analysant 1423 biopsies humaines normales et cancéreuses par immunohistochimie, nous avons observé, en comparaison avec les tissus bénins et normaux, une surproduction du proNGF dans les cellules épithéliales mammaires cancéreuses associée avec l’envahissement lymphatique. De façon intéressante, en utilisant des chambres de Boyden, nous avons validé, in vitro, nos observations concernant la métastase lymphatique, en montrant que le proNGF permet de stimuler la migration et l’invasion des cellules cancéreuses de façon autocrine. Enfin, l’invalidation de ses récepteurs a révélé le rôle essentiel de la sortiline pour conduire l’effet pro-invasif du proNGF alors que nos données ont montré que p75NTR n’intervenait pas. En conclusion, notre travail démontre que le proNGF est produit dans les tumeurs mammaires où il stimule l’invasion des cellules cancéreuses mammaires participant ainsi au processus métastatique. De plus amples investigations précliniques et cliniques devront être menées afin de déterminer la valeur pratique du proNGF comme un marqueur et/ou cibleNerve growth factor (NGF), the prototypical neurotrophin, has extensively been studied for its role in the nervous system where it activates neuron survival and differentiation through the tyrosine kinase receptor TrkA and the neurotrophin receptor p75NTR. More recently, it was described that the precursor of NGF (proNGF) can be secreted and elicits neuronal apoptosis by engaging with p75NTR and sortilin receptors. Outside of the nervous system, several studies have indicated the implication of neurotrophins in tumors and in particular, our laboratory was pioneer in demonstrating the pro-survival and mitogenic effects of NGF for breast cancer cells. Nevertheless, nothing was reported about the role of the proNGF in breast cancer and this is what we have studied here. We demonstrated that proNGF was produced and secreted by breast cancer cells. Western blots analysis revealed a 25 kDa band that was decreased after transfection with siRNA against proNGF. Immunocytochemical observation showed proNGF localization in intracellular vesicles which were released upon treatment with the inducer of secretion ionomycin and proNGF was detected by mass spectrometry in conditioned medium of breast cancer cell. In addition, immunohistochemical analysis of 1423 normal vs human tumor biopsies showed proNGF overproduction in the epithelial compartment of malignant tumors when compared with benign and normal tissues, with a relationship to lymph node metastasis. Interestingly, in vitro, using Boyden chambers and RNA interference we showed that proNGF was a potent autocrine stimulator of breast cancer cell migration and invasion. Besides, impairment of its receptors revealed the essential role of sortilin in conducting proNGF pro-invasive effect whereas p75NTR was found not involved. In conclusion, our work demonstrates that proNGF is produced in breast tumors where it can stimulate cancer cell invasion, hence participating to metastasis. Further preclinical and clinical investigations will now have to be performed to determine the practical value of proNGF as a marker and/or therapeutic target

Le ProNGF dans le cancer du sein

Le NGF (nerve growth factor), membre prototypique des neurotrophines, a été largement décrit pour son rôle dans le système nerveux où il active la survie et la différenciation neuronale via son récepteur tyrosine kinase TrkA et le récepteur commun à toutes les neurotrophines p75NTR. Plus récemment, il a été décrit que le précurseur du NGF, le proNGF, peut être sécrété et induire la mort neuronale en se liant à p75NTR et à la sortiline. Plusieurs études ont montré l implication des neurotrophines dans la pathologie tumorale et les travaux pionniers de notre laboratoire ont démontré plus particulièrement l effet mitogénique et l action anti-apoptotique du NGF dans le cancer du sein. Néanmoins, aucune donnée n a été rapportée concernant le rôle du proNGF dans ce cancer, c est pourquoi nous avons décidé de mener cette étude. Nous avons démontré que le proNGF est produit et sécrété par les cellules cancéreuses mammaires. Ainsi, des western-blots ont permis de mettre en évidence une bande de 25 kDa qui diminue après transfection de siRNA contre le proNGF. De plus, par immunocytochimie, nous avons observé que le proNGF est localisé dans des vésicules intracellulaires qui sont libérées suite à un traitement avec un inducteur de sécrétion comme la ionomycine. En outre, le proNGF est détecté par spectrométrie de masse dans le milieu conditionné des cellules cancéreuses. Par ailleurs, en analysant 1423 biopsies humaines normales et cancéreuses par immunohistochimie, nous avons observé, en comparaison avec les tissus bénins et normaux, une surproduction du proNGF dans les cellules épithéliales mammaires cancéreuses associée avec l envahissement lymphatique. De façon intéressante, en utilisant des chambres de Boyden, nous avons validé, in vitro, nos observations concernant la métastase lymphatique, en montrant que le proNGF permet de stimuler la migration et l invasion des cellules cancéreuses de façon autocrine. Enfin, l invalidation de ses récepteurs a révélé le rôle essentiel de la sortiline pour conduire l effet pro-invasif du proNGF alors que nos données ont montré que p75NTR n intervenait pas. En conclusion, notre travail démontre que le proNGF est produit dans les tumeurs mammaires où il stimule l invasion des cellules cancéreuses mammaires participant ainsi au processus métastatique. De plus amples investigations précliniques et cliniques devront être menées afin de déterminer la valeur pratique du proNGF comme un marqueur et/ou cibleNerve growth factor (NGF), the prototypical neurotrophin, has extensively been studied for its role in the nervous system where it activates neuron survival and differentiation through the tyrosine kinase receptor TrkA and the neurotrophin receptor p75NTR. More recently, it was described that the precursor of NGF (proNGF) can be secreted and elicits neuronal apoptosis by engaging with p75NTR and sortilin receptors. Outside of the nervous system, several studies have indicated the implication of neurotrophins in tumors and in particular, our laboratory was pioneer in demonstrating the pro-survival and mitogenic effects of NGF for breast cancer cells. Nevertheless, nothing was reported about the role of the proNGF in breast cancer and this is what we have studied here. We demonstrated that proNGF was produced and secreted by breast cancer cells. Western blots analysis revealed a 25 kDa band that was decreased after transfection with siRNA against proNGF. Immunocytochemical observation showed proNGF localization in intracellular vesicles which were released upon treatment with the inducer of secretion ionomycin and proNGF was detected by mass spectrometry in conditioned medium of breast cancer cell. In addition, immunohistochemical analysis of 1423 normal vs human tumor biopsies showed proNGF overproduction in the epithelial compartment of malignant tumors when compared with benign and normal tissues, with a relationship to lymph node metastasis. Interestingly, in vitro, using Boyden chambers and RNA interference we showed that proNGF was a potent autocrine stimulator of breast cancer cell migration and invasion. Besides, impairment of its receptors revealed the essential role of sortilin in conducting proNGF pro-invasive effect whereas p75NTR was found not involved. In conclusion, our work demonstrates that proNGF is produced in breast tumors where it can stimulate cancer cell invasion, hence participating to metastasis. Further preclinical and clinical investigations will now have to be performed to determine the practical value of proNGF as a marker and/or therapeutic target.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

The Valosin-containing Protein (VCP) Is a Target of Akt Signaling Required for Cell Survival

International audienceThe serine/threonine kinase Akt is a key mediator of cell survival and growth, but its precise mechanism of action, and more specifically, the nature of its signaling partners largely remain to be elucidated. We show, using a proteomics-based approach, that the valosin-containing protein (VCP), a member of the AAA ( ATPases associated with a variety of cellular activities) family, is a target of Akt signaling. SDS-PAGE of Akt co-immunoprecipitated proteins obtained from MCF-7 breast cancer cells revealed the increase of a 97-kDa band under Akt activation. Mass spectrometry analysis allowed the identification of VCP, and we have shown a serine/threonine phosphorylation on an Akt consensus site upon activation by growth factors. Site-directed mutagenesis identified Ser-351, Ser-745, and Ser-747 as Akt phosphorylation sites on VCP. Confocal microscopy indicated a co-localization between Akt and VCP upon Akt stimulation. Interestingly, small interfering RNA against VCP induced an inhibition of the growth factor-induced activation of NF-kappa B and a potent pro-apoptotic effect. Together, these data identify VCP as an essential target of Akt signaling

Proteomics Exploration Reveals That Actin Is a Signaling Target of the Kinase Akt*

International audienceThe serine/threonine kinase Akt is a key mediator of cell survival and cell growth that is activated by most growth factors, but its downstream signaling largely remains to be elucidated. To identify signaling partners of Akt, we analyzed proteins co-immunoprecipitated with Akt in MCF-7 breast cancer cells. Mass spectrometry analysis (MALDI-TOF and MS-MS) of SDS-PAGE-separated Akt co-immunoprecipitates allowed the identification of 10 proteins: ␣-actinin, valosin-containing protein, inhibitor B kinase, mortalin, tubulin ␤, cytokeratin 8, actin, 14-3-3, proliferating cell nuclear antigen, and heat shock protein HSP27. The identification of these putative Akt binding partners were validated with specific antibodies. Interestingly, the major protein band observed in Akt coimmunoprecipitates was found to be the cytoskeleton protein actin for which a 14-fold increase was observed in Akt-activated compared with non-activated conditions. The interaction between Akt and actin was further confirmed by reverse immunoprecipitation, and confocal microscopy demonstrated a co-localization specifically induced under growth factor stimulation. The use of wortmannin indicated a dependence on the phosphatidylinositol 3-kinase pathway. Using a phospho-Akt substrate antibody, the phosphorylation of actin on an Akt consensus site was detected upon growth factor stimulation, both in cellulo and in vitro, suggesting that actin is a substrate of Akt kinase activity. Interestingly, cortical remodeling of actin associated with cell migration was reversed by small interfering RNA directed against Akt, indicating the involvement of Akt in the dynamic reorganization of actin cytoskeleton germane to breast cancer cell migration. Together these data identify actin as a new functional target of Akt signaling. Molecular & Cellular Proteomics 6:114-124, 2007

Combining Imaging Flow Cytometry and Machine Learning for High-Throughput Schistocyte Quantification: A SVM Classifier Development and External Validation Cohort.

International audienceBACKGROUND: Schistocyte counts are a cornerstone of the diagnosis of thrombotic microangiopathy syndrome (TMA). Their manual quantification is complex and alternative automated methods suffer from pitfalls that limit their use. We report a method combining imaging flow cytometry (IFC) and artificial intelligence for the direct label-free and operator-independent quantification of schistocytes in whole blood. METHODS: We used 135,045 IFC images from blood acquisition among 14 patients to extract 188 features with IDEAS\textregistered software and 128 features from a convolutional neural network (CNN) with Keras framework in order to train a support vector machine (SVM) blood elements' classifier used for schistocytes quantification. FINDING: Keras features showed better accuracy (94.03%, CI: 93.75-94.31%) than ideas features (91.54%, CI: 91.21-91.87%) in recognising whole-blood elements, and together they showed the best accuracy (95.64%, CI: 95.39-95.88%). We obtained an excellent correlation (0.93, CI: 0.90-0.96) between three haematologists and our method on a cohort of 102 patient samples. All patients with schistocytosis (>1% schistocytes) were detected with excellent specificity (91.3%, CI: 82.0-96.7%) and sensitivity (100%, CI: 89.4-100.0%). We confirmed these results with a similar specificity (91.1%, CI: 78.8-97.5%) and sensitivity (100%, CI: 88.1-100.0%) on a validation cohort (n=74) analysed in an independent healthcare centre. Simultaneous analysis of 16 samples in both study centres showed a very good correlation between the 2 imaging flow cytometers (Y=1.001x). INTERPRETATION: We demonstrate that IFC can represent a reliable tool for operator-independent schistocyte quantification with no pre-analytical processing which is of most importance in emergency situations such as TMA. FUNDING: None

Pro-nerve growth factor induces autocrine stimulation of breast cancer cell invasion through tropomyosin-related kinase A (TrkA) and sortilin protein

The precursor of nerve growth factor (proNGF) has been described as a biologically active polypeptide able to induce apoptosis in neuronal cells, via the neurotrophin receptor p75^NTR and the sortilin receptor. Herein, it is shown that proNGF is produced and secreted by breast cancer cells, stimulating their invasion. Using Western blotting and mass spectrometry, proNGF was detected in a panel of breast cancer cells as well as in their conditioned media. Immunohistochemical analysis indicated an overproduction of proNGF in breast tumors, when compared with benign and normal breast biopsies, and a relationship to lymph node invasion in ductal carcinomas. Interestingly, siRNA against proNGF induced a decrease of breast cancer cell invasion that was restored by the addition of non-cleavable proNGF. The activation of TrkA, Akt, and Src, but not the MAP kinases, was observed. In addition, the proNGF invasive effect was inhibited by the Trk pharmacological inhibitor K252a, a kinase-dead TrkA, and siRNA against TrkA sortilin, neurotensin, whereas siRNA against p75^NTR and the MAP kinase inhibitor PD98059 had no impact. These data reveal the existence of an autocrine loop stimulated by proNGF and mediated by TrkA and sortilin, with the activation of Akt and Src, for the stimulation of breast cancer cell invasion

ProNGF correlates with Gleason score and is a potential driver of nerve infiltration in prostate cancer

Nerve infiltration is essential to prostate cancer progression, but the mechanism by which nerves are attracted to prostate tumors remains unknown. We report that the precursor of nerve growth factor (proNGF) is overexpressed in prostate cancer and involved in the ability of prostate cancer cells to induce axonogenesis. A series of 120 prostate cancer and benign prostate hyperplasia (BPH) samples were analyzed by IHC for proNGF. ProNGF was mainly localized in the cytoplasm of epithelial cells, with marked expression in cancer compared with BPH. Importantly, the proNGF level positively correlated with the Gleason score (n = 104, tB = 0.51). A higher level of proNGF was observed in tumors with a Gleason score of =8 compared with a Gleason score of 7 and 6 (P < 0.001). In vitro, proNGF was detected in LNCaP, DU145, and PC-3 prostate cancer cells and BPH-1 cells but not in RWPE-1 immortalized nontumorigenic prostate epithelial cells or primary normal prostate epithelial cells. Co-culture of PC12 neuronal-like cells or 50B11 neurons with PC-3 cells resulted in neurite outgrowth in neuronal cells that was inhibited by blocking antibodies against proNGF, indicating that prostate cancer cells can induce axonogenesis via secretion of proNGF. These data reveal that ProNGF is a biomarker associated with high-risk prostate cancers and a potential driver of infiltration by nerves

A new role of glutathione peroxidase 4 during human erythroblast enucleation

International audienceThe selenoprotein glutathione peroxidase 4 (GPX4), the only member of the glutathione peroxidase family able to directly reduce cell membrane-oxidized fatty acids and cholesterol, was recently identified as the central regulator of ferroptosis. GPX4 knockdown in mouse hematopoietic cells leads to hemolytic anemia and to increased spleen erythroid progenitor death. The role of GPX4 during human erythropoiesis is unknown. Using in vitro erythroid differentiation, we show here that GPX4-irreversible inhibition by 1S,3R-RSL3 (RSL3) and its short hairpin RNA-mediated knockdown strongly impaired enucleation in a ferroptosis-independent manner not restored by tocopherol or iron chelators. During enucleation, GPX4 localized with lipid rafts at the cleavage furrows between reticulocytes and pyrenocytes. Its inhibition impacted enucleation after nuclear condensation and polarization and was associated with a defect in lipid raft clustering (cholera toxin staining) and myosin-regulatory light-chain phosphorylation. Because selenoprotein translation and cholesterol synthesis share a common precursor, we investigated whether the enucleation defect could represent a compensatory mechanism favoring GPX4 synthesis at the expense of cholesterol, known to be abundant in lipid rafts. Lipidomics and filipin staining failed to show any quantitative difference in cholesterol content after RSL3 exposure. However, addition of cholesterol increased cholera toxin staining and myosin-regulatory light-chain phosphorylation, and improved enucleation despite GPX4 knockdown. In summary, we identified GPX4 as a new actor of human erythroid enucleation, independent of its function in ferroptosis control. We described its involvement in lipid raft organization required for contractile ring assembly and cytokinesis, leading in fine to nucleus extrusion