Functional Analysis of the Adrenomedullin Pathway in Malignant Pleural Mesothelioma

Our plain-English summary of Functional Analysis of the Adrenomedullin Pathway in Malignant Pleural Mesothelioma, by L. Greillier, A. Tounsi, C. Berenguer-Daizé, et al (Journal of Thoracic Oncology, 2016).

The study

• Authors: L. Greillier, A. Tounsi, C. Berenguer-Daizé, N. Dussault, C. Delfino, Z. Benyahia, M. Cayol, K. Mabrouk, S. Garcia, P.M. Martin, F. Barlesi, L. Ouafik
• Journal: Journal of Thoracic Oncology
• Published: 2016;11(1):94-107
• Cited by: 14 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

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.

Copyright © 2015 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

In plain English

This laboratory study, by researchers in Marseille, France, examined adrenomedullin (AM), a peptide, and its receptors in malignant pleural mesothelioma. It did not study asbestos exposure. The authors compared tumor tissue from 24 patients with mesothelioma with 5 samples of normal pleural tissue. AM messenger RNA levels were twofold to 10-fold higher in the tumor tissue. In mesothelioma cell lines grown in the laboratory, treatment with desferrioxamine, a chemical that mimics low oxygen (hypoxia), raised AM levels. Antibodies that block AM or its receptors reduced cell growth by as much as 40% in one cell line. They also completely blocked invasion in the two cell lines tested. In mice carrying implanted human mesothelioma tumors (MSTO-211H cells), an anti-AM antibody or an AM-blocking peptide significantly inhibited tumor growth. Mean tumor weights were 1.35 g with the antibody and 1.55 g with the peptide, compared with 2.80 g in control mice. Treated tumors showed fewer blood-vessel and lymphatic-vessel cells and more cells undergoing programmed cell death (apoptosis). The authors concluded that the AM system is a relevant target for mesothelioma treatment and warrants further preclinical and clinical research.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study combining analysis of human tumor tissue, experiments on cell lines, and mouse xenograft (implanted human tumor) experiments
• Who was studied: Tissue specimens from 24 patients with malignant pleural mesothelioma treated at Assistance Publique Hopitaux de Marseille (France), compared with 5 normal pleural tissue specimens (dates not given). The study also used the mesothelioma cell lines H2452 and H2052 (epithelial), MSTO-211H (biphasic), and MeT-5A (virus-transformed cells from noncancerous mesothelium), plus 30 male athymic nude mice injected with MSTO-211H cells.
• Exposure: No asbestos exposure was studied. The experimental treatments were AM itself, neutralizing antibodies against AM and its receptors (CLR, RAMP2, RAMP3), and the AM antagonist peptide AM22–52. The cells were also treated with desferrioxamine mesylate (DFX), a chemical that mimics hypoxia. In mice, the anti-AM antibody was given at 12 mg/kg every 3 days, compared with a control antibody.
• Main results:
  • In the human tissue samples, AM messenger RNA was twofold to 10-fold higher in mesothelioma tissue (about 20 to 110 fg/pg GAPDH mRNA) than in normal pleural tissue (about 10 fg/pg GAPDH mRNA).
  • Under chemically induced hypoxia (DFX treatment), AM mRNA rose threefold to fourfold in H2452 cells, twofold to 2.5-fold in MSTO-211H cells, and threefold to fivefold in H2052 cells after 24 hours. MeT-5A cells showed a sevenfold increase only after 48 hours.
  • Compared with a control antibody, anti-AM or anti-AM-receptor antibodies reduced proliferation by as much as 40% in MSTO-211H cells, 30% in H2452 cells, and 30% to 40% in H2052 cells. AM22–52 reduced proliferation by up to 35% in MSTO-211H cells but had no effect on H2452, H2052, or MeT-5A cells.
  • AM significantly increased migration of H2452 cells but not MSTO-211H cells, and did not significantly stimulate invasion of either line. Blocking antibodies inhibited migration of H2452 cells but not MSTO-211H cells, and completely impaired invasion in both.
  • In MSTO-211H and H2452 cells, AM led to prolonged activation (phosphorylation) of the CRAF/MEK/ERK signaling pathway. No such ERK activation by AM was seen in MeT-5A cells.
  • In mice with MSTO-211H tumors, mean tumor weights were 2.80 plus or minus 0.40 g with control antibody, 1.35 plus or minus 0.25 g with anti-AM antibody, and 1.55 plus or minus 0.20 g with AM22–52.
  • Tumors treated with anti-AM antibody or AM22–52 had significantly more cleaved caspase-3-positive (apoptotic) cells. They showed no significant difference in the Ki-67 proliferation index. They had clearly fewer CD31-positive blood-vessel cells and LYVE-1-positive lymphatic cells than control tumors.
• The authors' conclusion: The authors concluded that the AM system is a relevant target for treatment of malignant pleural mesothelioma and warrants additional preclinical and clinical research.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Angiogenesis, Immunohistochemistry, Hypoxia, MeT-5A Cells, Mesothelioma, Apoptosis.

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