Bibliographie IAL

Recherche bibliographique scientifique

Moteur de recherche scientifique en Médecine, Biologie et Sciences

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  • Abdallah, C, Lejamtel, C, Benzoubir, N, Battaglia, S, Sidahmed-Adrar, N, Desterke, C, Lemasson, M, Rosenberg, AR, Samuel, D, Bréchot, C, Pflieger, D, Le Naour, F & Bourgeade, M-F 2017, « Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice », Oncotarget.
    Résumé : Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.
    Mots-clés : 4E-BP1 phosphorylation, HCV core, hepatocellular carcinoma, phosphoproteomics, SILAC.

  • Aggoune, D, Sorel, N, Bonnet, M-L, Goujon, J-M, Tarte, K, Hérault, O, Domenech, J, Réa, D, Legros, L, Johnson-Ansa, H, Rousselot, P, Cayssials, E, Guerci-Bresler, A, Bennaceur-Griscelli, A, Chomel, J-C & Turhan, AG 2017, « Bone marrow mesenchymal stromal cell (MSC) gene profiling in chronic myeloid leukemia (CML) patients at diagnosis and in deep molecular response induced by tyrosine kinase inhibitors (TKIs) », Leukemia Research, vol. 60, p. 94-102.
    Résumé : Although it has been well-demonstrated that bone marrow mesenchymal stromal cells (MSCs) from CML patients do not belong to the Ph1-positive clone, there is growing evidence that they could play a role in the leukemogenesis process or the protection of leukemic stem cells from the effects of tyrosine kinase inhibitors (TKIs). The aim of the present study was to identify genes differentially expressed in MSCs isolated from CML patients at diagnosis (CML-MSCs) as compared to MSCs from healthy controls. Using a custom gene-profiling assay, we identified six genes over-expressed in CML-MSCs (BMP1, FOXO3, MET, MITF, NANOG, PDPN), with the two highest levels being documented for PDPN (PODOPLANIN) and NANOG. To determine whether this aberrant signature persisted in patients in deep molecular response induced by TKIs, we analyzed MSCs derived from such patients (MR-MSCs). This analysis showed that, despite the deep molecular responses, BMP1, MET, MITF, NANOG, and PDPN mRNA were upregulated in MR-MSCs. Moreover, BMP1, MITF, and NANOG mRNA expressions in MR-MSCs were found to be intermediate between control MSCs and CML-MSCs. These results suggest that CML-MSCs exhibit an abnormal gene expression pattern which might have been established during the leukemogenic process and persist in patients in deep molecular response.
    Mots-clés : Chronic myeloid leukemia, Hematopoietic niche, Mesenchymal Stromal Cells, TaqMan low-density array.

  • Chastagner, P, Rubinstein, E & Brou, C 2017, « Ligand-activated Notch undergoes DTX4-mediated ubiquitylation and bilateral endocytosis before ADAM10 processing », Science Signaling, vol. 10, no. 483.
    Résumé : The Notch signaling pathway, which is activated by cell-cell contact, is a major regulator of cell fate decisions. Mammalian Notch1 is present at the cell surface as a heterodimer of the Notch extracellular domain associated with the transmembrane and intracellular domains. After ligand binding, Notch undergoes proteolysis, releasing the Notch intracellular domain (NICD) that regulates gene expression. We monitored the early steps of activation with biochemical analysis, immunofluorescence analysis, and live-cell imaging of Notch1-expressing cells. We found that, upon ligand binding, Notch1 at the cell surface was ubiquitylated by the E3 ubiquitin ligase DTX4. This ubiquitylation event led to the internalization of the Notch1 extracellular domain by the ligand-expressing cell and the internalization of the membrane-anchored fragment of Notch1 and DTX4 by the Notch1-expressing cell, which we referred to as bilateral endocytosis. ADAM10 generates a cleavage product of Notch that is necessary for the formation of the NICD, which has been thought to occur at the cell surface. However, we found that blocking dynamin-mediated endocytosis of Notch1 and DTX4 reduced the colocalization of Notch1 with ADAM10 and the formation of the ADAM10-generated cleavage product of Notch1, suggesting that ADAM10 functions in an intracellular compartment to process Notch. Thus, this study suggests that a specific pool of ADAM10 acts on Notch in an endocytic compartment, rather than at the cell surface.

  • Chiappini, F, Coilly, A, Kadar, H, Gual, P, Tran, A, Desterke, C, Samuel, D, Duclos-Vallée, J-C, Touboul, D, Bertrand-Michel, J, Brunelle, A, Guettier, C & Le Naour, F 2017, « Metabolism dysregulation induces a specific lipid signature of nonalcoholic steatohepatitis in patients », Scientific Reports, vol. 7, p. 46658.
    Résumé : Nonalcoholic steatohepatitis (NASH) is a condition which can progress to cirrhosis and hepatocellular carcinoma. Markers for NASH diagnosis are still lacking. We performed a comprehensive lipidomic analysis on human liver biopsies including normal liver, nonalcoholic fatty liver and NASH. Random forests-based machine learning approach allowed characterizing a signature of 32 lipids discriminating NASH with 100% sensitivity and specificity. Furthermore, we validated this signature in an independent group of NASH patients. Then, metabolism dysregulations were investigated in both patients and murine models. Alterations of elongase and desaturase activities were observed along the fatty acid synthesis pathway. The decreased activity of the desaturase FADS1 appeared as a bottleneck, leading upstream to an accumulation of fatty acids and downstream to a deficiency of long-chain fatty acids resulting to impaired phospholipid synthesis. In NASH, mass spectrometry imaging on tissue section revealed the spreading into the hepatic parenchyma of selectively accumulated fatty acids. Such lipids constituted a highly toxic mixture to human hepatocytes. In conclusion, this study characterized a specific and sensitive lipid signature of NASH and positioned FADS1 as a significant player in accumulating toxic lipids during NASH progression.

  • Da Costa, LS & Arnoult, D 2017, « Organelle Separation and Cell Signaling », Methods in Molecular Biology (Clifton, N.J.), vol. 1557, p. 111-115.
    Résumé : Recent findings indicate that some signaling hubs coalesce at the surfaces of organelles through the accumulation of ubiquitylated components required for the signal transduction. For instance, ubiquitylated components of the NF-κB pathway accumulated at the endoplasmic reticulum while ubiquitylated components of the IRF3 pathway are found at the Golgi apparatus. Here we describe simple methods to observe and assess these ubiquitylated components by immunoblotting using differential centrifugation and in vitro assays.
    Mots-clés : Cell fractionation, Differential centrifugation, Immunoblotting, Signaling, Ubiquitination.

  • El Kharbili, M, Robert, C, Witkowski, T, Danty-Berger, E, Barbollat-Boutrand, L, Masse, I, Gadot, N, de la Fouchardière, A, McDonald, PC, Dedhar, S, Le Naour, F, Degoul, F & Berthier-Vergnes, O 2017, « Tetraspanin 8 is a novel regulator of ILK-driven β1 integrin adhesion and signaling in invasive melanoma cells », Oncotarget.
    Résumé : Melanoma is well known for its propensity for lethal metastasis and resistance to most current therapies. Tumor progression and drug resistance depend to a large extent on the interplay between tumor cells and the surrounding matrix. We previously identified Tetraspanin 8 (Tspan8) as a critical mediator of melanoma invasion, whose expression is absent in healthy skin. The present study investigated whether Tspan8 may influence cell-matrix anchorage and regulate downstream molecular pathways leading to an aggressive behavior. Using silencing and ectopic expression strategies, we showed that Tspan8-mediated invasion of melanoma cells resulted from defects in cell-matrix anchorage by interacting with β1 integrins and by interfering with their clustering, without affecting their surface or global expression levels. These effects were associated with impaired phosphorylation of integrin-linked kinase (ILK) and its downstream target Akt-S473, but not FAK. Specific blockade of Akt or ILK activity strongly affected cell-matrix adhesion. Moreover, expression of a dominant-negative form of ILK reduced β1 integrin clustering and cell-matrix adhesion. Finally, we observed a tumor-promoting effect of Tspan8 in vivo and a mutually exclusive expression pattern between Tspan8 and phosphorylated ILK in melanoma xenografts and human melanocytic lesions. Altogether, the in vitro, in vivo and in situ data highlight a novel regulatory role for Tspan8 in melanoma progression by modulating cell-matrix interactions through β1 integrin-ILK axis and establish Tspan8 as a negative regulator of ILK activity. These findings emphasize the importance of targeting Tspan8 as a means of switching from low- to firm-adhesive states, mandatory to prevent tumor dissemination.
    Mots-clés : ILK, integrin, matrix, melanoma, tetraspanin 8.
    Note Note

  • Ferratge, S, Boyer, J, Arouch, N, Chevalier, F & Uzan, G 2017, « Circulating endothelial progenitors in vascular repair », Bio-Medical Materials and Engineering, vol. 28, no. s1, p. S65-S74.
    Résumé : Endothelial Colony Forming Cells (ECFCs) are obtained in culture from Circulating Endothelial Progenitor Cells. They display all characteristics of endothelial cells and they display stem cells features. Cord blood-derived ECFCs (CB-ECFCs) have a high clonogenic and proliferative potentials, and exhibit vascular repair capabilities useful for the treatment of ischemic diseases. However, the link between immaturity and functional properties of CB-ECFCs is still poorly defined. We showed that these cells have a high clonogenic potential and are capable to be efficiently reprogrammed into induced pluripotent stem cells. Moreover, we analyzed the expression of a broad panel of genes involved in embryonic stem cell properties. We define a novel stem cell transcriptional signature for CB-ECFCs fora better characterization and stratification according to their stem cell profile. We then improved the yield of CB-ECFC production for obtaining cells more functional in fewer passages. We used Glycosaminoglycans (GAG), components from the extracellular matrix which potentiate heparin binding growth factor activities. GAG mimetics were designed, having the capacity to increase the yield of ECFC during the isolation process, to increase the number of colonies, improve adhesion, proliferation, migration and self-renewal. GAG mimetics have thus great interest for vascular regeneration in combination with ECFC. Our results show that CB-ECFC are immature cells harboring specific functions such as formation of colonies, proliferation and formation of vascular structures in vitro and in vivo.
    Mots-clés : circulating endothelial progenitors, plasticity, stem cells, Vascular repair.

  • Ferratge, S, Ha, G, Carpentier, G, Arouche, N, Bascetin, R, Muller, L, Germain, S & Uzan, G 2017, « Initial clonogenic potential of human endothelial progenitor cells is predictive of their further properties and establishes a functional hierarchy related to immaturity », Stem Cell Research, vol. 21, p. 148-159.
    Résumé : Endothelial progenitor cells (EPCs) generate in vitro Endothelial Colony Forming Cells (ECFCs) combining features of endothelial and stem/progenitor cells. Their angiogenic properties confer them a therapeutic potential for treating ischemic lesions. They may be isolated from umbilical cord blood (CB-ECFCs) or peripheral adult blood (AB-ECFCs). It is generally accepted that CB-ECFCs are more clonogenic, proliferative and angiogenic than AB-ECFCs. Nevertheless, only a few studies have focused on the functional heterogeneity of CB-ECFCs from different individuals. Moreover, AB-ECFC loss of function is yet to be precisely described. We have focused on these two issues that are critical for clinical perspectives. The detailed clonogenic profile of CB-ECFCs and AB-ECFCs was obtained and revealed a high inter individual heterogeneity and the absence of correlation with age. Most CB-ECFCs yielded initial colonies and had functional properties similar to those of AB-ECFCs. Conversely, a high clonogenicity was associated with an enhanced proliferative and angiogenic potential and stemness gene overexpression, confirming that immaturity, lost by AB-ECFCs, was a prerequisite to functionality. We thus demonstrated the importance of selecting CB-ECFCs according to specific criteria, and we propose using the initial clonogenicity as a relevant marker of their potential efficacy on vascular repair.
    Mots-clés : Cord blood, Endothelial Colony Forming Cells, Endothelial progenitor cells, Functional hierarchy, Immaturity, Peripheral adult blood, Senescence.

  • Gaillard, M & Dagher, I 2017, « Minimally Invasive Liver Preconditioning for Hepatocyte Transplantation in Rats », Methods in Molecular Biology (Clifton, N.J.), vol. 1506, p. 193-200.
    Résumé : In the context of cell transplantation in the liver parenchyma, preconditioning is essential to enhance cell engraftment and liver repopulation. The authors have developed a minimally invasive technique of temporary portal embolization using an absorbable material, called reversible portal vein embolization. We hereby describe the method for isolating hepatocytes from a donor rat before transplanting hepatocytes after reversible portal vein embolization in the recipient.
    Mots-clés : Hepatocyte isolation, Hepatocyte transplantation, Liver perfusion, Liver preconditioning, Portal vein embolization.

  • Grigorov, B, Molle, J, Rubinstein, E, Zoulim, F & Bartosch, B 2017, « CD81 large extracellular loop-containing fusion proteins with a dominant negative effect on HCV cell spread and replication », The Journal of General Virology, vol. 98, no. 7, p. 1646-1657.
    Résumé : The roles of CD81 in the hepatitis C virus (HCV) life cycle are multiple but remain ill characterized. CD81 is known to interact with the HCV glycoproteins as an attachment factor. It also has an important role in the post-attachment entry process. Its interaction with claudin-1, for example, is vital for viral uptake and trafficking. Furthermore, CD81 and its role in membrane organization and trafficking are thought to play a pivotal role in HCV replication. Some of these functions are particularly limited to human CD81; others can be substituted with CD81 molecules from other species. However, with the exception of the large extracellular loop sequence, the structure-function analysis of CD81 in the HCV infectious cycle remains ill characterized. We describe here the fusion molecules between the large extracellular loops of human or mouse CD81 and lipid-raft-associated or unassociated GPI anchors. These fusion molecules have strong antiviral activity in a dominant negative fashion, independent of membrane raft association. Their expression in the hepatoma cell line Huh7.5 blocks HCV uptake, transmission and replication. These molecules will be useful to decipher the various roles of CD81 in the HCV life cycle and transmission in more detail.
    Mots-clés : Animals, Antigens, CD81, Cell Line, Tumor, HEK293 Cells, HeLa Cells, Hepacivirus, Hepatitis C, HIV-1, Humans, Membrane Microdomains, Mice, Protein Binding, Viral Envelope Proteins, Virus Attachment, Virus Internalization, Virus Replication.

  • Hamidouche, Z, Rother, K, Przybilla, J, Krinner, A, Clay, D, Hopp, L, Fabian, C, Stolzing, A, Binder, H, Charbord, P & Galle, J 2017, « Bistable Epigenetic States Explain Age-Dependent Decline in Mesenchymal Stem Cell Heterogeneity », Stem Cells (Dayton, Ohio), vol. 35, no. 3, p. 694-704.
    Résumé : The molecular mechanisms by which heterogeneity, a major characteristic of stem cells, is achieved are yet unclear. We here study the expression of the membrane stem cell antigen-1 (Sca-1) in mouse bone marrow mesenchymal stem cell (MSC) clones. We show that subpopulations with varying Sca-1 expression profiles regenerate the Sca-1 profile of the mother population within a few days. However, after extensive replication in vitro, the expression profiles shift to lower values and the regeneration time increases. Study of the promoter of Ly6a unravels that the expression level of Sca-1 is related to the promoter occupancy by the activating histone mark H3K4me3. We demonstrate that these findings can be consistently explained by a computational model that considers positive feedback between promoter H3K4me3 modification and gene transcription. This feedback implicates bistable epigenetic states which the cells occupy with an age-dependent frequency due to persistent histone (de-)modification. Our results provide evidence that MSC heterogeneity, and presumably that of other stem cells, is associated with bistable epigenetic states and suggest that MSCs are subject to permanent state fluctuations. Stem Cells 2017;35:694-704.
    Mots-clés : Aging, Epigenetics, FACS, Mesenchymal stem cells, Methylation.
    Note Note

  • Manzoni, G, Marinach, C, Topçu, S, Briquet, S, Grand, M, Tolle, M, Gransagne, M, Lescar, J, Andolina, C, Franetich, J-F, Zeisel, MB, Huby, T, Rubinstein, E, Snounou, G, Mazier, D, Nosten, F, Baumert, TF & Silvie, O 2017, « Plasmodium P36 determines host cell receptor usage during sporozoite invasion », eLife, vol. 6.
    Résumé : Plasmodium sporozoites, the mosquito-transmitted forms of the malaria parasite, first infect the liver for an initial round of replication before the emergence of pathogenic blood stages. Sporozoites represent attractive targets for antimalarial preventive strategies, yet the mechanisms of parasite entry into hepatocytes remain poorly understood. Here we show that the two main species causing malaria in humans, Plasmodium falciparum and Plasmodium vivax, rely on two distinct host cell surface proteins, CD81 and the Scavenger Receptor BI (SR-BI), respectively, to infect hepatocytes. By contrast, CD81 and SR-BI fulfil redundant functions during infection by the rodent parasite P. berghei. Genetic analysis of sporozoite factors reveals the 6-cysteine domain protein P36 as a major parasite determinant of host cell receptor usage. Our data provide molecular insights into the invasion pathways used by different malaria parasites to infect hepatocytes, and establish a functional link between a sporozoite putative ligand and host cell receptors.
    Mots-clés : hepatocyte, human, infectious disease, malaria, microbiology, mouse, P. berghei, P. falciparum, P. vivax, P. yoelii, sporozoite.

  • Maslah, N, Cassinat, B, Verger, E, Kiladjian, J-J & Velazquez, L 2017, « The role of LNK/SH2B3 genetic alterations in myeloproliferative neoplasms and other hematological disorders », Leukemia.
    Résumé : Malignant hematological diseases are mainly because of the occurrence of molecular abnormalities leading to the deregulation of signaling pathways essential for precise cell behavior. High-resolution genome analysis using microarray and large-scale sequencing have helped identify several important acquired gene mutations that are responsible for such signaling deregulations across different hematological malignancies. In particular, the genetic landscape of classical myeloproliferative neoplasms (MPNs) has been in large part completed with the identification of driver mutations (targeting the cytokine receptor/Janus-activated kinase 2 (JAK2) pathway) that determine MPN phenotype, as well as additional mutations mainly affecting the regulation of gene expression (epigenetics or splicing regulators) and signaling. At present, most efforts concentrate in understanding how all these genetic alterations intertwine together to influence disease evolution and/or dictate clinical phenotype in order to use them to personalize diagnostic and clinical care. However, it is now evident that factors other than somatic mutations also play an important role in MPN disease initiation and progression, among which germline predisposition (single-nucleotide polymorphisms and haplotypes) may strongly influence the occurrence of MPNs. In this context, the LNK inhibitory adaptor protein encoded by the LNK/SH2B adaptor protein 3 (SH2B3) gene is the target of several genetic variations, acquired or inherited in MPNs, lymphoid leukemia and nonmalignant hematological diseases, underlying its importance in these pathological processes. As LNK adaptor is a key regulator of normal hematopoiesis, understanding the consequences of LNK variants on its protein functions and on driver or other mutations could be helpful to correlate genotype and phenotype of patients and to develop therapeutic strategies to target this molecule. In this review we summarize the current knowledge of LNK function in normal hematopoiesis, the different SH2B3 mutations reported to date and discuss how these genetic variations may influence the development of hematological malignancies.Leukemia advance online publication, 23 May 2017; doi:10.1038/leu.2017.139.

  • Nault, J-C, Couchy, G, Balabaud, C, Morcrette, G, Caruso, S, Blanc, J-F, Bacq, Y, Calderaro, J, Paradis, V, Ramos, J, Scoazec, J-Y, Gnemmi, V, Sturm, N, Guettier, C, Fabre, M, Savier, E, Chiche, L, Labrune, P, Selves, J, Wendum, D, Pilati, C, Laurent, A, De Muret, A, Le Bail, B, Rebouissou, S, Imbeaud, S, GENTHEP Investigators,, Bioulac-Sage, P, Letouzé, E & Zucman-Rossi, J 2017, « Molecular Classification of Hepatocellular Adenoma Associates With Risk Factors, Bleeding, and Malignant Transformation », Gastroenterology, vol. 152, no. 4, p. 880-894.e6.
    Résumé : BACKGROUND & AIMS: Hepatocellular adenomas (HCAs) are benign liver tumors that can be assigned to molecular subtypes based on inactivating mutations in hepatocyte nuclear factor 1A, activating mutations in β-catenin, or activation of inflammatory signaling pathways. We aimed to update the classification system for HCA and associate the subtypes with disease risk factors and complications. METHODS: We analyzed expression levels of 20 genes and sequenced exon regions of 8 genes (HNF1A, IL6ST, CTNNB1, FRK, STAT3, GNAS, JAK1, and TERT) in 607 samples of 533 HCAs from 411 patients, collected from 28 centers mainly in France from 2000 and 2014. We performed gene expression profile, RNA sequence, whole-exome and genome sequence, and immunohistochemical analyses of select samples. Molecular data were associated with risk factors, histopathology, bleeding, and malignant transformation. RESULTS: Symptomatic bleeding occurred in 14% of the patients (85% of cases were female, median age, 38 years); 7% of the nodules were borderline between HCA and hepatocellular carcinoma, and 3% of patients developed hepatocellular carcinoma from HCA. Based on molecular features, we classified HCA into 8 subgroups. One new subgroup, composed of previously unclassified HCA, represented 4% of HCAs overall and was associated with obesity and bleeding. These tumors were characterized by activation of sonic hedgehog signaling, due to focal deletions that fuse the promoter of INHBE with GLI1. Analysis of genetic heterogeneity among multiple HCAs, from different patients, revealed a molecular subtype field effect; multiple tumors had different mutations that deregulated similar pathways. Specific molecular subtypes of HCA associated with various HCA risk factors, including imbalances in estrogen or androgen hormones. Specific molecular subgroup of HCA with β-catenin and sonic hedgehog activation associated with malignant transformation and bleeding, respectively. CONCLUSIONS: Using sequencing and gene expression analyses, we identified a subgroup of HCA characterized by fusion of the INHBE and GLI1 genes and activation of sonic hedgehog pathway. Molecular subtypes of HCAs associated with different patients' risk factors for HCA, disease progression, and pathology features of tumors. This classification system might be used to select treatment strategies for patients with HCA.
    Mots-clés : Benign, HCC, SHH, Tumor Progression.
    Note Note

  • Rocha-Perugini, V, Martínez Del Hoyo, G, González-Granado, JM, Ramírez-Huesca, M, Zorita, V, Rubinstein, E, Boucheix, C & Sánchez-Madrid, F 2017, « CD9 regulates MHC-II trafficking in Monocyte-derived Dendritic Cells », Molecular and Cellular Biology.
    Résumé : Antigen presentation by dendritic cells (DCs) stimulates naïve CD4(+) T cells, triggering T cell activation and the adaptive arm of the immune response. Newly synthesized major histocompatibility complex class II molecules (MHC-II) accumulate at MHC-II-enriched endosomal compartments, and are transported to the plasma membrane of DCs after binding to antigenic peptides to enable antigen presentation. In DCs, MHC-II molecules are included in tetraspanin-enriched microdomains (TEMs). However, the role of tetraspanin CD9 in these processes remains largely undefined. Here, we show that CD9 regulates the T-cell stimulatory capacity of GM-CSF-dependent bone-marrow derived DCs (BMDCs), without affecting antigen-presentation by Flt3L-dependent BMDCs. CD9 knock-out (KO) GM-CSF-dependent BMDCs, which resemble monocyte-derived DCs (MoDCs) induce lower T cell activation than wild-type DCs, and this effect is related to a reduction in MHC-II surface expression in CD9-deficient MoDCs. Importantly, MHC-II targeting to the plasma membrane is largely impaired in immature MoDCs from CD9 KO, in which MHC-II remains arrested in acidic intracellular compartments enriched in LAMP-1, and MHC-II internalization is also blocked. Moreover, CD9 participates in MHC-II trafficking in mature MoDCs, regulating its endocytosis and recycling. Our results demonstrate that the tetraspanin CD9 specifically regulates antigenic presentation in MoDCs through the regulation of MHC-II intracellular trafficking.

  • Saint-Pol, J, Billard, M, Dornier, E, Eschenbrenner, E, Danglot, L, Boucheix, C, Charrin, S & Rubinstein, E 2017, « New Insights into the Tetraspanin Tspan5 Using Novel Monoclonal Antibodies », The Journal of Biological Chemistry.
    Résumé : Tspan5 is a member of a subgroup of tetraspanins referred to as TspanC8. These tetraspanins directly interact with the metalloprotease ADAM10, regulate its exit from the endoplasmic reticulum and subsequent trafficking, and differentially regulate its ability to cleave various substrates and activate Notch signaling. The study of Tspan5 has been limited by the lack of good antibodies. This study provides new insights into Tspan5 using new monoclonal antibodies (mAbs), including two mAbs recognizing both Tspan5 and the highly similar tetraspanin Tspan17. Using these mAbs, we show that endogenous Tspan5 associates with ADAM10 in human cell lines and in mouse tissues where it is most abundant such as the brain, the lung, the kidney or the intestine. We also uncover two TspanC8-specific motifs in the large extracellular domain of Tspan5 that are important for ADAM10 interaction and exit from the endoplasmic reticulum. One of the anti-Tspan5 mAb does not recognize Tspan5 associated with ADAM10, providing a convenient way to measure the fraction of Tspan5 not associated with ADAM10. This fraction is minor in the cell lines tested, and increases upon transfection of cells with TspanC8 tetraspanins such as Tspan15 or Tspan33 that inhibit Notch signalling. Finally, two antibodies inhibit ligand-induced Notch signalling, and this effect is stronger in cells depleted of the TspanC8 Tspan14, further indicating that Tspan5 and Tspan14 can compensate for each other in Notch signalling.
    Mots-clés : ADAM, ADAM10, intracellular trafficking, metalloprotease, monoclonal antibody, Notch pathway, tetraspanin, Tspan5.

  • Saint-Pol, J, Eschenbrenner, E, Dornier, E, Boucheix, C, Charrin, S & Rubinstein, E 2017, « Regulation of the trafficking and the function of the metalloprotease ADAM10 by tetraspanins », Biochemical Society Transactions, vol. 45, no. 4, p. 937-944.
    Résumé : By interacting directly with partner proteins and with one another, tetraspanins organize a network of interactions referred to as the tetraspanin web. ADAM10 (A Disintegrin And Metalloprotease 10), an essential membrane-anchored metalloprotease that cleaves off the ectodomain of a large variety of cell surface proteins including cytokines, adhesion molecules, the precursor of the β-amyloid peptide APP or Notch, has emerged as a major component of the tetraspanin web. Recent studies have shown that ADAM10 associates directly with all members (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33) of a subgroup of tetraspanins having eight cysteines in the large extracellular domain and referred to as TspanC8. All TspanC8 regulate ADAM10 exit from the endoplasmic reticulum, but differentially regulate its subsequent trafficking and its function, and have notably a different impact on Notch signaling. TspanC8 orthologs in invertebrates also regulate ADAM10 trafficking and Notch signaling. It may be possible to target TspanC8 tetraspanins to modulate in a tissue- or substrate-restricted manner ADAM10 function in pathologies such as cardiovascular diseases, cancer or Alzheimer's disease.
    Mots-clés : ADAM10, metalloproteases, tetraspanins.

  • Sloma, I, Mitjavila-Garcia, M, Feraud, O, Griscelli, F, Oudrhiri, N, El Marsafy, S, Gobbo, E, Divers, D, Proust, A, Smadja, DM, Desterke, C, Carles, A, Ma, Y, Hirst, M, Marra, MA, Eaves, CJ, Bennaceur-Griscelli, A & Turhan, AG 2017, « Whole genome analysis reveals unexpected dynamics of mutant subclone development in a patient with JAK2-V617F-positive chronic myeloid leukemia », Experimental Hematology.
    Résumé : We report here the first use of whole genome sequencing (WGS) to examine the initial clonal dynamics in an unusual patient with chronic myeloid leukemia (CML) who presented in chronic phase (CP) with doubly marked BCR-ABL1(+)/JAK2(V617F)-mutant cells and over a 9 year period progressed into an accelerated phase (AP) and then terminal blast phase (BP). WGS showed the diagnostic cells also contained mutations in ASXL1, SEC23B, MAD1L1 and RREB1, as well as 12,000 additional uncommon DNA variants. WGS of endothelial cells generated from circulating precursors revealed many of these were shared with the CML clone. Surprisingly, WGS of induced pluripotent stem cells (iPSCs) derived from the AP cells revealed only 6 additional coding somatic mutations despite retention by their hematopoietic progeny of the parental AP cell levels of BCR-ABL1 expression and sensitivity to imatinib and pimozide. Limited analysis of BP cells showed independent subclonal progression to homozygosity of the MAD1L1 and RREB1 variants. MAD1L1 and SEC23B mutations were also identified in 2/101 cases of myeloproliferative neoplasms but not in 42 healthy subjects. These findings challenge historic concepts of clonal evolution in CML.

  • Torossian, F, Guerton, B, Anginot, A, Alexander, KA, Desterke, C, Soave, S, Tseng, H-W, Arouche, N, Boutin, L, Kulina, I, Salga, M, Jose, B, Pettit, AR, Clay, D, Rochet, N, Vlachos, E, Genet, G, Debaud, C, Denormandie, P, Genet, F, Sims, NA, Banzet, S, Levesque, J-P, Lataillade, J-J & Le Bousse-Kerdilès, M-C 2017, « Macrophage-derived oncostatin M contributes to human and mouse neurogenic heterotopic ossifications », JCI insight, vol. 2, no. 21.
    Résumé : Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury-induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.

  • Vatel, O, Aumont, C, Mathy, V, Petit, M, Feriel, J, Sloma, I, Bennaceur-Griscelli, A & Turhan, AG 2017, « Drug reaction with eosinophilia and systemic symptoms (DRESS) induced by imatinib in chronic myeloid leukemia », Leukemia & Lymphoma, vol. 58, no. 2, p. 473-474.

  • Zeng, Y, Liu, B, Rubio, M-T, Wang, X, Ojcius, DM, Tang, R, Durrbach, A, Ru, Z, Zhou, Y & Lone, Y-C 2017, « Creation of an immunodeficient HLA-transgenic mouse (HUMAMICE) and functional validation of human immunity after transfer of HLA-matched human cells », PloS One, vol. 12, no. 4, p. e0173754.
    Résumé : Research on human immunology has been hindered by the lack of optimal small animal models, given that the protective immune responses of human and non-human species show significant differences. However, due to ethical constraints[1] and the high cost of clinical trials, it is urgent to improve the current animal models that can mimic faithfully human physiology, particularly the human immune system (HIS). HIS mice had been generated recently by engrafting human hematopoietic stem cells (hHSCs) or human peripheral mononuclear cells (hPBMCs) into highly immuno-deficient mice such as NSG, NOG or NRG mice. However, a major experimental drawback for studies using these models is the rapid onset of Graft-versus-Host Disease (GvHD). In the present study, we overcome this limitation by generating new immuno-deficient mice named "HUMAMICE" (HLA-A2+/+/DR1+/+/H-2-β2m-/-/IAβ-/-/Rag2-/-/IL2rγ-/-/Perf-/- mice), which expressed human HLA molecules instead of mouse MHC molecules (H-2), and whose immuno-deficient status was reversed by transferring functional HLA-matched PBMCs thus producing mice with an immuno-competent status with a functional human immune system. We showed that in this HLA-matched context, the hPBMC-transfer led to high lymphocytes engraftment rates without GvHD over three months in this novel mouse model. Furthermore, to evaluate the utility of the hPBMC-HUMAMICE, we immunized them with commercial vaccine of Hepatitis B virus (HBsAg, Hepvac@) which resulted in robust and reproducible production of high levels of HBsAg-specific antibodies, implying that both transferred T and B lymphocytes were functional in HUMAMICE. These responses are comparable to those observed in human clinical trials with this identical vaccine. In conclusion, these findings indicated that the HLA-matched-hPBMC-HUMAMICE represents a promising model for dissecting human immune responses in various human diseases, including infectious diseases, cancers and tumors, and to facilitate the development of novel vaccines and cellular therapies.

  • Zhu, Y, Ailane, N, Sala-Valdés, M, Haghighi-Rad, F, Billard, M, Nguyen, V, Saffroy, R, Lemoine, A, Rubinstein, E, Boucheix, C & Greco, C 2017, « Multi-factorial modulation of colorectal carcinoma cells motility - partial coordination by the tetraspanin Co-029/tspan8 », Oncotarget, vol. 8, no. 16, p. 27454-27470.
    Résumé : Colorectal carcinoma cells Isreco1 display an ability to migrate controlled by a complex set of signals issued from the membrane. By comparing cells infected by mycoplasmas and mycoplasmas free cells, we have established that basal 2D migration is dependent on a double signal mediated by the collagen receptors integrins alpha1/2 and the Toll-Like receptor TLR2. The signal issued from mycoplasmas can be replaced by a TLR2 ligand and the functional effect is neutralized by silencing of MyD88. Following previous observation that downregulation of E-cadherin/p120 catenin increases cell motility, we now report that EGFR or CD44 inhibition have a similar effect on cell motility that is restricted to tetraspanin Co-029/tspan8 transduced IsrecoI cells (Is1-Co029). The modulation of cell migration linked to EGFR or CD44 can be neutralized by antagonizing Co-029 with the mAb Ts29.1 or by RNA interference. Altogether these data point to a crucial role of Co-029 in the modulation of colon cancer cell motility which could be related to the protumoral effect reported for this tetraspanin. Among surface molecules able to mediate Co-029 function, E-cadherin, EGFR and CD44 appear as likely candidates.
    Mots-clés : cell motility, Co-029/tspan8, colorectal carcinoma, EGFR, mycoplasmas.


  • Akil, A, Peng, J, Omrane, M, Gondeau, C, Desterke, C, Marin, M, Tronchère, H, Taveneau, C, Sar, S, Briolotti, P, Benjelloun, S, Benjouad, A, Maurel, P, Thiers, V, Bressanelli, S, Samuel, D, Bréchot, C & Gassama-Diagne, A 2016, « Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV », Nature Communications, vol. 7, p. 12203.
    Résumé : The accumulation of lipid droplets (LD) is frequently observed in hepatitis C virus (HCV) infection and represents an important risk factor for the development of liver steatosis and cirrhosis. The mechanisms of LD biogenesis and growth remain open questions. Here, transcriptome analysis reveals a significant upregulation of septin 9 in HCV-induced cirrhosis compared with the normal liver. HCV infection increases septin 9 expression and induces its assembly into filaments. Septin 9 regulates LD growth and perinuclear accumulation in a manner dependent on dynamic microtubules. The effects of septin 9 on LDs are also dependent on binding to PtdIns5P, which, in turn, controls the formation of septin 9 filaments and its interaction with microtubules. This previously undescribed cooperation between PtdIns5P and septin 9 regulates oleate-induced accumulation of LDs. Overall, our data offer a novel route for LD growth through the involvement of a septin 9/PtdIns5P signalling pathway.

  • Candelier, J-J 2016, « The hydatidiform mole », Cell Adhesion & Migration, vol. 10, no. 1-2, p. 226-235.
    Résumé : The hydatidiform mole (HM) is a placental pathology of androgenetic origin. Placental villi have an abnormal hyperproliferation event and hydropic degeneration. Three situations can be envisaged at its origin: 1. The destruction/expulsion of the female pronucleus at the time of fertilization by 1 or 2 spermatozoa with the former being followed by an endoreplication of the male pronucleus leading to a complete hydatidiform mole (CHM) 2. A triploid zygote (fertilization by 2 spermatozoa) leading to a partial hydatidiform mole (PHM) but can also lead to haploid and diploid clones. The diploid clone may produce a normal fetus while the haploid clone after endoreplication generates a CHM 3. A nutritional defect during the differentiation of the oocytes or the deterioration of the limited oxygen pressure during the first trimester of gestation may lead to the formation of a HM. In countries with poor medical health care system, moles (mainly the CHM) can become invasive or, in rare cases, lead to gestational choriocarcinomas.
    Mots-clés : choriocarcinoma, epigenetic, fertilization, Hydatidiform Mole, invasive mole, trophoblast.

  • Dianat, N, Weber, A & Dubart-Kupperschmitt, A 2016, « [Human pluripotent stem cells and liver disorders] », Biologie Aujourd'hui, vol. 210, no. 1, p. 19-26.
    Résumé : The liver is associated with many diseases including metabolic and cholestatic diseases, cirrhosis as well as chronic and acute hepatitis. However, knowledge about the mechanisms involved in the pathophysiology of these diseases remains limited due to the restricted access to liver biopsies and the lack of cellular models derived from patients. The liver is the main organ responsible for the elimination of xenobiotics and thus hepatocytes have a key role in toxicology and pharmacokinetics. The induced pluripotent stem cells generated from patients with monogenic metabolic disorders, for which the corresponding gene is identified, are relevant in vitro models for the study of the mechanisms involved in generation of pathologies and also for drug screening. Towards this aim, robust protocols for generating liver cells, such as hepatocytes and cholangiocytes, are essential. Our study focused on familial hypercholesterolemia disease modeling, as well as on establishing a protocol for generation of functional cholangiocytes from pluripotent stem cells.
    Mots-clés : Cell Differentiation, Hepatocytes, Humans, Hyperlipoproteinemia Type II, Induced Pluripotent Stem Cells, Liver, Liver Diseases, Models, Biological.

  • Dornier, E, Coumailleau, F, Ottavi, J-F, Moretti, J, Boucheix, C, Mauduit, P, Schweisguth, F & Rubinstein, E 2016, « Correction: TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals », The Journal of Cell Biology, vol. 213, no. 4, p. 495-496.

  • El-Kehdy, H, Pourcher, G, Zhang, W, Hamidouche, Z, Goulinet-Mainot, S, Sokal, E, Charbord, P, Najimi, M & Dubart-Kupperschmitt, A 2016, « Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation », Stem Cells International, vol. 2016, p. 6323486.
    Résumé : In line with the search of effective stem cell population that would progress liver cell therapy and because the rate and differentiation potential of mesenchymal stem cells (MSC) decreases with age, the current study investigates the hepatogenic differentiation potential of human fetal liver MSCs (FL-MSCs). After isolation from 11-12 gestational weeks' human fetal livers, FL-MSCs were shown to express characteristic markers such as CD73, CD90, and CD146 and to display adipocytic and osteoblastic differentiation potential. Thereafter, we explored their hepatocytic differentiation potential using the hepatogenic protocol applied for adult human liver mesenchymal cells. FL-MSCs differentiated in this way displayed significant features of hepatocyte-like cells as demonstrated in vitro by the upregulated expression of specific hepatocytic markers and the induction of metabolic functions including CYP3A4 activity, indocyanine green uptake/release, and glucose 6-phosphatase activity. Following transplantation, naive and differentiated FL-MSC were engrafted into the hepatic parenchyma of newborn immunodeficient mice and differentiated in situ. Hence, FL-MSCs appeared to be interesting candidates to investigate the liver development at the mesenchymal compartment level. Standardization of their isolation, expansion, and differentiation may also support their use for liver cell-based therapy development.

  • Féraud, O, Valogne, Y, Melkus, MW, Zhang, Y, Oudrhiri, N, Haddad, R, Daury, A, Rocher, C, Larbi, A, Duquesnoy, P, Divers, D, Gobbo, E, Brunet de la Grange, P, Louache, F, Bennaceur-Griscelli, A & Mitjavila-Garcia, MT 2016, « Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines », PloS One, vol. 11, no. 3, p. e0149291.
    Résumé : Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.

  • François, H, Durrbach, A, Beaudreuil, S, Charpentier, B & Lecru, L 2016, « [Role of cannabinoid receptors in renal diseases] », Nephrologie & Therapeutique.
    Résumé : Chronic kidney disease remains a major challenge for public health systems and corresponds to the replacement of renal functional tissue by extracellular matrix proteins such as collagens and fibronectin. There is no efficient treatment to date for chronic kidney disease except nephroprotective strategies. The cannabinoid system and more specifically the cannabinoid receptors 1 (CB1) and 2 (CB2) may represent a new therapeutic target in chronic kidney disease. Experimental data obtained in models of diabetes and obesity suggested that CB1 blockade and CB2 stimulation may slow the development of diabetic nephropathy. In human kidneys, CB1 expression is increased in various chronic nephropathies and correlates with renal function. Moreover, endogenous CB1 and CB2 ligands are greatly increased during renal fibrogenesis. A microarray analysis performed in an experimental model of renal fibrosis found that the gene encoding for the CB1 receptor was among the most upregulated genes. We also demonstrated that renal fibrogenesis could be reduced by CB1 inhibition and CB2 stimulation in an experimental model through a direct mechanism involving CB1 on myofibroblasts, which are the major effector cells during renal fibrosis. Therefore, CB1 blockers may represent a novel therapeutic target in chronic kidney disease and diabetes.
    Mots-clés : Cannabinoid receptor, Chronic kidney disease, Diabète, Diabetes, Fibrose rénale, Insuffisance rénale chronique, Récepteur cannabinoïde, Renal fibrosis.

  • Hannoun, Z, Steichen, C, Dianat, N, Weber, A & Dubart-Kupperschmitt, A 2016, « The potential of induced pluripotent stem cell derived hepatocytes », Journal of Hepatology, vol. 65, no. 1, p. 182-199.
    Résumé : Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers.
    Mots-clés : Directed differentiation, Disease modelling, Drug screening, Genomic integrity, Human induced pluripotent stem cells (hiPSCs), Regenerative medicine, Toxicology studies.

  • Kaščáková, S, Kewish, CM, Rouzière, S, Schmitt, F, Sobesky, R, Poupon, J, Sandt, C, Francou, B, Somogyi, A, Samuel, D, Jacquemin, E, Dubart-Kupperschmitt, A, Nguyen, TH, Bazin, D, Duclos-Vallée, J-C, Guettier, C & Le Naour, F 2016, « Rapid and reliable diagnosis of Wilson disease using X-ray fluorescence », The Journal of Pathology. Clinical Research, vol. 2, no. 3, p. 175-186.
    Résumé : Wilson's disease (WD) is a rare autosomal recessive disease due to mutations of the gene encoding the copper-transporter ATP7B. The diagnosis is hampered by the variability of symptoms induced by copper accumulation, the inconstancy of the pathognomonic signs and the absence of a reliable diagnostic test. We investigated the diagnostic potential of X-ray fluorescence (XRF) that allows quantitative analysis of multiple elements. Studies were performed on animal models using Wistar rats (n = 10) and Long Evans Cinnamon (LEC) rats (n = 11), and on human samples including normal livers (n = 10), alcohol cirrhosis (n = 8), haemochromatosis (n = 10), cholestasis (n = 6) and WD (n = 22). XRF experiments were first performed using synchrotron radiation to address the elemental composition at the cellular level. High-resolution mapping of tissue sections allowed measurement of the intensity and the distribution of copper, iron and zinc while preserving the morphology. Investigations were further conducted using a laboratory X-ray source for irradiating whole pieces of tissue. The sensitivity of XRF was highlighted by the discrimination of LEC rats from wild type even under a regimen using copper deficient food. XRF on whole formalin-fixed paraffin embedded needle biopsies allowed profiling of the elements in a few minutes. The intensity of copper related to iron and zinc significantly discriminated WD from other genetic or chronic liver diseases with 97.6% specificity and 100% sensitivity. This study established a definite diagnosis of Wilson's disease based on XRF. This rapid and versatile method can be easily implemented in a clinical setting.
    Mots-clés : copper, diagnosis, Wilson disease, X‐ray fluorescence spectroscopy.

  • Le Coz, V, Zhu, C, Devocelle, A, Vazquez, A, Boucheix, C, Azzi, S, Gallerne, C, Eid, P, Lecourt, S & Giron-Michel, J 2016, « IGF-1 contributes to the expansion of melanoma-initiating cells through an epithelial-mesenchymal transition process », Oncotarget, vol. 7, no. 50, p. 82511-82527.
    Résumé : Melanoma is a particularly virulent human cancer, due to its resistance to conventional treatments and high frequency of metastasis. Melanomas contain a fraction of cells, the melanoma-initiating cells (MICs), responsible for tumor propagation and relapse. Identification of the molecular pathways supporting MICs is, therefore, vital for the development of targeted treatments. One factor produced by melanoma cells and their microenvironment, insulin-like growth factor-1 (IGF- 1), is linked to epithelial-mesenchymal transition (EMT) and stemness features in several cancers.We evaluated the effect of IGF-1 on the phenotype and chemoresistance of B16-F10 cells. IGF-1 inhibition in these cells prevented malignant cell proliferation, migration and invasion, and lung colony formation in immunodeficient mice. IGF-1 downregulation also markedly inhibited EMT, with low levels of ZEB1 and mesenchymal markers (N-cadherin, CD44, CD29, CD105) associated with high levels of E-cadherin and MITF, the major regulator of melanocyte differentiation. IGF-1 inhibition greatly reduced stemness features, including the expression of key stem markers (SOX2, Oct-3/4, CD24 and CD133), and the functional characteristics of MICs (melanosphere formation, aldehyde dehydrogenase activity, side population). These features were associated with a high degree of sensitivity to mitoxantrone treatment.In this study, we deciphered new connections between IGF-1 and stemness features and identified IGF-1 as instrumental for maintaining the MIC phenotype. The IGF1/IGF1-R nexus could be targeted for the development of more efficient anti-melanoma treatments. Blocking the IGF-1 pathway would improve the immune response, decrease the metastatic potential of tumor cells and sensitize melanoma cells to conventional treatments.
    Mots-clés : chemoresistance, EMT, IGF-1, melanoma-initiating cells, metastasis.

  • Ojeda-Uribe, M, Morel, O, Ungureanu, C, Desterke, C, Le Bousse-Kerdilès, M-C & Boulahdour, H 2016, « Assessment of sites of marrow and extramedullary hematopoiesis by hybrid imaging in primary myelofibrosis patients », Cancer Medicine.
    Résumé : We investigated noninvasive procedures by hybrid imaging to assess the sites of active or inactive hematopoiesis in patients with primary myelofibrosis (PMF). To this end, we used two radionuclides, technetium 99m ((99m) Tc) and indium 111-chloride ((111) In-Cl3 ), coupled with single-photon emission tomography/computed tomography (SPECT/CT). We studied five patients with PMF and one with secondary myelofibrosis (MF). The classical pattern of lower fixation of both tracers at the axial skeleton where the myelofibrotic process occurs and the reactivation of sites of active hematopoiesis at the distal skeleton were confirmed. Coupling both radionuclides to SPECT/CT imaging allowed for more precise visualization of the sites of extramedullary hematopoiesis as those observed in the spleen and liver. Splenic high uptake of (111) In-Cl3 coupled with SPECT/CT represents a pathognomonic feature of PMF. We conclude that, the hybrid imaging procedures that we studied might constitute an alternative noninvasive method for the screening of the whole-body marrow and, by this way, to assess the impact of targeted therapies in PMF patients in whom it is well known that the distribution of the hematopoietic active areas is disturbed. Hybrid imaging could also be useful for diagnostic purposes in cases of early PMF or in suspected cases of myelofibrosis secondary to polycythemia vera or essential thrombocythemia.
    Mots-clés : Extramedullary hematopoiesis, hybrid imaging, primary myelofibrosis, radionuclides, SPECT, spleen.

  • Petit Cocault, L, Fleury, M, Clay, D, Larghero, J, Vanneaux, V & Souyri, M 2016, « Monoclonal antibody 1.6.1 against human MPL receptor allows HSC enrichment of CB and BM CD34(+)CD38(-) populations », Experimental Hematology, vol. 44, no. 4, p. 297-302.e1.
    Résumé : Thrombopoietin (TPO) and its receptor Mpl (CD110) play a crucial role in the regulation of hematopoietic stem cells (HSCs). Functional study of Mpl-expressing HSCs has, however, been hampered by the lack of efficient monoclonal antibodies, explaining the very few data available on Mpl(+) HSCs during human embryonic development and after birth. Investigating the main monoclonal antibodies used so far to sort CD110(+) cells from cord blood (CB) and adult bone marrow (BM), we found that only the recent monoclonal antibody 1.6.1 engineered by Immunex Corporation was specific. Using in vitro functional assays, we found that this antibody can be used to sort a CD34(+)CD38(-)CD110(+) population enriched in hematopoietic progenitor stem cells, both in CB and in adult BM. In vivo injection into NSG mice further indicated that the CB CD34(+)CD38(-)CD110(+) population is highly enriched in HSCs compared with both CD34(+)CD38(-)CD110(-) and CD34(+)CD38(-) populations. Together our results validate MAb1.6.1 as an important tool, which has so far been lacking, in the HSC field.
    Mots-clés : human HSC, Human Mpl.

  • Pourcher, G, El-Kehdy, H, Kanso, F, Groyer-Picard, M-T, Gaillard, M, Trassard, O, Blazsek, I, Agostini, H, Dubart-Kupperschmitt, A & Dagher, I 2016, « Volumetric Portal Embolization: A New Concept to Improve Liver Regeneration and Hepatocyte Engraftment », Transplantation, vol. 100, no. 2, p. 344-354.
    Résumé : BACKGROUND: Hepatocyte transplantation has been proposed as an alternative to orthotopic liver transplantation to treat metabolic liver diseases. This approach requires preconditioning of the host liver to enhance engraftment of transplanted hepatocytes. Different methods are currently used in preclinical models: partial hepatectomy, portal ligature or embolization, and radiotherapy or chemotherapeutic drugs. However, these methods carry high risks of complications and are problematic for use in clinical practice. Here, we developed an innovative method called volumetric (distal, partial, and random) portal embolization (VPE), which preserves total liver volume. METHODS: Embolization was performed in the portal trunk of C57BL6 adult mice with polyester microspheres, to ensure a bilateral and distal distribution. The repartition of microspheres was studied by angiographic and histological analyses. Liver regeneration was evaluated by Ki67 labeling. Optimal conditions for VPE were determined, and the resulting regeneration was compared with that after partial hepatectomy (70%). Labeled adult hepatocytes were then transplanted, and engraftment was compared between embolized (n = 19) and nonembolized mice (n = 8). Engraftment was assessed in vivo and histologically by tracking labeled cells at day 5. RESULTS: The best volumetric embolization conditions, which resulted in the regeneration of 5% of total liver, were 8 × 10 ten-micron microspheres infused with a 29 G needle directly into the portal trunk at 3.3 μL/s. In these conditions, transplanted hepatocytes engraftment was significantly higher than that in control conditions (3 vs 0.65%). CONCLUSIONS: The VPE is a new, minimally invasive, and efficient technique to prepare the host liver for cell transplantation.
    Mots-clés : Animals, Biomarkers, Cell Survival, Cell Tracking, Embolization, Therapeutic, Female, Graft Survival, Hepatectomy, Hepatocytes, Injections, Intravenous, Ki-67 Antigen, Liver, Liver Regeneration, Male, Mice, Inbred C57BL, Microspheres, Organ Size, Polyesters, Portal Vein, Radiography, Time Factors.

  • Strassel, C, Bull, A, Moog, S, Receveur, N, Mallo, L, Mangin, P, Eckly, A, Freund, M, Dubart-Kupperschmitt, A, Gachet, C & Lanza, F 2016, « Lentiviral gene rescue of a Bernard-Soulier mouse model to study platelet glycoprotein Ibβ function », Journal of thrombosis and haemostasis: JTH, vol. 14, no. 7, p. 1470-1479.
    Résumé : Essentials A signaling role of glycoprotein (GP)Ibβ is postulated but not formally demonstrated in platelets. Lentiviral-mediated rescue in knock-out mice can be used to evaluate GPIbβ function in vivo. Transduction of the native subunit corrected the main defects associated with GPIb-IX deficiency Deletion of intracellular 159-170 segment increased thrombosis, 150-160 removal increased bleeding. SUMMARY: Background The platelet glycoprotein (GP)Ib-V-IX complex is required for normal hemostasis and megakaryopoiesis. A role in GPIb-dependent responses has been ascribed to the less well characterized GPIbβ subunit using a specific antibody and GPIb-IX transfected cells. Objectives Our aim was to evaluate, in vivo, the role of the GPIbβ in hemostasis and thrombosis. Methods GPIbβ(null) Sca-1(+) progenitors transduced with viral particles harboring hGPIbβ were transplanted into lethally irradiated GPIbβ(-/-) recipient mice. Results hGPIbβ transplanted into the bone marrow of GPIbβ(null) mice rescued GPIb-IX expression in 97% of circulating platelets. These platelets efficiently bound von Willebrand factor (VWF) and extended filopodia on a VWF matrix, demonstrating the restoration of GPIb-dependent adhesive and signaling properties. These mice exhibited less severe macrothrombocytopenia and had normal tail bleeding times as compared with GPIbβ(null) mice. This strategy was employed to manipulate and evaluate the role of the GPIbβ intracellular domain. Removal of the membrane proximal segment (Δ(150-160) ) decreased GPIb-IX expression by 43%, confirming its involvement in receptor assembly and biosynthesis, and resulted in increased bleeding times and decreased thrombosis in a mechanical injury model in the aorta. On the other hand, deletion of the C-flanking 159-170 segment allowed normal GPIb-IX expression, VWF-dependent responses and bleeding times, but resulted in enhanced arterial thrombosis. Conclusion This pointed to a repressor role of GPIbβ in thrombus formation in vivo that was not predicted in studies of heterologous cells. These results highlight the utility of this lentiviral strategy for the structure-function evaluation of GPIb-IX in platelets.
    Mots-clés : Bernard-Soulier syndrome, blood platelets, platelet function tests, platelet glycoprotein GPIb-IX complex, thrombosis, virion.

  • Tranchart, H, Koffi, GM, Gaillard, M, Lainas, P, Poüs, C, Gonin, P, Nguyen, TH, Dubart-Kupperschmitt, A & Dagher, I 2016, « Liver regeneration following repeated reversible portal vein embolization in an experimental model », The British Journal of Surgery, vol. 103, no. 9, p. 1209-1219.
    Résumé : BACKGROUND: Portal vein embolization (PVE) is used routinely to prevent postoperative liver failure as a result of anticipated insufficient future liver remnant volume following resection. The authors have recently developed a technique for temporary PVE. The aim of this study was to assess the effect of repeated reversible PVE on hepatocyte proliferation and subsequent liver hypertrophy in rodents. METHODS: Four treatments were compared (n = 21 rats per group): single reversible PVE, two PVEs separated by 14 days, partial portal vein ligation or sham procedure. The feasibility and tolerance of the procedure were assessed. Volumetric imaging by CT was used to estimate the evolution of liver volumes. After death, the weight of liver lobes was measured and hepatocyte proliferation evaluated by immunostaining. RESULTS: Embolization of portal branches corresponding to 70 per cent of total portal flow was performed successfully in all animals. Repeated PVE induced additional hepatocyte proliferation. Repeated embolization resulted in superior hepatocyte proliferation in the non-occluded segments compared with portal vein ligation (31·1 versus 22·2 per cent; P = 0·003). The non-occluded to total liver volume ratio was higher in the repeated PVE group than in the single PVE and sham groups (P = 0·050 and P = 0·001 respectively). CONCLUSION: Repeated reversible PVE successfully induced additional hepatocyte proliferation and subsequent liver hypertrophy. Surgical relevance Portal vein embolization (PVE) is used routinely to prevent postoperative liver failure as a result of anticipated insufficient future liver remnant volume following resection. In the present study, a technique of repeated temporary PVE was developed in a rat model; this induced additional hepatocyte proliferation and an increase in liver volume compared with single embolization. This novel approach might help induce major hypertrophy of the future remnant liver, which could increase the rate of patients amenable to major liver resections.

  • Zeng, Y, Gao, T, Zhao, G, Jiang, Y, Yang, Y, Yu, H, Kou, Z, Lone, Y, Sun, S & Zhou, Y 2016, « Generation of human MHC (HLA-A11/DR1) transgenic mice for vaccine evaluation », Human Vaccines & Immunotherapeutics, vol. 12, no. 3, p. 829-836.
    Résumé : The rapid occurrence of emerging infectious diseases demonstrates an urgent need for a new preclinical experimental model that reliably replicates human immune responses. Here, a new homozygous humanized human leukocyte antigen (HLA)-A11/DR1 transgenic mouse (HLA-A11(+/+)/DR01(+/+)/H-2-β2m(-/-)/IAβ(-/-)) was generated by crossing HLA-A11 transgenic (Tg) mice with HLA-A2(+/+)/DR01(+/+)/H-2-β2m(-/-)/IAβ(-/-) mice. The HLA-A11-restricted immune response of this mouse model was then examined. HLA-A11 Tg mice expressing a chimeric major histocompatibility complex (MHC) molecule comprising the α1, α2, and β2m domains of human HLA-A11 and the α3 transmembrane and cytoplasmic domains of murine H-2D(b) were generated. The correct integration of HLA-A11 and HLA-DR1 into the genome of the HLA-A11/DR1 Tg mice (which lacked the expression of endogenous H-2-I/II molecules) was then confirmed. Immunizing mice with a recombinant HBV vaccine or a recombinant HIV-1 protein resulted in the generation of IFN-γ-producing cytotoxic T lymphocyte (CTL) and antigen-specific antibodies. The HLA-A11-restricted CTL response was directed at HLA immunodominant epitopes. These mice represent a versatile animal model for studying the immunogenicity of HLA CTL epitopes in the absence of a murine MHC response. The established animal model will also be useful for evaluating and optimizing T cell-based vaccines and for studying differences in antigen processing between mice and humans.
    Mots-clés : HLA, HLA-A11/DR1 transgenic mice, MHC, MHC-restricted epitopes, Vaccine.


  • Ambroise, G, Portier, A, Roders, N, Arnoult, D & Vazquez, A 2015, « Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells », Oncotarget.
    Résumé : The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes.
    Mots-clés : Apoptosis, Burkitt’s Lymphoma, Mitochondria, PUMA, translocation.

  • Beaudreuil, S, Lorenzo, HK & Durrbach, A 2015, « The anti-CD40 auto-antibody: a biomarker or a factor for the permeability of recurrent focal segmental glomerulosclerosis? », Annals of Translational Medicine, vol. 3, no. 9, p. 115.

  • Benzoubir, N, Mussini, C, Lejamtel, C, Dos Santos, A, Guillaume, C, Desterke, C, Samuel, D, Bréchot, C, Bourgeade, M-F & Guettier, C 2015, « Gamma-Smooth Muscle Actin Expression Is Associated with Epithelial-Mesenchymal Transition and Stem-Like Properties in Hepatocellular Carcinoma », PloS One, vol. 10, no. 6, p. e0130559.
    Résumé : BACKGROUND AND AIMS: The prognosis of hepatocellular carcinoma (HCC) is hampered by frequent tumour recurrence and metastases. Epithelial-Mesenchymal Transition (EMT) is now recognized as a key process in tumour invasion, metastasis and the generation of cancer initiating cells. The morphological identification of EMT in tumour samples from the expression of novel mesenchymal markers could provide relevant prognostic information and aid in understanding the metastatic process. METHODS: The expression of Smooth Muscle Actins was studied using immunofluorescence and immunohistochemistry assays in cultured liver cells during an induced EMT process and in liver specimens from adult and paediatric HCC series. RESULTS: We report here that in HCC cell lines treated with TGF-β and in HCC specimens, the expression of αSMA, a known mesenchymal marker of EMT, could never be detected. In addition, our in vitro studies identified the enteric form of SMA, γSMA, as being a marker of EMT. Moreover, this SMA isoform was expressed in 46% of 58 tumours from 42 adult HCC patients and in 90% of 16 tumours from 12 paediatric HCC patients. Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19. CONCLUSION: Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells. This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression.

  • Blasimme, A, Anegon, I, Concordet, J-P, De Vos, J, Dubart-Kupperschmitt, A, Fellous, M, Fouchet, P, Frydman, N, Giovannangeli, C, Jouannet, P, Serre, J-L, Steffann, J, Rial-Sebbag, E, Thomsen, M & Cambon-Thomsen, A 2015, « Genome Editing and Dialogic Responsibility: "What's in a Name?" », The American journal of bioethics: AJOB, vol. 15, no. 12, p. 54-57.
    Mots-clés : Humans, Social Behavior, Terminology as Topic.

  • Candelier, J-J 2015, « [Complete hydatidiform mole] », Medecine Sciences: M/S, vol. 31, no. 10, p. 861-868.
    Résumé : "The battle of the sexes begins in the zygote" W. Reik and J. Walter. Complete hydatidiform mole (CHM) is a pathology of the placenta with androgenetic diploid origin (chromosomes only from paternal origin). Placental villi present an abnormal hyperproliferation and hydropic degeneration associated with the absence of embryo. Three mechanisms can be envisaged at its origin: (1) destruction/expulsion of the female pronucleus at the time of fertilization by one or two spermatozoa, the former being followed by an endoreplication of the male pronucleus (homozygous mole), (2) a triploid zygote (fertilization by two spermatozoa) leading to a haploid and a diploid clones. The diploid clone may produce a normal fetus while the haploid clone, after endoreplication, generates a complete hydatidiform mole, (3) a nutritional defect during the differentiation of the oocytes of the female embryo that will affect the integrity and maturity of her oocytes during her adult life and lead to hydatidiform mole. In countries with a poor medical health care system, moles can be invasive or, in rare cases, lead to gestational choriocarcinomas.
    Mots-clés : Adult, Epigenesis, Genetic, Female, Humans, Hydatidiform Mole, Incidence, Male, Placenta, Pregnancy, Uterine Neoplasms.

  • Coullin, P, Diatta, AL, Boufettal, H, Feingold, J, Leguern, E & Candelier, JJ 2015, « The involvement of the trans-generational effect in the high incidence of the hydatidiform mole in Africa », Placenta, vol. 36, no. 1, p. 48-51.
    Résumé : INTRODUCTION: While the incidence of various chromosomal anomalies observed, including triploid partial moles is independent of the socio-economic level, higher incidences of complete hydatidiform mole "CHM" is generally associated with under developed areas. Moreover, studies have shown that some nutritional deficiencies are related to the abnormal development of oocytes and placenta. In Senegal and Morocco, the annual seasonal cycle contains one period with food shortages and the incidence of complete moles is significant. Accordingly, accurate statistical analyses have been performed in these two countries. METHODS: Each month during a one year period, we investigated the occurrence of normal conceptions, molar conceptions and the conception of the future patients in Senegal and Morocco. The comparisons of the conception dates for these three types of conception were analyzed using the Chi-squared test. RESULTS: 94% of the patients were conceived just prior to the period in the year with food shortages. Consequently, the development of the female embryos occurred under nutritional constraints, which negatively affect the recruitment of the vital factors required for the normal synthesis of DNA, proteins and placental differentiation. DISCUSSIONS: A nutritional deficiency in the mother at conception of their daughter (future patient) is implicated in the higher incidence of CHM in their daughters' filiation. These nutritional deficiencies during the first weeks of pregnancy will have repercussions on the normal development of the oocytes. Accordingly, these developmental impairments take place during the embryonic life of the future mothers of complete moles and not during the conception of the moles themselves.
    Mots-clés : Female, Humans, Hydatidiform Mole, Incidence, Maternal Nutritional Physiological Phenomena, Morocco, Nutrition, Nutritional Status, Oocyte, Placental development, Pregnancy, Senegal, trophoblast, Uterine Neoplasms.

  • Desterke, C, Martinaud, C, Guerton, B, Pieri, L, Bogani, C, Clay, D, Torossian, F, Lataillade, J-J, Hasselbach, HC, Gisslinger, H, Demory, J-L, Dupriez, B, Boucheix, C, Rubinstein, E, Amsellem, S, Vannucchi, AM & Le Bousse-Kerdilès, M-C 2015, « Tetraspanin CD9 participates in dysmegakaryopoiesis and stromal interactions in primary myelofibrosis », Haematologica, vol. 100, no. 6, p. 757-767.
    Résumé : Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the "bad seed in bad soil" hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis.

  • Desterke, C, Martinaud, C, Ruzehaji, N & Le Bousse-Kerdilès, M-C 2015, « Inflammation as a Keystone of Bone Marrow Stroma Alterations in Primary Myelofibrosis », Mediators of Inflammation, vol. 2015, p. 415024.
    Résumé : Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm where severity as well as treatment complexity is mainly attributed to a long lasting disease and presence of bone marrow stroma alterations as evidenced by myelofibrosis, neoangiogenesis, and osteosclerosis. While recent understanding of mutations role in hematopoietic cells provides an explanation for pathological myeloproliferation, functional involvement of stromal cells in the disease pathogenesis remains poorly understood. The current dogma is that stromal changes are secondary to the cytokine "storm" produced by the hematopoietic clone cells. However, despite therapies targeting the myeloproliferation-sustaining clones, PMF is still regarded as an incurable disease except for patients, who are successful recipients of allogeneic stem cell transplantation. Although the clinical benefits of these inhibitors have been correlated with a marked reduction in serum proinflammatory cytokines produced by the hematopoietic clones, further demonstrating the importance of inflammation in the pathological process, these treatments do not address the role of the altered bone marrow stroma in the pathological process. In this review, we propose hypotheses suggesting that the stroma is inflammatory-imprinted by clonal hematopoietic cells up to a point where it becomes "independent" of hematopoietic cell stimulation, resulting in an inflammatory vicious circle requiring combined stroma targeted therapies.
    Mots-clés : Bone Marrow, Data Mining, DNA Methylation, Hematopoietic Stem Cells, Humans, Inflammation, primary myelofibrosis, Stromal Cells, Transforming Growth Factor beta.
  • García-Cano, J, Ambroise, G, Pascual-Serra, R, Carrión, MC, Serrano-Oviedo, L, Ortega-Muelas, M, Cimas, FJ, Sabater, S, Ruiz-Hidalgo, MJ, Sanchez Perez, I, Mas, A, Jalón, FA, Vazquez, A & Sánchez-Prieto, R 2015, « Exploiting the potential of autophagy in cisplatin therapy: A new strategy to overcome resistance », Oncotarget, vol. 6, no. 17, p. 15551-15565.
    Résumé : Resistance to cisplatin is a major challenge in the current cancer therapy. In order to explore new therapeutic strategies to cisplatin resistance, we evaluated, in a model of lung cancer (H1299 and H460 cell lines), the nature of the pathways leading to cell death. We observed that H1299 displayed a natural resistance to cisplatin due to an inability to trigger an apoptotic response that correlates with the induction of autophagy. However, pharmacological and genetic approaches showed how autophagy was a mechanism associated to cell death rather than to resistance. Indeed, pro-autophagic stimuli such as mTOR or Akt inhibition mediate cell death in both cell lines to a similar extent. We next evaluated the response to a novel platinum compound, monoplatin, able to promote cell death in an exclusive autophagy-dependent manner. In this case, no differences were observed between both cell lines. Furthermore, in response to monoplatin, two molecular hallmarks of cisplatin response (p53 and MAPKs) were not implicated, indicating the ability of this pro-autophagic compound to overcome cisplatin resistance. In summary, our data highlight how induction of autophagy could be used in cisplatin resistant tumours and an alternative treatment for p53 mutated patient in a synthetic lethally approach.
    Mots-clés : Apoptosis, autophagy, cisplatin, monoplatin, synthetic lethality.

  • Genêt, F, Kulina, I, Vaquette, C, Torossian, F, Millard, S, Pettit, AR, Sims, NA, Anginot, A, Guerton, B, Winkler, IG, Barbier, V, Lataillade, J-J, Le Bousse-Kerdilès, M-C, Hutmacher, DW & Levesque, J-P 2015, « Neurological heterotopic ossification following spinal cord injury is triggered by macrophage-mediated inflammation in muscle », The Journal of Pathology, vol. 236, no. 2, p. 229-240.
    Résumé : Neurological heterotopic ossification (NHO) is the abnormal formation of bone in soft tissues as a consequence of spinal cord or traumatic brain injury. NHO causes pain, ankyloses, vascular and nerve compression and delays rehabilitation in this high-morbidity patient group. The pathological mechanisms leading to NHO remain unknown and consequently there are no therapeutic options to prevent or reduce NHO. Genetically modified mouse models of rare genetic forms of heterotopic ossification (HO) exist, but their relevance to NHO is questionable. Consequently, we developed the first model of spinal cord injury (SCI)-induced NHO in genetically unmodified mice. Formation of NHO, measured by micro-computed tomography, required the combination of both SCI and localized muscular inflammation. Our NHO model faithfully reproduced many clinical features of NHO in SCI patients and both human and mouse NHO tissues contained macrophages. Muscle-derived mesenchymal progenitors underwent osteoblast differentiation in vitro in response to serum from NHO mice without additional exogenous osteogenic stimuli. Substance P was identified as a candidate NHO systemic neuropeptide, as it was significantly elevated in the serum of NHO patients. However, antagonism of substance P receptor in our NHO model only modestly reduced the volume of NHO. In contrast, ablation of phagocytic macrophages with clodronate-loaded liposomes reduced the size of NHO by 90%, supporting the conclusion that NHO is highly dependent on inflammation and phagocytic macrophages in soft tissues. Overall, we have developed the first clinically relevant model of NHO and demonstrated that a combined insult of neurological injury and soft tissue inflammation drives NHO pathophysiology.
    Mots-clés : Animals, Cardiotoxins, Disease Models, Animal, Female, heterotopic ossification, Humans, Inflammation, macrophage, Macrophages, Mice, Inbred C57BL, Muscle, Skeletal, Myositis, Ossification, Heterotopic, Paraplegia, Spinal Cord Injuries, spinal cord injury, Stem Cells.

  • Hasmim, M, Bruno, S, Azzi, S, Gallerne, C, Michel, JG, Chiabotto, G, Lecoz, V, Romei, C, Spaggiari, GM, Pezzolo, A, Pistoia, V, Angevin, E, Gad, S, Ferlicot, S, Messai, Y, Kieda, C, Clay, D, Sabatini, F, Escudier, B, Camussi, G, Eid, P, Azzarone, B & Chouaib, S 2015, « Isolation and characterization of renal cancer stem cells from patient-derived xenografts », Oncotarget.
    Résumé : As rapidly developing patient-derived xenografts (PDX) could represent potential sources of cancer stem cells (CSC), we selected and characterized non-cultured PDX cell suspensions from four different renal carcinomas (RCC). Only the cell suspensions from the serial xenografts (PDX-1 and PDX-2) of an undifferentiated RCC (RCC-41) adapted to the selective CSC medium. The cell suspension derived from the original tumor specimen (RCC-41-P-0) did not adapt to the selective medium and strongly expressed CSC-like markers (CD133 and CD105) together with the non-CSC tumor marker E-cadherin. In comparison, PDX-1 and PDX-2 cells exhibited evolution in their phenotype since PDX-1 cells were CD133high/CD105-/Ecadlow and PDX-2 cells were CD133low/CD105-/Ecad-. Both PDX subsets expressed additional stem cell markers (CD146/CD29/OCT4/NANOG/Nestin) but still contained non-CSC tumor cells. Therefore, using different cell sorting strategies, we characterized 3 different putative CSC subsets (RCC-41-PDX-1/CD132+, RCC-41-PDX-2/CD133-/EpCAMlow and RCC-41-PDX-2/CD133+/EpCAMbright). In addition, transcriptomic analysis showed that RCC-41-PDX-2/CD133- over-expressed the pluripotency gene ERBB4, while RCC-41-PDX-2/CD133+ over-expressed several tumor suppressor genes. These three CSC subsets displayed ALDH activity, formed serial spheroids and developed serial tumors in SCID mice, although RCC-41-PDX-1/CD132+ and RCC-41-PDX-2/CD133+ displayed less efficiently the above CSC properties. RCC-41-PDX-1/CD132+ tumors showed vessels of human origin with CSC displaying peri-vascular distribution. By contrast, RCC-41-PDX-2 originated tumors exhibiting only vessels of mouse origin without CSC peri-vascular distribution.Altogether, our results indicate that PDX murine microenvironment promotes a continuous redesign of CSC phenotype, unmasking CSC subsets potentially present in a single RCC or generating ex novo different CSC-like subsets.
    Mots-clés : cancer stem cells, CD133, clear cell renal cell carcinoma, EpCAM, patient-derived xenografts.

  • Jouannet, S, Saint-Pol, J, Fernandez, L, Nguyen, V, Charrin, S, Boucheix, C, Brou, C, Milhiet, P-E & Rubinstein, E 2015, « TspanC8 tetraspanins differentially regulate the cleavage of ADAM10 substrates, Notch activation and ADAM10 membrane compartmentalization », Cellular and molecular life sciences: CMLS.
    Résumé : The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aβ, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization.
    Mots-clés : ADAM10, Ectodomain shedding, Membrane compartmentalization, Microdomain, Notch, Tetraspanin.

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