Phosphatase PTP-SHP2 activation by the adrenomedullin system in vascular endothelial cells allowing tumor vessels stabilization

L’adrénomédulline (AM) est un des principaux facteurs de croissance impliqués dans la formations de nouveaux vaisseaux. L’AM est responsable de la formation de jonctions adhérentes stables entre cellules endothéliales vasculaires via le maintien d’un état déphosphorylé du complexe d’adhésion VE-cadhérine/caténines. La phosphorylation de tyrosines est un évènement régulé par un équilibre entre protéine tyrosine kinases et protéine tyrosine phosphatases (PTP). Peu de choses sont encore connues sur le rôle des PTPs dans les voies de signalisation de l’AM au niveau des cellules endothéliales. La SHP2 a été décrite comme étant capable de déphosphoryler le complexe d’adhésion. Son association avec la β-caténine lui permet de contrôler le niveau de phosphorylation du complexe et de maintenir l’association entre VE-cadhérine et caténines. Nous avons ainsi émis l’hypothèse selon laquelle l’AM puisse agir sur la SHP2 permettant ainsi le contrôle de la formation du complexe d’adhésion VE-cadhérine-β-caténine. Nos travaux ont mis en évidence une augmentation de l’activation de la SHP2 induite par l’AM dans les cellules endothéliales entrainant sa localisation au niveau de la membrane et la stabilisation de l'adhésion cellulaire induite par la VE-cadhérine en réduisant le niveau de phosphorylation de cette dernière. Le blocage de la SHP2 entraine des effets opposés avec une inhibition de la déphosphorylation induite par l’AM de la VE-cadhérine sur les tyrosines 731 et 658. En résumé, l’AM régule l’activité de la SHP2 via sa phosphorylation sur la tyrosine 542 ce qui entraine une stabilisation des contacts cellules-cellules via une diminution de la phosphorylation de la VE-cadhérine.Adrenomedullin (AM) is one of the main factors in the formation of tumor neo-vessels. It's responsible for stable adherent junction formation between vascular endothelial (VE) cells by maintaining VE-cadherin/catenins adhesion complex in a dephosphorylated status. Indeed, AM blockade induces phosphorylation of VE-cadherin in tyrosine 731, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby leading to EC adhesion loss and tumor vessels disruption. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in AM signaling in ECs. SHP2 is capable of dephosphorylating the complex. Its association with β-catenin allows it to control the dephosphorylated steady state of the complex and to maintain the VE-cadherin/β-catenin association. To study the mechanism of AM on the inter-endothelial junction stabilization, we hypothesized that AM may act on SHP2 allowing a control upon formation of VE-cadherin-β-catenin complex. In this study, we found that SHP2 activity is markedly increased by AM. In ECs, AM-induced phospho-SHP2 Y542 activity to localize at the human umbilical vein endothelial cell membrane and stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation. SHP2 inhibition causes opposite effects with inhibiting AM-induced dephosphorylation of VE-cadherin at Y731 and Y658. In summary, AM regulates SHP2 activity through phosphorylation of Y542, which stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin

Stromal fibroblasts present in breast carcinomas promote tumor growth and angiogenesis through adrenomedullin secretion

International audienceTumor-or cancer-associated fibroblasts (TAFs or CAFs) are active players in tumorigenesis and exhibit distinct angiogenic and tumorigenic properties. Adrenomedullin (AM), a multifunctional peptide plays an important role in angiogenesis and tumor growth through its receptors calcitonin receptor-like receptor/receptor activity modifying protein-2 and-3 (CLR/RAMP2 and CLR/RAMP3). We show that AM and AM receptors mRNAs are highly expressed in CAFs prepared from invasive breast carcinoma when compared to normal fibroblasts. Immunostaining demonstrates the presence of immunoreactive AM and AM receptors in the CAFs (n = 9). The proliferation of CAFs is decreased by anti-AM antibody (αAM) and anti-AM receptors antibody (aAMR) treatment, suggesting that AM may function as a potent autocrine/paracrine growth factor. Systemic administration of aAMR reduced neovascularization of in vivo Matrigel plugs containing CAFs as demonstrated by reduced numbers of the vessel structures, suggesting that AM is one of the CAFs-derived factors responsible for endothelial cell-like and pericytes recruitment to built a neovascularization. We show that MCF-7 admixed with CAFs generated tumors of greater volume significantly different from the MCF-7 xenografts in nude mice due in part to the induced angiogenesis. aAMR and AM 22-52 therapies significantly suppressed the growth of CAFs/MCF-7 tumors. Histological examination of tumors treated with AM 22-52 and aAMR showed evidence of disruption of tumor vasculature with depletion of vascular endothelial cells, induced apoptosis and decrease of tumor cell proliferation. Our findings highlight the importance of CAFs-derived AM pathway in growth of breast carcinoma and in neovascularization by supplying and amplifying signals that are essential for pathologic angiogenesis

Adrenomedullin Blockade Induces Regression of Tumor Neovessels through Interference with Vascular Endothelial- Cadherin Signalling

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Adrenomedullin blockade induces regression of tumor neovessels through interference with vascular endothelial-cadherin signalling.

International audienceThe cellular and molecular mechanisms by which adrenomedullin (AM) blockade suppresses tumor neovessels are not well defined. Herein, we show that AM blockade using anti-AM and anti-AM receptors antibodies targets vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), and induces regression of unstable nascent tumor neovessels. The underlying mechanism involved, and shown in vitro and in vivo in mice, is the disruption of the molecular engagement of the endothelial cell-specific junctional molecules vascular endothelial-cadherin (VE-cadherin)/β-catenin complex. AM blockade increases endothelial cell permeability by inhibiting cell-cell contacts predominantly through disruption of VE-cadherin/β-catenin/Akt signalling pathway, thereby leading to vascular collapse and regression of tumor neovessels. At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function. Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin. In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation. We found that AM blockade induces β-catenin phosphorylation on Ser33/Ser37/Thr41 sites in both ECs and VSMCs both in vitro and in vivo in mice. These data suggest that AM blockade selectively induces regression of unstable tumor neovessels, through disruption of VE-cadherin signalling. Targeting AM system may present a novel therapeutic target to selectively disrupt assembly and induce regression of nascent tumor neovessels, without affecting normal stabilized vasculature

Role of the Tyrosine Phosphatase SHP-2 in Mediating Adrenomedullin Proangiogenic Activity in Solid Tumors

International audienceVE-cadherin is an essential adhesion molecule in endothelial adherens junctions, and the integrity of these complexes is thought to be regulated by VE-cadherin tyrosine phosphorylation. We have previously shown that adrenomedullin (AM) blockade correlates with elevated levels of phosphorylated VE-cadherin (pVE-cadherin Y731 ) in endothelial cells, associated with impaired barrier function and a persistent increase in vascular endothelial cell permeability. However, the mechanism underlying this effect is unknown. In this article, we demonstrate that the AM-mediated dephosphorylation of pVE-cadherin Y731 takes place through activation of the tyrosine phosphatase SHP-2, as judged by the rise of its active fraction phosphorylated at tyrosine 542 (pSHP-2 Y542 ) in HUVECs and glioblastoma-derived-endothelial cells. Both pre-incubation of HUVECs with SHP-2 inhibitors NSC-87877 and SHP099 and SHP-2 silencing hindered AM-induced dephosphorylation of pVE-cadherin Y731 in a dose dependent-manner, showing the role of SHP-2 in the regulation of endothelial cell contacts. Furthermore, SHP-2 inhibition impaired AM-induced HUVECs differentiation into cord-like structures in vitro and impeded AM-induced neovascularization in in vivo Matrigel plugs bioassays. Subcutaneously transplanted U87-glioma tumor xenograft mice treated with AM-receptors-blocking antibodies showed a decrease in pSHP-2 Y542 associated with VE-cadherin in nascent tumor vasculature when compared to control IgG-treated xenografts. Our findings show that AM acts on VE-cadherin dynamics through pSHP-2 Y542 to finally modulate cell-cell junctions in the angiogenesis process, thereby promoting a stable and functional tumor vasculature

Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines

International audienceAbstract Introduction Patients with pediatric low-grade gliomas (pLGGs), the most common primary brain tumors in children, can often benefit from MAPK inhibitor (MAPKi) treatment. However, rapid tumor regrowth, also referred to as rebound growth, may occur once treatment is stopped, constituting a significant clinical challenge. Methods Four patient-derived pediatric glioma models were investigated to model rebound growth in vitro based on viable cell counts in response to MAPKi treatment and withdrawal. A multi-omics dataset (RNA sequencing and LC-MS/MS based phospho-/proteomics) was generated to investigate possible rebound-driving mechanisms. Following in vitro validation, putative rebound-driving mechanisms were validated in vivo using the BT-40 orthotopic xenograft model. Results Of the tested models, only a BRAF V600E -driven model (BT-40, with additional CDKN2A/B del) showed rebound growth upon MAPKi withdrawal. Using this model, we identified a rapid reactivation of the MAPK pathway upon MAPKi withdrawal in vitro, also confirmed in vivo. Furthermore, transient overactivation of key MAPK molecules at transcriptional (e.g. FOS ) and phosphorylation (e.g. pMEK) levels, was observed in vitro. Additionally, we detected increased expression and secretion of cytokines (CCL2, CX3CL1, CXCL10 and CCL7) upon MAPKi treatment, maintained during early withdrawal. While increased cytokine expression did not have tumor cell intrinsic effects, presence of these cytokines in conditioned media led to increased attraction of microglia cells in vitro. Conclusion Taken together, these data indicate rapid MAPK reactivation upon MAPKi withdrawal as a tumor cell intrinsic rebound-driving mechanism. Furthermore, increased secretion of microglia-recruiting cytokines may play a role in treatment response and rebound growth upon withdrawal, warranting further evaluation

BH3 mimetics targeting BCL-XL impact the senescent compartment of pilocytic astrocytoma

BACKGROUND: Pilocytic astrocytoma (PA) is the most common pediatric brain tumor and a mitogen-activated protein kinase (MAPK)-driven disease. Oncogenic MAPK-signaling drives the majority of cells into oncogene-induced senescence (OIS). While OIS induces resistance to anti-proliferative therapies, it represents a potential vulnerability exploitable by senolytic agents. METHODS: We established new patient-derived PA cell lines that preserve molecular features of the primary tumors and can be studied in OIS and proliferation depending on expression ore repression of the SV40 large T antigen. We determined expression of anti-apoptotic BCL-2 members in these models and primary PA. Dependence of senescent PA cells on anti-apoptotic BCL-2 members was investigated using a comprehensive set of BH3-mimetics. RESULTS: Senescent PA cells upregulate BCL-XL upon senescence induction and show dependency on BCL-XL for survival. BH3 mimetics with high affinity for BCL-XL (BCL-XLi) reduce metabolic activity and induce mitochondrial apoptosis in senescent PA cells at nano-molar concentrations. In contrast, BH3 mimetics without BCL-XLi activity, conventional chemotherapy and MEK inhibitors show no effect. CONCLUSIONS: Our data demonstrates that BCL-XL is critical for survival of senescent PA tumor cells and provides proof-of-principle for the use of clinically available BCL-XL-dependent senolytics

Original Research Molecular diagnostics enables detection of actionable targets: the Pediatric Targeted Therapy 2.0 registry

Background: Precision oncology requires diagnostic accuracy and robust detection of actionable alterations. The Pediatric Targeted Therapy (PTT) 2.0 program aims at improving diagnostic accuracy by addition of molecular analyses to the existing histological diagnosis and detection of actionable alterations for relapsed paediatric oncology patients, in cases with limited availability of tumour material.Methods: Paediatric patients diagnosed with relapse or progression of a central nervous system tumour (n = 178), a sarcoma (n = 41) or another solid tumour (n = 44) were included. DNA methylation array, targeted gene panel sequencing on tumour and blood (130 genes), RNA sequencing in selected cases and a pathway-specific immunohistochemistry (IHC) panel were performed using limited formalin-fixed paraffin embedded tissue from any disease episode available. The clinical impact of reported findings was assessed by a serial questionnaire-based follow-up.Results: Integrated molecular diagnostics resulted in refined or changed diagnosis in 117/263 (44%) tumours. Actionable targets were detected in 155/263 (59%) cases. Constitutional DNA variants with clinical relevance were identified in 16/240 (7%) of patients, half of which were previously unknown. Clinical follow-up showed that 26/263 (10%) of patients received mechanism-of-action based treatment matched to the molecular findings.Conclusion: Next-generation diagnostics adds robust and relevant information on diagnosis, actionable alterations and cancer predisposition syndromes even when tissue from the current disease episode is limited. 2022 Elsevier Ltd. All rights reserved

Integrative multi-omics reveals two biologically distinct groups of pilocytic astrocytoma

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Pilocytic astrocytoma (PA), the most common pediatric brain tumor, is driven by aberrant mitogen-activated protein kinase signaling most commonly caused by BRAF gene fusions or activating mutations. While 5-year overall survival rates exceed 95%, tumor recurrence or progression constitutes a major clinical challenge in incompletely resected tumors. Here, we used similarity network fusion (SNF) analysis in an integrative multi-omics approach employing RNA transcriptomic and mass spectrometry-based proteomic profiling to molecularly characterize PA tissue samples from 62 patients. Thereby, we uncovered that PAs segregated into two molecularly distinct groups, namely, Group 1 and Group 2, which were validated in three non-overlapping cohorts. Patients with Group 1 tumors were significantly younger and showed worse progression-free survival compared to patients with group 2 tumors. Ingenuity pathways analysis (IPA) and gene set enrichment analysis (GSEA) revealed that Group 1 tumors were enriched for immune response pathways, such as interferon signaling, while Group 2 tumors showed enrichment for action potential and neurotransmitter signaling pathways. Analysis of immune cell-related gene signatures showed an enrichment of infiltrating T Cells in Group 1 versus Group 2 tumors. Taken together, integrative multi-omics of PA identified biologically distinct and prognostically relevant tumor groups that may improve risk stratification of this single pathway driven tumor type.This project funded in part by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Project-ID 418179183—KFO 337, RO 3577/7-1 (A.R.), RE 2857/4-1 (M.R.) and supported by Biomed Valley and Day One Therapeutics (T.M.), Fundação Amélia de Melo (C.C.F.) and Fundação Millennium bcp (C.C.F.)info:eu-repo/semantics/publishedVersio

Renouveler la macroéconomie postkeynésienne ? Les modèles stock-flux cohérent et multi-agents

Le numéro 16 de la Revue de la régulation nous donne à nouveau l’occasion d’un échange avec le courant postkeynésien (après le n° 10). Il illustre les liens tissés entre ces approches et permet à des chercheurs d’orientations différentes, souvent complémentaires, de travailler ensemble. Le dossier, coordonné par Mickaël Clévenot et Edwin Le Héron, invite à explorer les nouvelles voies de la modélisation macroéconomique postkeynésienne. >> lire la suite Issue 16 of the Revue de la regulation offers another occasion (after Issue 10) for exchanges with the post-Keynesian school of thought. It illustrates the connections between these frameworks and allows researchers with different, but often complementary, orientations to work together. The dossier, edited by Mickaël Clévenot and Edwin Le Heron, invites us to explore emerging directions in post-Keynesian macroeconomic modeling. >> read mor