Laurent Greillier1, Asma Tounsi2, Caroline Berenguer-Daizé2, Nadège Dussault2, Christine Delfino2, Zohra Benyahia2, Mylène Cayol2, Kamel Mabrouk3, Stéphane Garcia4, Pierre-Marie Martin5, Fabrice Barlesi6, L'Houcine Ouafik5. 1. Aix-Marseille University, CRO2 UMR 911, Marseille, France; INSERM, CRO2 UMR 911, Marseille, France; Assistance Publique Hopitaux de Marseille, Service d'Oncologie Multidisciplinaire et Innovations Thérapeutiques, Marseille, France. Electronic address: laurent.greillier@ap-hm.fr. 2. Aix-Marseille University, CRO2 UMR 911, Marseille, France; INSERM, CRO2 UMR 911, Marseille, France. 3. Aix-Marseille University, LCP UMR 6264, CROPS, Marseille, France. 4. Assistance Publique Hopitaux de Marseille, Service d'Anatomie et de Cytologie Pathologiques, Marseille, France. 5. Aix-Marseille University, CRO2 UMR 911, Marseille, France; INSERM, CRO2 UMR 911, Marseille, France; Assistance Publique Hopitaux de Marseille, Service de Transfert d'Oncologie Biologique, Marseille, France. 6. Aix-Marseille University, CRO2 UMR 911, Marseille, France; INSERM, CRO2 UMR 911, Marseille, France; Assistance Publique Hopitaux de Marseille, Service d'Oncologie Multidisciplinaire et Innovations Thérapeutiques, Marseille, France.
Abstract
INTRODUCTION: Malignant pleural mesothelioma (MPM) grows aggressively within the thoracic cavity and has a very low cure rate, thus highlighting the need for identification of new therapeutic targets. Adrenomedullin (AM) is a multifunctional peptide that is highly expressed in several tumors and plays an important role in angiogenesis and tumor growth after binding to its receptors, calcitonin receptor-like receptor/receptor activity-modifying protein 2 (CLR/RAMP2) and calcitonin receptor-like receptor/receptor activity-modifying protein 3 (CLR/RAMP3). METHODS: Real time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the steady-state levels of AM, CLR, RAMP2 and RAMP3 messenger RNA (mRNA) transcripts in normal pleural tissue (n=5) and MPM (n=24). The expression of these candidates at protein level was revealed by immunohistochemistry. We also characterized the expression and regulation by hypoxia of AM system in MPM cell lines and MeT-5A cells. In vitro and in vivo studies were performed to determine the functional role of AM system in MPM. RESULTS: In this study, real-time quantitative reverse transcriptase polymerase chain reaction showed twofold to 10-fold higher levels of AM messenger RNA in MPM tissue than in normal pleural tissue. The MPM cell lines H2452, H2052, and human mesothelioma cell line MSTO-211H showed a significant increase in expression of AM messenger RNA under hypoxic conditions. Our results also show that AM stimulates cell proliferation in vitro through the Raf1 proto-oncogene, serine/threonine kinase (CRAF)/ Mitogen-activated protein kinase kinase 1 (MEK)/Extracellular regulated MAPKinase (ERK) pathway. Furthermore, the proliferation, migration, and invasion of MPM cells were decreased after treatment with anti-AM (αAM) and anti-AM receptor antibodies, thus indicating that MPM cells are regulated by AM. The action of AM was specific and mediated by CLR/RAMP2 and CLR/RAMP3 receptors. In vivo, αAM and AM22-52 antagonist therapies blocked angiogenesis and induced apoptosis in MSTO-211H xenografts, thereby resulting in tumor regression. Histologic examination of tumors treated with AM22-52 and αAM antibody showed evidence of disruption of tumor vasculature with depletion of vascular endothelial cells and a significant decrease in lymphatic endothelial cells. CONCLUSIONS: Our findings highlight the importance of the AM pathway in growth of MPM and in neovascularization by supplying and amplifying signals that are essential for pathologic neoangiogenesis and lymphangiogenesis.
INTRODUCTION:Malignant pleural mesothelioma (MPM) grows aggressively within the thoracic cavity and has a very low cure rate, thus highlighting the need for identification of new therapeutic targets. Adrenomedullin (AM) is a multifunctional peptide that is highly expressed in several tumors and plays an important role in angiogenesis and tumor growth after binding to its receptors, calcitonin receptor-like receptor/receptor activity-modifying protein 2 (CLR/RAMP2) and calcitonin receptor-like receptor/receptor activity-modifying protein 3 (CLR/RAMP3). METHODS: Real time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the steady-state levels of AM, CLR, RAMP2 and RAMP3 messenger RNA (mRNA) transcripts in normal pleural tissue (n=5) and MPM (n=24). The expression of these candidates at protein level was revealed by immunohistochemistry. We also characterized the expression and regulation by hypoxia of AM system in MPM cell lines and MeT-5A cells. In vitro and in vivo studies were performed to determine the functional role of AM system in MPM. RESULTS: In this study, real-time quantitative reverse transcriptase polymerase chain reaction showed twofold to 10-fold higher levels of AM messenger RNA in MPM tissue than in normal pleural tissue. The MPM cell lines H2452, H2052, and humanmesothelioma cell line MSTO-211H showed a significant increase in expression of AM messenger RNA under hypoxic conditions. Our results also show that AM stimulates cell proliferation in vitro through the Raf1 proto-oncogene, serine/threonine kinase (CRAF)/ Mitogen-activated protein kinase kinase 1 (MEK)/Extracellular regulated MAPKinase (ERK) pathway. Furthermore, the proliferation, migration, and invasion of MPM cells were decreased after treatment with anti-AM (αAM) and anti-AM receptor antibodies, thus indicating that MPM cells are regulated by AM. The action of AM was specific and mediated by CLR/RAMP2 and CLR/RAMP3 receptors. In vivo, αAM and AM22-52 antagonist therapies blocked angiogenesis and induced apoptosis in MSTO-211H xenografts, thereby resulting in tumor regression. Histologic examination of tumors treated with AM22-52 and αAM antibody showed evidence of disruption of tumor vasculature with depletion of vascular endothelial cells and a significant decrease in lymphatic endothelial cells. CONCLUSIONS: Our findings highlight the importance of the AM pathway in growth of MPM and in neovascularization by supplying and amplifying signals that are essential for pathologic neoangiogenesis and lymphangiogenesis.
Authors: Jean-Olivier Zirimwabagabo; Ameera B A Jailani; Paris Avgoustou; Matthew J Tozer; Karl R Gibson; Paul A Glossop; James E J Mills; Roderick A Porter; Paul Blaney; Ning Wang; Timothy M Skerry; Gareth O Richards; Joseph P A Harrity Journal: J Med Chem Date: 2021-03-05 Impact factor: 7.446