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PDF file 1160K, AM is a growth factor survival for LECs

PDF file 114K, described the legends of all the supplementary figures

Purpose: To study the role of the adrenomedullin system [adrenomedullin and its receptors (AMR), CLR, RAMP2, and RAMP3] in prostate cancer androgen-independent growth.Experimental Design: Androgen-dependent and -independent prostate cancer models were used to investigate the role and mechanisms of adrenomedullin in prostate cancer hormone-independent growth and tumor-associated angiogenesis and lymphangiogenesis.Results: Adrenomedullin and AMR were immunohistochemically localized in the carcinomatous epithelial compartment of prostate cancer specimens of high grade (Gleason score >7), suggesting a role of the adrenomedullin system in prostate cancer growth. We used the androgen-independent Du145 cells, for which we demonstrate that adrenomedullin stimulated cell proliferation in vitro through the cAMP/CRAF/MEK/ERK pathway. The proliferation of Du145 and PC3 cells is decreased by anti-adrenomedullin antibody (αAM), supporting the fact that adrenomedullin may function as a potent autocrine/paracrine growth factor for prostate cancer androgen-independent cells. In vivo, αAM therapy inhibits the growth of Du145 androgen-independent xenografts and interestingly of LNCaP androgen-dependent xenografts only in castrated animals, suggesting strongly that adrenomedullin might play an important role in tumor regrowth following androgen ablation. Histologic examination of αAM-treated tumors showed evidence of disruption of tumor vascularity, with depletion of vascular as well as lymphatic endothelial cells and pericytes, and increased lymphatic endothelial cell apoptosis. Importantly, αAM potently blocks tumor-associated lymphangiogenesis, but does not affect established vasculature and lymphatic vessels in normal adult mice.Conclusions: We conclude that expression of adrenomedullin upon androgen ablation in prostate cancer plays an important role in hormone-independent tumor growth and in neovascularization by supplying/amplifying signals essential for pathologic neoangiogenesis and lymphangiogenesis. Clin Cancer Res; 19(22); 6138–50. ©2013 AACR.

PDF file 97K, AM induced activation of MAPK pathway and Normal Vascularization is not disrupted by {alpha}AM treatment

Introduction: Metastatic melanoma is an aggressive tumor and can constitute a real therapeutic challenge despite the significant progress achieved with targeted therapies and immunotherapies, thus highlighting the need for the identification of new therapeutic targets. Adrenomedullin (AM) is a peptide with significant expression in multiple types of tumors and is multifunctional. AM impacts angiogenesis and tumor growth and binds to calcitonin receptor-like receptor/receptor activity-modifying protein 2 or 3 (CLR/RAMP2; CLR/RAMP3). Methods: In vitro and in vivo studies were performed to determine the functional role of AM in melanoma growth and tumor-associated angiogenesis and lymphangiogenesis. Results: In this study, AM and AM receptors were immunohistochemically localized in the tumoral compartment of melanoma tissue, suggesting that the AM system plays a role in melanoma growth. We used A375, SK-MEL-28, and MeWo cells, for which we demonstrate an expression of AM and its receptors; hypoxia induces the expression of AM in melanoma cells. The proliferation of A375 and SK-MEL-28 cells is decreased by anti-AM antibody (αAM) and anti-AMR antibodies (αAMR), supporting the fact that AM may function as a potent autocrine/paracrine growth factor for melanoma cells. Furthermore, migration and invasion of melanoma cells increased after treatment with AM and decreased after treatment with αAMR, thus indicating that melanoma cells are regulated by AM. Systemic administration of αAMR reduced neovascularization of in vivo Matrigel plugs containing melanoma cells, as demonstrated by reduced numbers of vessel structures, which suggests that AM is one of the melanoma cells-derived factors responsible for endothelial cell-like and pericyte recruitment in the construction of neovascularization. In vivo, αAMR therapy blocked angiogenesis and lymphangiogenesis and decreased proliferation in MeWo xenografts, thereby resulting in tumor regression. Histological examination of αAMR-treated tumors showed evidence of the disruption of tumor vascularity, with depletion of vascular endothelial cells and a significant decrease in lymphatic endothelial cells. Conclusions: The expression of AM by melanoma cells promotes tumor growth and neovascularization by supplying/amplifying signals for neoangiogenesis and lymphangiogenesis.

International audience; 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 protooncogene, serine/threonine kinase (CRAF)/Mitogen-activated protein kinase kinase 1 (MEIC)/Extracellular regulated MAPKinase (ERK) pathway. Furthermore, the proliferation, migration, and invasion of MPM cells were decreased after treatment with anti-AM (alpha AM) 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, aAM and AM(22-52) antagonist therapies blocked angiogenesis and induced apoptosis in MSTO-211H xenografts, thereby resulting in tumor regression. Histologic examination of tumors treated with AM(22-52) and aAM 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.

// Zohra Benyahia 1 , Nadege Dussault 1 , Mylene Cayol 1 , Romain Sigaud 1 , Caroline Berenguer-Daize 1 , Christine Delfino 1 , Asma Tounsi 1 , Stephane Garcia 2 , Pierre-Marie Martin 1 , Kamel Mabrouk 3 , L’Houcine Ouafik 1, 4 1 Aix Marseille Univ, The Institut National pour la Recherche Medicale, Centre de Recherche en Oncologie et Oncopharmacologie, UMR 911, 13005, Marseille, France 2 Assistance Publique Hopitaux de Marseille, Laboratoire d’Anatomie Pathologique, 13015, Marseille, France 3 Aix Marseille Univ, CNRS, ICR, UMR 7273 CROPS, 13397, Marseille, France 4 Assistance Publique Hopitaux de Marseille, Service de Transfert d’Oncologie Biologique, 13015, Marseille, France Correspondence to: L’Houcine Ouafik email: lhoucine.ouafik@univ-amu.fr Keywords: adrenomedullin, breast cancer, myofibroblasts, invasion, tumor growth Received: July 20, 2016 Accepted: January 03, 2017 Published: February 02, 2017 ABSTRACT Tumor- or cancer-associated fibroblasts (TAFs or CAFs) are active players in tumorigenesis and exhibit distinct angiogenic and tumorigenic properties. Adrenomedullin (AM), a multifunctional peptide plays an important role in angiogenesis and tumor growth through its receptors calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). We show that AM and AM receptors mRNAs are highly expressed in CAFs prepared from invasive breast carcinoma when compared to normal fibroblasts. Immunostaining demonstrates the presence of immunoreactive AM and AM receptors in the CAFs (n = 9). The proliferation of CAFs is decreased by anti-AM antibody (αAM) and anti-AM receptors antibody (αAMR) treatment, suggesting that AM may function as a potent autocrine/paracrine growth factor. Systemic administration of αAMR reduced neovascularization of in vivo Matrigel plugs containing CAFs as demonstrated by reduced numbers of the vessel structures, suggesting that AM is one of the CAFs-derived factors responsible for endothelial cell-like and pericytes recruitment to built a neovascularization. We show that MCF-7 admixed with CAFs generated tumors of greater volume significantly different from the MCF-7 xenografts in nude mice due in part to the induced angiogenesis. αAMR and AM 22-52 therapies significantly suppressed the growth of CAFs/MCF-7 tumors. Histological examination of tumors treated with AM 22-52 and aAMR showed evidence of disruption of tumor vasculature with depletion of vascular endothelial cells, induced apoptosis and decrease of tumor cell proliferation. Our findings highlight the importance of CAFs-derived AM pathway in growth of breast carcinoma and in neovascularization by supplying and amplifying signals that are essential for pathologic angiogenesis.

// Ghizlane Khalfaoui-Bendriss 1, 2, * , Nadege Dussault 1, 2, * , Samantha Fernandez-Sauze 1, 2 , Caroline Berenguer-Daize 1, 2 , Romain Sigaud 1, 2 , Christine Delfino 1, 2 , Mylene Cayol 1, 2 , Philippe Metellus 1, 2 , Olivier Chinot 1, 2 , Kamel Mabrouk 3 , Pierre-Marie Martin 1, 2, 4 , L’Houcine Ouafik 1, 2, 4 1 Aix Marseille Universite, CRO2, UMR_S 911, Faculte de Medecine, Marseille, France 2 Inserm, U911-CRO2, Marseille, France 3 Aix-Marseille Universite, CNRS, UMR 7273, Institut de Chimie Radicalaire (ICR) Marseille, France 4 AP-HM, CHU Nord, Service de Transfert d’Oncologie Biologique, Marseille, France * These authors have contributed equally to this work Correspondence to: L’Houcine Ouafik, e-mail: lhoucine.ouafik@univ-amu.fr Keywords: adrenomedullin, tumor neovessels, angiogenesis, VE-cadherin, β-catenin Received: November 27, 2014 Accepted: January 19, 2015 Published: February 05, 2015 ABSTRACT The cellular and molecular mechanisms by which adrenomedullin (AM) blockade suppresses tumor neovessels are not well defined. Herein, we show that AM blockade using anti-AM and anti-AM receptors antibodies targets vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), and induces regression of unstable nascent tumor neovessels. The underlying mechanism involved, and shown in vitro and in vivo in mice, is the disruption of the molecular engagement of the endothelial cell-specific junctional molecules vascular endothelial-cadherin (VE-cadherin)/β-catenin complex. AM blockade increases endothelial cell permeability by inhibiting cell-cell contacts predominantly through disruption of VE-cadherin/β-catenin/Akt signalling pathway, thereby leading to vascular collapse and regression of tumor neovessels. At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr 731 , which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function. Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr 416 , supporting a role of Src to phosphorylate Tyr 731 -VE-cadherin. In this model, Src inhibition impairs αAM and αAMR-induced Tyr 731 -VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr 731 -VE-cadherin phosphorylation state is dependent on Src activation. We found that AM blockade induces β-catenin phosphorylation on Ser 33 /Ser 37 /Thr 41 sites in both ECs and VSMCs both in vitro and in vivo in mice. These data suggest that AM blockade selectively induces regression of unstable tumor neovessels, through disruption of VE-cadherin signalling. Targeting AM system may present a novel therapeutic target to selectively disrupt assembly and induce regression of nascent tumor neovessels, without affecting normal stabilized vasculature.

Purpose: To study the role of the adrenomedullin system [adrenomedullin and its receptors (AMR), CLR, RAMP2, and RAMP3] in prostate cancer androgen-independent growth. Experimental Design: Androgen-dependent and -independent prostate cancer models were used to investigate the role and mechanisms of adrenomedullin in prostate cancer hormone-independent growth and tumor-associated angiogenesis and lymphangiogenesis. Results: Adrenomedullin and AMR were immunohistochemically localized in the carcinomatous epithelial compartment of prostate cancer specimens of high grade (Gleason score >7), suggesting a role of the adrenomedullin system in prostate cancer growth. We used the androgen-independent Du145 cells, for which we demonstrate that adrenomedullin stimulated cell proliferation in vitro through the cAMP/CRAF/MEK/ERK pathway. The proliferation of Du145 and PC3 cells is decreased by anti-adrenomedullin antibody (αAM), supporting the fact that adrenomedullin may function as a potent autocrine/paracrine growth factor for prostate cancer androgen-independent cells. In vivo, αAM therapy inhibits the growth of Du145 androgen-independent xenografts and interestingly of LNCaP androgen-dependent xenografts only in castrated animals, suggesting strongly that adrenomedullin might play an important role in tumor regrowth following androgen ablation. Histologic examination of αAM-treated tumors showed evidence of disruption of tumor vascularity, with depletion of vascular as well as lymphatic endothelial cells and pericytes, and increased lymphatic endothelial cell apoptosis. Importantly, αAM potently blocks tumor-associated lymphangiogenesis, but does not affect established vasculature and lymphatic vessels in normal adult mice. Conclusions: We conclude that expression of adrenomedullin upon androgen ablation in prostate cancer plays an important role in hormone-independent tumor growth and in neovascularization by supplying/amplifying signals essential for pathologic neoangiogenesis and lymphangiogenesis. Clin Cancer Res; 19(22); 6138–50. ©2013 AACR.

Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Glioblastoma (GBM) and pilocytic astrocytoma (PA), both angiogenic tumours display strong contrast enhancement associated with peripheral oedema in GBM but not in PA indicating differences in vascular permeability in these two types of gliomas. Here we show that expression of adrenomedullin (AM) mRNA is induced in GBM whereas is barely detectable in PA. In situ analysis of tumour specimens undergoing neovascularisation shows that the production of AM is specifically induced in a subset of GBM cells distinguished by their immediate proximity to necrotic foci (presumably hypoxic regions), suggesting a hypoxic induction of AM expression in GBM. Vascular endothelial growth factor (VEGF) mRNA levels are increased in GBM and moderate in PA. Immunohistochemical study showed that cytoplasmic AM, VEGF and HIF-1α nuclear immunoreactivity were recorded in GBM located near large necrotic areas whereas they were not expressed by PA tumour cells. Interestingly, double fluorescence immunostaining demonstrated that 85% of AM immunoreactivity colocalised with VEGF. AM transduces its effects through calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). Real-time quantitative RT-PCR showed expression of RAMP2, RAMP3 and CLR in PA and GBM, suggesting that AM may function as an autocrine/paracrine growth factor for GBM cells. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify beside VEGF, AM as a potential tumour angiogenesis factor in vivo which constitutes a potential interesting molecular target in GBM treatment.

Rationnel L’AM est une proteine impliquee dans la croissance tumorale et l’angiogenese de plusieurs cancers. Ces phenomenes sont actives par la fixation de l’AM a des recepteurs membranaires (heterodimeres CLR/RAMP2 et CLR/RAMP3), exprimes par les cellules tumorales et endotheliales. Nos precedents travaux montrent que l’AM et ses recepteurs sont significativement plus exprimes dans les biopsies pleurales de MPM que dans le tissu pleural normal (Greillier et al, CPLF 2012). L’objectif de cette etude etait d’evaluer sur le plan fonctionnel la voie de l’AM dans le MPM. Methodes Deux lignees cellulaires de MPM (H2452 et MSTO-211H) ont ete utilisees pour des experimentations in vitro et in vivo (xenogreffes sur modele murin). Resultats In vitro, les capacites de proliferation, de migration et d’invasion des cellules neoplasiques sont significativement augmentees par l’AM, et significativement reduites par les anticorps inhibiteurs, ciblant l’AM ou ses recepteurs. In vivo, ces memes anticorps diminuent de maniere significative la croissance tumorale des xenogreffes. L’analyse histologique de ces dernieres montre une rarefaction des cellules neoplasiques et un remaniement de la vascularisation tumorale, avec une depletion en cellules endotheliales et en pericytes. Conclusions L’expression conjointe de l’AM et de ses recepteurs suggerait l’existence d’une boucle autocrine/paracrine impliquee dans le developpement du MPM. Cette hypothese est confortee par les resultats obtenus in vitro et in vivo. L’inhibition de la voie de l’AM apparait comme une cible therapeutique prometteuse, de part son effet anti-tumoral direct et son effet anti-angiogenique.

Adrenomedullin (AM) is a multifunctional regulatory peptide with important angiogenic and mitogenic properties. Here we identify a region of stable secondary structure in the 5'-untranslated region (5' UTR) of human AM mRNA. Reverse transcriptase-polymerase chain reaction of the 5' UTR consistently resulted, in addition to the product with the expected size of 155 base pair (bp), in a second product with an approximately 65-bp deletion from the central region of the 5' UTR, suggesting the presence of a secondary structure. The presence of a stem-loop structure was confirmed by probing the 5' UTR with RNases with selectivity for single- or double-stranded RNA. We investigated the role of this stem-loop structure in expression of luciferase reporter gene in cultured cell lines. Reporter assays using a chimeric mRNA that combined luciferase and the 5' UTR of AM mRNA demonstrated a dramatic decrease of the reporter activity owing to a decreased translation, whereas the deletion of the stem-loop structure localized between nt +31 and +95 from the cap site led to the recovery of activity. Gel migration shift assays using cytosolic extracts from mammalian cell lines demonstrate a specific binding of a cytosolic protein to riboprobes containing the 5' UTR of AM but not to riboprobes either corresponding to other areas of the message or containing the 5' UTR but lacking the region of secondary structure. Although we conclude that the 5' UTR of the human AM mRNA can modulate the translation of AM mRNA in vivo, and that the predicted stem-loop structure is necessary for this inhibition, the functional consequences of the cis element-binding activity remain to be determined.

Udgivelsesdato: 2005-Sep Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3' untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3' UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3' UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3' UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules.