title: Functional Analysis of the Adrenomedullin Pathway in Malignant Pleural Mesothelioma.
abstract: BACKGROUND
  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.
authors.affiliation: 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.
authors.forename: Laurent
authors.initials: L
authors.lastname: Greillier
authors.affiliation: Aix-Marseille University, CRO2 UMR 911, Marseille, France; INSERM, CRO2 UMR 911, Marseille, France.
authors.forename: Asma
authors.initials: A
authors.lastname: Tounsi
authors.affiliation: Aix-Marseille University, CRO2 UMR 911, Marseille, France; INSERM, CRO2 UMR 911, Marseille, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Berenguer-Daizé
authors.affiliation: Aix-Marseille University, CRO2 UMR 911, Marseille, France; INSERM, CRO2 UMR 911, Marseille, France.
authors.forename: Nadège
authors.initials: N
authors.lastname: Dussault
authors.affiliation: Aix-Marseille University, CRO2 UMR 911, Marseille, France; INSERM, CRO2 UMR 911, Marseille, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Delfino
authors.affiliation: Aix-Marseille University, CRO2 UMR 911, Marseille, France; INSERM, CRO2 UMR 911, Marseille, France.
authors.forename: Zohra
authors.initials: Z
authors.lastname: Benyahia
authors.affiliation: Aix-Marseille University, CRO2 UMR 911, Marseille, France; INSERM, CRO2 UMR 911, Marseille, France.
authors.forename: Mylène
authors.initials: M
authors.lastname: Cayol
authors.affiliation: Aix-Marseille University, LCP UMR 6264, CROPS, Marseille, France.
authors.forename: Kamel
authors.initials: K
authors.lastname: Mabrouk
authors.affiliation: Assistance Publique Hopitaux de Marseille, Service d'Anatomie et de Cytologie Pathologiques, Marseille, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Garcia
authors.affiliation: 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.
authors.forename: Pierre-Marie
authors.initials: PM
authors.lastname: Martin
authors.affiliation: 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.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Barlesi
authors.affiliation: 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.
authors.forename: L'Houcine
authors.initials: L
authors.lastname: Ouafik
mesh_terms: D053607:Adrenomedullin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D058286:Calcitonin Receptor-Like Protein / Q000235:genetics / Q000378:metabolism; D002465:Cell Movement; D049109:Cell Proliferation; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007124:Immunoenzyme Techniques; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008654:Mesothelioma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D000086002:Mesothelioma, Malignant; D051379:Mice; D008819:Mice, Nude; D009389:Neovascularization, Pathologic; D010997:Pleural Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D058263:Receptor Activity-Modifying Protein 2 / Q000235:genetics / Q000378:metabolism; D058264:Receptor Activity-Modifying Protein 3 / Q000235:genetics / Q000378:metabolism; D058265:Receptors, Adrenomedullin / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D014408:Biomarkers, Tumor; C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; C545503:RAMP2 protein, human; C545504:RAMP3 protein, human; D012333:RNA, Messenger; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058265:Receptors, Adrenomedullin; D053607:Adrenomedullin
keywords: Adrenomedullin; Invasion; Mesothelioma; Neoangiogenesis- and lymphangiogenesis-associated tumors; Tumor growth