0383 : In vitro 3D model of in vitro angiogenesis using human endothelial cells and pericytes

Human tissue is three-dimensional, and requires convective transport of nutrients and waste through capillary networks to meet metabolic demands Angiogenesis is the formation of new blood vessels from the existing vasculature. It is a multi-step process that include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane. The literatture in 3D in vitro models using endothelial cells is wide, using various types of EC (essentially Human Umbilical Vein Endothelial Cells), but blood vessels are composed of two interacting cells types: endothelial cells form the inner of the vessel wall, and mural cells that wrap the first ones. Pericytes are the mural cells of microvessels. They serve as scaffolding, and they communicate with endothelial cells by direct physical contacts and paracrine signaling pathways. Presently, there are no three-dimensional in vitro models of 3D Matrices which contain human pericyte-coated capillaries. Therefore, we aim at including pericytes in a 3D vascular morphogenesis assay in order to create a 3D in vitro model more close to physiologic conditions. We’ll show and discuss our first analyzes and results, the goal of which is to provide new in vitro tools in order to better understand vascular biology, for later studies of endothelial cells-pericytes interactions, extracellular matrix-pericytes interactions, and eventually, further elucidate the role of pericytes in the microvasculature

Expression and biological effects of neurotrophins in breast cancer

Le laboratoire avait précédemment montré le rôle mitogène et anti-apoptotique du NGF pour les cellules de cancer du sein via l'activation de ses récepteurs TrkA et p75NTR. Dans le présent travail, nous montrons que le NGF (nerve growth factor) est exprimé par une large majorité de biopsies cancéreuses mammaires et nous apportons la démonstration définitive de son rôle dans la croissance du cancer du sein in vivo en modèle préclinique. Ces nouvelles données indiquent que le NGF peut être présenté comme une cible thérapeutique potentielle dans le cancer du sein. Ces résultats nous ont amené à nous intéresser aux autres membres de la famille du NGF, les neurotrophines brain-derived neurotrophic factor (BDNF, NT-3 et NT-4/5) qui exercent leurs effets en se liant aux récepteurs à activité tyrosine kinase TrkB et TrkC ainsi qu'au récepteur commun aux neurotrophines, p75NTR. Nous montrons pour la première fois l'expression et la sécrétion du BDNF et de la NT-4/5 par les cellules de cancer du sein. L'étude de leurs récepteurs indique que TrkB est présent sous une forme tronquée sans domaine kinasique intracellulaire appelée TrkB-T1 dont le rôle reste à déterminer, nous confirmons par ailleurs la présence de p75NTR et montrons qu'il est régulé positivement par les œstrogènes. En revanche, la NT-3 et son récepteur TrkC sont peu ou pas détectés dans le cancer du sein. La stimulation exogène des cellules de cancer du sein par le BDNF et la NT-4/5 leur confère une résistance à l'apoptose via la stimulation du récepteur p75NTR. De plus, à l'aide d'anticorps bloquant, nous avons mis en évidence que le BDNF et la NT-4/5 libérés sont biologiquement actifs puisque leur inhibition augmente le nombre de cellules apoptotiques in vitro et dans des souris porteuses de tumeurs mammaires. Le BDNF et la NT-4/5 sont donc des facteurs de survie dans les cellules de cancer du sein. L'ensemble de ce travail a permis de mieux comprendre le rôle pro-tumoral de cette famille de facteurs de croissance dans la cancérogenèse mammaire.The laboratory had previously shown the mitogenic and anti-apoptotic roles of NGF for breast cancer ce Ils through activation of its tyrosine kinase receptors TrkA and the neurotrophin receptor p75NTR. ln this work, we show that NGF (nerve growth factor) is expressed by a large majority of breast cancer biopsies and we bring the demonstration of its role in the growth of breast cancer in vivo using a preclinical mode!. These new data indicate that NGF can be considered as a potential therapeutic target in breast cancer. These results led us t study the other members of NGF family, the neurotrophins brain-derived neurotrophic factor (BDNF, NT-3 an NT-4/5) which act by binding to the tyrosine kinase receptors TrkB and TrkC and to p75NTR. We show for the first time the expression and secretion of BON F and NT -4/5 by breast cancer cells. The study of their receptor indicates that TrkB is present in a truncated form without intracellular kinase domain ca lied TrkB- T1, whose role remains to be determined, we also confirm the presence of p75NTR and show that it is positively regulated by estrogen. ln contrast, NT -3 and its receptor TrkC were not detected in breast cancer cells. The exogenous stimulation of breast cancer ce Us by the BDNF and NT-4/5 confers resistance to apoptosis via stimulation of p75NTR. ln addition, using blocking antibodies, we highlighted that the BDNF and NT-4/5 released are biologically active since their inhibition increases the number of apoptotic cancer cells in vitro and in mice carrying breast tumor xenografts. ln conclusion, BDNF and NT -4/5 are survival factors for breast cancer cells. This work has led to a better com rehension of the ro-tumour role of neurotro hins in breast cance

Expression et effets biologiques des neurotrophines dans le cancer du sein

LILLE1-BU (590092102) / SudocSudocFranceF

Inhibition of tumor necrosis factor-induced phenotypes by short intracellular versions of latent membrane protein-1.

International audienceTumor necrosis factor (TNF) is a potent multi-functional cytokine with a homeostatic role in host defence. In case of deregulation, TNF is implicated in numerous pathologies. The latent membrane protein-1 (LMP1) is expressed by Epstein-Barr virus during viral latency and displaying properties of a constitutively activated member of the TNF receptor family. Both TNFR1 and LMP1 share a similar set of proximal adapters and signalling pathways although they display different biological responses. We previously demonstrated that the intracellular part of LMP1, LMP1-CT, a dominant-negative form of LMP1, inhibits LMP1 signalling. Here, we developed shorter versions derived from C-terminal part of LMP1 to investigate their roles on LMP1 and TNF signalling. We constructed several mutants of LMP1 containing a part of cytoplasmic signalling region fused to the green fluorescent protein. These mutants selectively impair signalling by LMP1 and TNF but not by IL-1beta which uses other adapters. Dominant-negative effect was due to binding and sequestration of LMP1 adapters RIP, TRAF2 and TRADD as assessed by coimmunoprecipitation experiments and confocal analysis. Expression of these mutants impairs the recruitment of these adapters by TNFR1 and TNF-associated phenotypes. These mutants did not display cytostatic properties but were able to modulate TNF-induced phenotypes, apoptosis or cell survival, depending on the cell context. Interestingly, these mutants are able to inhibit a pro-inflammatory response in endothelial cells. These data demonstrate that LMP1 derived molecules can be used to design compounds with potential therapeutic roles in diseases due to TNF overactivation

PARP1 impact on DNA repair of platinum adducts: Preclinical and clinical read-outs

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A HIF-regulated VHL-PTP1B-Src signaling axis identifies a therapeutic target in renal cell carcinoma

Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials