Biology of Human Tumors Antagonism of EG-VEGF Receptors as Targeted Therapy for Choriocarcinoma Progression In Vitro and In Vivo Wael Traboulsi 1,2,3 , Fr  ed eric Sergent 1,2,3 , Houssine Boufettal 4 , Sophie Brouillet 1,2,3,5 , Rima Slim 6 , Pascale Hoffmann 1,2,3,5 , Mohammed Benlahﬁd 4 , Qun Y. Zhou 7 , Gianfranco Balboni 8 , Valentina Onnis 8 , Pierre A. Bolze 9,10,11 , Aude Salomon 1,2,3 , Philippe Sauthier 6 , Fran¸ cois Mallet 11,12 , Touria Aboussaouira 4 , Jean J. Feige 1,2,3 , Mohamed Benharouga 2,3,13 , and Nadia Alfaidy 1,2,3 Abstract Purpose: Choriocarcinoma (CC) is the most malignant gesta- tional trophoblastic disease that often develops from complete hydatidiform moles (CHM). Neither the mechanism of CC devel- opment nor its progression is yet characterized. We recently identiﬁed endocrine gland–derived vascular endothelial growth factor (EG-VEGF) as a novel key placental growth factor that controls trophoblast proliferation and invasion. EG-VEGF acts via two receptors, PROKR1 and PROKR2. Here, we demonstrate that EG-VEGF receptors can be targeted for CC therapy. Experimental Design: Three approaches were used: (i) a clin- ical investigation comparing circulating EG-VEGF in control (n ¼ 20) and in distinctive CHM (n ¼ 38) and CC (n ¼ 9) cohorts, (ii) an in vitro study investigating EG-VEGF effects on the CC cell line JEG3, and (iii) an in vivo study including the development of a novel CC mouse model, through a direct injection of JEG3- luciferase into the placenta of gravid SCID-mice. Results: Both placental and circulating EG-VEGF levels were increased in CHM and CC (5) patients. EG-VEGF increased JEG3 proliferation, migration, and invasion in two-dimensional (2D) and three-dimensional (3D) culture systems. JEG3 injec- tion in the placenta caused CC development with large metas- tases compared with their injection into the uterine horn. Treatment of the animal model with EG-VEGF receptor's antagonists signiﬁcantly reduced tumor development and progression and preserved pregnancy. Antibody-array and immunohistological analyses further deciphered the mecha- nism of the antagonist's actions. Conclusions: Our work describes a novel preclinical animal model of CC and presents evidence that EG-VEGF receptors can be targeted for CC therapy. This may provide safe and less toxic therapeutic options compared with the currently used multi-agent chemotherapies. Clin Cancer Res; 23(22); 7130–40. Ó2017 AACR. Introduction Gestational choriocarcinoma (CC) is a malignant trophoblas- tic tumor that develops upon normal or abnormal pregnancy. The latter includes complete hydatidiform moles (CHM) or partial hydatidiform moles (PHM), spontaneous abortions, or ectopic pregnancies (1, 2). CHM develops when one or two spermatozoa fertilize an oocyte in the absence of the maternal nucleus, while PHM results from dispermic fertilization of a nucleated oocyte (1, 2). Both CHM and PHM patients are at high risk of developing CC, nevertheless this risk is much higher after CHM (20%) than after PHM (1.5%; refs. 1, 2). CC has an estimated incidence of 2 to 7 in 100,000 pregnancies in North America and Europe. This incidence is higher in Africa (3, 4) and even higher in Asia, where it reaches 5 to 202 in 100,000 pregnancies (5, 6). CC is highly metastatic due to the intrinsic invasive property of trophoblast cells (7). Most patients with non-metastatic gestational CC are successfully treated with single-agent chemotherapy (8–10). Metastatic cases are only curable using multi-agent chemothera- py, known to induce considerable adverse effects (10–12). CC diagnosis and progression is based on the measurement of the human chorionic gonadotropin (hCG) released by syncytiotro- phoblast cells (1, 2). Nevertheless, hCG measurements have recently been reported to be associated with false positive diag- noses and to unnecessary invasive therapeutic procedures, 1 Institut National de la Sant e et de la Recherche M edicale, Unit e Grenoble, Grenoble, France. 2 University Grenoble-Alpes, Grenoble, France. 3 Commissariat  a l'Energie Atomique et aux Energies Alternatives (CEA), Biosciences and Biotechnology Institute of Grenoble, Grenoble, France. 4 Faculty of Medicine and Pharmacy, University Hassan II Casablanca and Ibn Rochd Hospital of Casablanca, Obstetrics and Gynecology Department, Casablanca, Morocco. 5 University Hospital of Gre- noble, Department of Obstetrics and Gynaecology, and Laboratoire d'Aide  a la Procr eation-CECOS, La Tronche, France. 6 Department of Human Genetics, McGill University Health Centre Research Institute, Montr eal, Quebec, Canada. 7 Depart- ment of Pharmacology, University of California, Irvine, California. 8 Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy. 9 University of Lyon 1, University Hospital Lyon Sud, Department of Gynecological Surgery and Oncology, Obstetrics, Lyon, France. 10 French Reference Center for Gestational Trophoblastic Diseases, University Hospital Lyon Sud, Chemin du Grand Revoyet, Pierre B enite, Lyon, France. 11 Joint Unit Hospices Civils de Lyon-bioMerieux, Cancer Biomarkers Research Group, University Hospital Lyon Sud, Lyon, France. 12 EA 7426 Pathophysiology of Injury-induced Immunosuppression, University of Lyon 1 Hos- pices Civils de Lyon bioM erieux, H^ opital Edouard Herriot, Lyon, France. 13 Centre National de la Recherche Scientiﬁque, Unit e Mixte de Recherche, Laboratoire de Chimie et Biologie des M etaux, Grenoble, France. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Nadia Alfaidy, INSERM U1036, 17 rue des Martyrs, Grenoble 38054, France. Phone: 0033632073234; Fax: 0033438785058; E-mail: nadia.alfaidy-benharouga@cea.fr or alfaidynadia@gmail.com doi: 10.1158/1078-0432.CCR-17-0811 Ó2017 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 23(22) November 15, 2017 7130 on June 15, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst September 12, 2017; DOI: 10.1158/1078-0432.CCR-17-0811