Evidence-Based View of Safety and Effectiveness of Prokineticin Receptors Antagonists during Pregnancy

Endocrine gland derived vascular endothelial growth factor (EG-VEGF) is a canonical member of the prokineticin (PROKs) family. It acts via the two G-protein coupled receptors, namely PROKR1 and PROKR2. We have recently demonstrated that EG-VEGF is highly expressed in the human placenta; contributes to placental vascularization and growth and that its aberrant expression is associated with pregnancy pathologies including preeclampsia and fetal growth restriction. These findings strongly suggested that antagonization of its receptors may constitute a potential therapy for the pregnancy pathologies. Two specific antagonists of PROKR1 (PC7) and for PROKR2 (PKRA) were reported to reverse PROKs adverse effects in other systems. In the view of using these antagonists to treat pregnancy pathologies, a proof of concept study was designed to determine the biological significances of PC7 and PKRA in normal pregnancy outcome. PC7 and PKRA were tested independently or in combination in trophoblast cells and during early gestation in the gravid mouse. Both independent and combined treatments uncovered endogenous functions of EG-VEGF. The independent use of antagonists distinctively identified PROKR1 and PROKR2-mediated EG-VEGF signaling on trophoblast differentiation and invasion; thereby enhancing feto-placental growth and pregnancy outcome. Thus, our study provides evidence for the potential safe use of PC7 or PKRA to improve pregnancy outcome

Rôle de l’EG-VEGF (Endocrine Gland Derived-Vascular Endothelial Growth Factor) dans le développement et la progression tumorale placentaire : cas du choriocarcinome

Choriocarcinoma is a highly malignant trophoblastic tumor that often develop from molar pregnancies also called hydatidiform mole (HM). Nevertheless, HM progression towards choriocarcinoma remains uncharacterized. Involvement of angiogenic factors in this process is proposed. Here, we investigated the role of a new placental angiogenic factor, EG-VEGF (Endocrine Gland-Derived Endothelial Growth Factor) in choricarcinoma pathogenesis. EG-VEGF acts via two GPCR receptors PROKR-1 and PROKR-2. Three approaches were used to verify this hypothesis. A clinical approach using sera and placental samples collected from HM (n=38) and Choriocarcinoma patients (n=3) and from normal pregnant women (n=18); all collected during the first trimester of pregnancy. An In vitro approach using JEG3 cells, a human choriocarcinoma cell line and normal first trimester trophoblast cells (NTC). An in vivo approach that aimed at developing an animal model of choriocarcinoma in which therapeutic agents have been tested. Circulating EG-VEGF levels were significantly higher in HM and choriocarcinoma compared to normal patients. Placental EG-VEGF, PROKR1 and PROKR2 expression exhibited the same pattern. In JEG3 cells, EG-VEGF increased i) the expression of PROKR-1 and PROKR-2, ii). Their migration, proliferation invasion and spheroid formation using both 2 and 3D culture systems. These effects were abolished using PROKR1 and PROKR2 antagonists, iii) phosphorylation of different proteins involved in tumor progression as well as secretion of MMP-2 and MMP-9. Choriocarcinoma model has been developed by injection of JEG3 cells orthotopically within the placenta of SCID mice (Patent in progress). Within 12 days, injected gravid mice developed a choricarcinoma that metastasis in multiple organs. Importantly, injection of EG-VEGF receptors antagonists significantly reduced tumor development and its progression. Also, we have characterized the mechanism by which EG-VEGF contributes to tumor progression. This mechanism involves the cleavage of the key junctional protein, the VE-cadherin, following its phosphorylation at the tyrosine 685, by EG-VEGF. In total my thesis project i) demonstrated the direct involvement of the EG-VEGF in the development and progression of choriocarcinoma ii) elucidated the mechanism of this progression and iii) proposes a potential therapeutic for choriocarcinoma through the antagonisation of its receptors.Le choriocarcinome est une tumeur trophoblastique hautement maligne qui se développe souvent suite à des grossesses molaires dénommées moles hydatiformes (MH). La progression d’une MH vers un choriocarcinome reste à ce jour non caractérisée. L’implication de facteurs angiogéniques dans ce processus a été proposée. Nous avons étudié le rôle d'un nouveau facteur angiogénique spécifique du placenta, l’EG-VEGF (Endocrine Gland-Derived Endothelial Growth Factor) dans la pathogenèse du choriocarcinome. EG-VEGF agit via deux récepteurs (RCPG), PROKR-1 et PROKR-2. Trois approches ont été utilisées pour vérifier cette hypothèse. Une approche clinique, utilisant des échantillons placentaires et des sera collectés chez des patientes MH (n = 38) et avec choriocarcinome (n = 3) et chez des femmes normales (n = 18), tous prélevés au cours du premier trimestre de la grossesse. Une approche in vitro, utilisant les cellules JEG3, lignée humaine de cellules de choriocarcinome et des cellules trophoblastiques normales du premier trimestre (CTN). Une approche in vivo qui a visé à développer un modèle animal du choriocarcinome qui a servi à l’étude de la progression du choriocarcinome. Les niveaux circulants d’EG-VEGF étaient significativement plus élevés dans les MH et le choriocarcinome comparés aux niveaux chez les patientes normales. Au niveau placentaire, à la fois les niveaux d’expression de l’EG-VEGF et ses récepteurs ont été augmentés. Dans les cellules JEG3, EG-VEGF augmente i) l'expression de PROKR-1 et PROKR-2, ii). La migration, l'invasion, la prolifération et la formation de sphéroïdes dans des systèmes de culture 2 et 3D. Ces effets étaient significativement diminués en présence des antagonistes des deux récepteurs, iii) la phosphorylation de diverses protéines impliquées dans la progression tumorale, ainsi que la sécrétion de MMP-2 et MMP-9. Le modèle de choriocarcinome a été développé par injection de cellules JEG3 en orthotopie dans le placenta de la souris SCID (brevet en cours). En 12 jours, les souris gestantes injectées ont développés un choriocarcinome qui a métastasé dans différents organes. L'injection des antagonistes des récepteurs de l’EG-VEGF réduit significativement le développement et la progression de la tumeur. Par ailleurs, nous avons caractérisé le mécanisme par lequel EG-VEGF contribuerait à la progression tumorale. Ce mécanisme implique le clivage de la protéine de la jonction endothéliale, la VE-Cadhérine, suite à sa phosphorylation sur tyrosine 685 par l’EG-VEGF. Au total, mon projet de thèse i) démontre l’implication directe de l’EG-VEGF dans le développement et la progression du choriocarcinome ii) élucide le mécanisme de cette progression et iii) propose une piste thérapeutique via l’antagonisation de ses récepteurs

Role of EG-VEGF (Endocrine Gland-Derived Vascular Endothelial Growth Factor) in placental tumor development and progression : case of choriocarcinoma

Le choriocarcinome est une tumeur trophoblastique hautement maligne qui se développe souvent suite à des grossesses molaires dénommées moles hydatiformes (MH). La progression d’une MH vers un choriocarcinome reste à ce jour non caractérisée. L’implication de facteurs angiogéniques dans ce processus a été proposée. Nous avons étudié le rôle d'un nouveau facteur angiogénique spécifique du placenta, l’EG-VEGF (Endocrine Gland-Derived Endothelial Growth Factor) dans la pathogenèse du choriocarcinome. EG-VEGF agit via deux récepteurs (RCPG), PROKR-1 et PROKR-2. Trois approches ont été utilisées pour vérifier cette hypothèse. Une approche clinique, utilisant des échantillons placentaires et des sera collectés chez des patientes MH (n = 38) et avec choriocarcinome (n = 3) et chez des femmes normales (n = 18), tous prélevés au cours du premier trimestre de la grossesse. Une approche in vitro, utilisant les cellules JEG3, lignée humaine de cellules de choriocarcinome et des cellules trophoblastiques normales du premier trimestre (CTN). Une approche in vivo qui a visé à développer un modèle animal du choriocarcinome qui a servi à l’étude de la progression du choriocarcinome. Les niveaux circulants d’EG-VEGF étaient significativement plus élevés dans les MH et le choriocarcinome comparés aux niveaux chez les patientes normales. Au niveau placentaire, à la fois les niveaux d’expression de l’EG-VEGF et ses récepteurs ont été augmentés. Dans les cellules JEG3, EG-VEGF augmente i) l'expression de PROKR-1 et PROKR-2, ii). La migration, l'invasion, la prolifération et la formation de sphéroïdes dans des systèmes de culture 2 et 3D. Ces effets étaient significativement diminués en présence des antagonistes des deux récepteurs, iii) la phosphorylation de diverses protéines impliquées dans la progression tumorale, ainsi que la sécrétion de MMP-2 et MMP-9. Le modèle de choriocarcinome a été développé par injection de cellules JEG3 en orthotopie dans le placenta de la souris SCID (brevet en cours). En 12 jours, les souris gestantes injectées ont développés un choriocarcinome qui a métastasé dans différents organes. L'injection des antagonistes des récepteurs de l’EG-VEGF réduit significativement le développement et la progression de la tumeur. Par ailleurs, nous avons caractérisé le mécanisme par lequel EG-VEGF contribuerait à la progression tumorale. Ce mécanisme implique le clivage de la protéine de la jonction endothéliale, la VE-Cadhérine, suite à sa phosphorylation sur tyrosine 685 par l’EG-VEGF. Au total, mon projet de thèse i) démontre l’implication directe de l’EG-VEGF dans le développement et la progression du choriocarcinome ii) élucide le mécanisme de cette progression et iii) propose une piste thérapeutique via l’antagonisation de ses récepteurs.Choriocarcinoma is a highly malignant trophoblastic tumor that often develop from molar pregnancies also called hydatidiform mole (HM). Nevertheless, HM progression towards choriocarcinoma remains uncharacterized. Involvement of angiogenic factors in this process is proposed. Here, we investigated the role of a new placental angiogenic factor, EG-VEGF (Endocrine Gland-Derived Endothelial Growth Factor) in choricarcinoma pathogenesis. EG-VEGF acts via two GPCR receptors PROKR-1 and PROKR-2. Three approaches were used to verify this hypothesis. A clinical approach using sera and placental samples collected from HM (n=38) and Choriocarcinoma patients (n=3) and from normal pregnant women (n=18); all collected during the first trimester of pregnancy. An In vitro approach using JEG3 cells, a human choriocarcinoma cell line and normal first trimester trophoblast cells (NTC). An in vivo approach that aimed at developing an animal model of choriocarcinoma in which therapeutic agents have been tested. Circulating EG-VEGF levels were significantly higher in HM and choriocarcinoma compared to normal patients. Placental EG-VEGF, PROKR1 and PROKR2 expression exhibited the same pattern. In JEG3 cells, EG-VEGF increased i) the expression of PROKR-1 and PROKR-2, ii). Their migration, proliferation invasion and spheroid formation using both 2 and 3D culture systems. These effects were abolished using PROKR1 and PROKR2 antagonists, iii) phosphorylation of different proteins involved in tumor progression as well as secretion of MMP-2 and MMP-9. Choriocarcinoma model has been developed by injection of JEG3 cells orthotopically within the placenta of SCID mice (Patent in progress). Within 12 days, injected gravid mice developed a choricarcinoma that metastasis in multiple organs. Importantly, injection of EG-VEGF receptors antagonists significantly reduced tumor development and its progression. Also, we have characterized the mechanism by which EG-VEGF contributes to tumor progression. This mechanism involves the cleavage of the key junctional protein, the VE-cadherin, following its phosphorylation at the tyrosine 685, by EG-VEGF. In total my thesis project i) demonstrated the direct involvement of the EG-VEGF in the development and progression of choriocarcinoma ii) elucidated the mechanism of this progression and iii) proposes a potential therapeutic for choriocarcinoma through the antagonisation of its receptors

In Vivo Quantitative Assessment of Gestational Choriocarcinoma Development and Progression Using Luminescent Trophoblast Cells

International audienc

.EG-VEGF, BV8, and their receptor expression in human bronchi and their modification in cystic fibrosis: Impact of CFTR mutation (delF508)

International audienceEnhanced lung angiogenesis has been reported in cystic fibrosis (CF). Recently, two highly homologous ligands, endocrine gland vascular endothelial growth factor (EG-VEGF) and mammalian Bv8, have been described as new angiogenic factors. Both ligands bind and activate two closely related G protein-coupled receptors, the prokineticin receptor (PROKR) 1 and 2. Yet, the expression, regulation, and potential role of EG-VEGF, BV8, and their receptors in normal and CF lung are still unknown. The expression of the receptors and their ligands was examined using molecular, biochemical, and immunocytochemistry analyses in lungs obtained from CF patients vs. control and in normal and CF bronchial epithelial cells. Cystic fibrosis transmembrane conductance regulator (CFTR) activity was evaluated in relation to both ligands, and concentrations of EG-VEGF were measured by ELISA. At the mRNA level, EG-VEGF, BV8, and PROKR2 gene expression was, respectively, approximately five, four, and two times higher in CF lungs compared with the controls. At the cellular level, both the ligands and their receptors showed elevated expressions in the CF condition. Similar results were observed at the protein level. The EG-VEGF secretion was apical and was approximately two times higher in CF compared with the normal epithelial cells. This secretion was increased following the inhibition of CFTR chloride channel activity. More importantly, EG-VEGF and BV8 increased the intracellular concentration of Ca2+ and cAMP and stimulated CFTR-chloride channel activity. Altogether, these data suggest local roles for epithelial BV8 and EG-VEGF in the CF airway peribronchial vascular remodeling and highlighted the role of CFTR activity in both ligand biosynthesis and secretion

Vascular endothelial growth factor (VEGF) and Endocrine gland-VEGF (EG-VEGF) are down regulated in head and neck cancer

International audienceObjective To characterise the role of VEGF, EG-VEGF and its receptors in the development and progression of HNC. Design Human serum and tissues samples were collected from healthy, epulis and HNC patients and used for ELISA assays and immunohistochemistry studies, respectively. Setting Ibn Rochd Hospital of Casablanca (Morocco), INSERM and University of Grenoble Alpes (France). Participants We used serum from 64 patients with head and neck cancers and from 71 controls without general pathology. Tissues samples were collected from seven patients with OSCC and from seven patients with Epulis. Main outcome measures We compared circulating VEGF and EG-VEGF in normal and HNC patients and determined the expression, localisation and quantification of VEGF, EG-VEGF and its receptors; PROKR1 and PROKR2 as well as Ki67, CD31 and CD34 in OSCC and Epulis patients. Results Both EG-VEGF and VEGF circulating levels were significantly decreased in the HNC (P < .01). OSCC patients expressed less EG-VEGF and VEGF proteins, higher PROKR1 and PROKR2 with no change in CD31 and CD34 levels. A significant increase in Ki67 was observed in OSCC. Conclusions We demonstrated that circulating VEGF and EG-VEGF are downregulated in HNC patients and in OSCC tissue. EG-VEGF receptors were increased in OSCC, along with a stabilisation of two key markers of angiogenesis. These findings strongly suggest that downregulation of angiogenesis in HNC might explain its moderate metastatic feature

Prokineticins in central and peripheral control of human reproduction.

International audienceProkineticin 1 (PROK1) and (PROK2), are two closely related proteins that were identified as the mammalian homologs of their two amphibian homologs, mamba intestinal toxin (MIT-1) and Bv8. PROKs activate two G-protein linked receptors (prokineticin receptor 1 and 2, PROKR1 and PROKR2). Both PROK1 and PROK2 have been found to regulate a stunning array of biological functions. In particular, PROKs stimulate gastrointestinal motility, thus accounting for their family name "prokineticins". PROK1 acts as a potent angiogenic mitogen, thus earning its other name, endocrine gland-derived vascular endothelial factor. In contrast, PROK2 signaling pathway has been shown to be a critical regulator of olfactory bulb morphogenesis and sexual maturation. During the last decade, strong evidences established the key roles of prokineticins in the control of human central and peripheral reproductive processes. PROKs act as main regulators of the physiological functions of the ovary, uterus, placenta, and testis, with marked dysfunctions in various pathological conditions such as recurrent pregnancy loss, and preeclampsia. PROKs have also been associated to the tumor development of some of these organs. In the central system, prokineticins control the migration of GnRH neurons, a key process that controls reproductive functions. Importantly, mutations in PROK2 and PROKR2 are associated to the development of Kallmann syndrome, with direct consequences on the reproductive system. This review describes the finely tuned actions of prokineticins in the control of the central and peripheral reproductive processes. Also, it discusses future research directions for the use of these cytokines as diagnostic markers for several reproductive diseases

RGD-Based Fluorescence to Assess Placental Angiogenesis

International audienc

A novel anti-CD146 antibody specifically targets cancer cells by internalizing the molecule

International audienceCD146 is an adhesion molecule present on many tumors (melanoma, kidney, pancreas, breast, ...). In addition, it has been shown to be expressed on vascular endothelial and smooth muscle cells. Generating an antibody able to specifically recognize CD146 in cancer cells (designated as tumor CD146), but not in normal cells, would thus be of major interest for targeting tumor CD146 without affecting the vascular system. We thus generated antibodies against the extracellular domain of the molecule produced in cancer cells and selected an antibody that specifically recognizes tumor CD146. This antibody (TsCD146 mAb) was able to detect CD146-positive tumors in human biopsies and in vivo, by PET imaging, in a murine xenograft model. In addition, TsCD146 mAb antibody was able to specifically detect CD146-positive cancer microparticles in the plasma of patients. TsCD146 mAb displayed also therapeutic effects since it was able to reduce the growth of human CD146-positive cancer cells xenografted in nude mice. This effect was due to a decrease in the proliferation and an increase in the apoptosis of CD146-positive cancer cells after TsCD146-mediated internalization of the cell surface CD146. Thus, TsCD146 mAb could be of major interest for diagnostic and therapeutic strategies against CD146-positive tumors in a context of personalized medicine. www.impactjournals.com/oncotarget