Adrenomedullin Blockade Suppresses Growth of Human … · Cancer Therapy: Preclinical Adrenomedullin Blockade Suppresses Growth of Human Hormone–Independent Prostate Tumor Xenograft

Cancer Therapy: Preclinical

Adrenomedullin Blockade Suppresses Growth of HumanHormone–Independent Prostate Tumor Xenograft in Mice

Caroline Berenguer-Daiz�e1,2, Francoise Boudouresque1,2, Cyrille Bastide3, Asma Tounsi1,2,Zohra Benyahia1,2, Julie Acunzo1,2, Nad�ege Dussault1,2, Christine Delfino1,2, Nathalie Baeza1,2,Laurent Daniel1,2, Myl�ene Cayol1,2, Dominique Rossi3, Assou El Battari1,2, Denis Bertin4,Kamel Mabrouk4, Pierre-Marie Martin1,2,5, and L'Houcine Ouafik1,2,5

AbstractPurpose: 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 epi-

thelial compartment of prostate cancer specimens of high grade (Gleason score >7), suggesting a role of theadrenomedullin system inprostate cancer growth.Weused the androgen-independentDu145 cells, forwhich

wedemonstrate 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 (aAM),

supporting the fact that adrenomedullin may function as a potent autocrine/paracrine growth factor for

prostate cancer androgen-independent cells. In vivo, aAM therapy inhibits the growth of Du145 androgen-

independent xenografts and interestingly of LNCaP androgen-dependent xenografts only in castrated

animals, suggesting strongly that adrenomedullinmight play an important role in tumor regrowth following

androgen ablation. Histologic examination of aAM-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, aAM potently blocks tumor-associated lymphangiogen-

esis, 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.

IntroductionProstate cancer is the most diagnosed malignant growth

in men and is the second leading cause of male cancer-related deaths in the majority of Western countries. Thecancerous glandusually containsmultiple independent and

genetically distinct lesions, demonstrating heterogeneity ofthe disease (1, 2). Because pathologic growth of the prostateis controlled largely by steroid androgens, treatment oflocally advanced or metastatic disease relies heavily onhormonal therapies that target the androgen receptor. Amajor limitation of hormonal therapy, however, is that itoffers only temporary relief; the cancer eventually reappearsas an androgen-independent lesion characterized by aggres-sive growth and invasion of distal organs, predominantlythe bone (3). Whether it is clonal expansion with adapta-tion of the substitive pathway, development of androgen-independent prostate cancer shows clearly that factors otherthan, or together with, low levels of androgen must exist toprovide survival and growth instructions to the androgen-independent cells.

Adrenomedullin is a multifunctional peptide with prop-erties ranging from inducing vasorelaxation to acting as aregulator of cellular growth and angiogenesis (4–7). Adre-nomedullin binds and mediates its activity through the

Authors' Affiliations: 1Aix-Marseille Universit�e and 2Insitut national de lasanté et de la recherchemedicale (INSERM), CRO2UMR 911, 13005; 3AP-HM, CHU Nord, Service Urologie, 13015; 4Aix-Marseille Universit�e, LCPUMR 6264, CROPS, 13397; and 5AP-HM, CHU Nord, Service de Transfertd'Oncologie Biologique, 13015, Marseille, France

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: L'Houcine Ouafik, Institut national de la sant�e etde la recherche medicale (INSERM) CRO2 UMR 911, Faculty of Medicine,27 Boulevard Jean MOULIN, 13385 Marseille Cedex 05, France. Phone:491-324-447; Fax: 491-254-232; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-0691

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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G protein–coupled receptor and calcitonin receptor–likereceptor (CLR), with specificity for adrenomedullin beingconferred by the receptor activity modifying protein -2(RAMP2) and -3 (RAMP3; ref. 8). The ability ofCLR/RAMP2and CLR/RAMP3 to respond with high affinity to adreno-medullin implies the existence of two molecularly distinctadrenomedullin receptors referred to as AM1 and AM2

receptors, respectively (9). Adrenomedullin is widelyexpressed in a variety of tumor types (10) and was shownto be mitogenic for many human cancer cell lines in vitro(4, 11–15). Several in vivo studies have shown a reduction oftumor angiogenesis and growth upon the treatment withneutralizing adrenomedullin antibodies (13), adrenome-dullin receptor antagonist (16, 17), or adrenomedullinreceptor interference (14).Others and we have reported the expression of adreno-

medullin in human prostate carcinoma and prostate cancercell lines (11, 18–20). The expression of adrenomedullinincreased in a hormone-independent manner after a pri-mary response to castration in the LuCaPmodel in vivo (21).Adrenomedullin is an angiogenic factor that is induced inthe absence of androgens (22), promotes prostate cancercell growth in vitro and in vivo (22), and is required for themaintenance of the neuroendocrine phenotype in LNCaPcells (22). In the absence of androgen, administration ofadrenomedullin to LNCaP xenograft–bearing animals pro-motes tumor growth, suggesting a pivotal role of adreno-medullin in tumor resurgence following primary responseto androgen ablation (22).These observations suggest that adrenomedullin

expression upon androgen receptors blockade might beinvolved in prostate cancer hormone-independent tumorgrowth. Accordingly, the aim of our study was to inves-tigate the potential role of endogenous adrenomedullin

in the growth of hormone-independent prostate cancerby evaluating the effect of anti-adrenomedullin polyclon-al antibody (aAM) on androgen-insensitive prostate can-cer cells. We demonstrate that adrenomedullin plays asignificant role in the proliferation and invasion of andro-gen-insensitive prostate cancer cells via CLR/RAMP2 andCLR/RAMP3 receptors; and that aAM decreases the pro-liferation and invasion of androgen-independent Du145and PC3 cells in vitro and inhibits the growth of Du145xenografts and LNCaP xenografts (only in castrated mice)by targeting tumor cell growth and tumor-associatedangiogenesis and lymphangiogenesis.

Materials and MethodsHuman prostate specimens

We analyzed human prostate specimens from 8 patientswith prostate cancer of high-grade adenocarcinomas (Glea-son score >7) at the Department of Urology (AP-HM,Marseille, France). Paraffin-embedded tumor specimenswere collected from consenting patients, assigned a deiden-tifying number, and provided by the AP-HM Tumor TissueBank (ISO 9001:2008) in accordance with the protocolapproved by the relevant institutional committees (Aix-Marseille Universit�e). Sections of paraffin-embedded sam-ples (4 mm) of human prostate cancer specimens wereanalyzed for adrenomedullin, CLR, RAMP2, RAMP3, andneuron-specific enolase (NSE) proteins as described previ-ously (11). Optimal dilution for anti-adrenomedullin andanti-CLR antibodies was 1/2,000, anti-RAMP2 antibodywas 1/750, anti-RAMP3 antibody was 1/1,500, and anti-NSE antibody (Dako) was 1/200. As a control for immu-nostaining, the antibodies preabsorbed by human syntheticadrenomedullin peptide (50 mmol/L; Bachem), CLR,RAMP2, and RAMP3 peptides (50 mmol/L, CROPS labora-tory) were used instead of the primary antibodies.

Cell cultureThe human prostate cancer androgen-dependent cell line

LNCaP [American Type Culture Collection (ATCC); CRL-1740] and androgen-independent cell lines Du145 (ATCC;HTB-81) and PC3 (ATCC; CRL-1435), originally authenti-cated by short tandem repeat (STR) analysis by ATCC, wereobtained from ATCC and cultured in RPMI 1640 (Invitro-gen Life Technologies Inc.) as described previously (11).Lymphatic endothelial cells (LEC) were obtained fromLonza and cultured in endothelial basal medium 2(EBM-2) medium supplemented with 2% FBS under moist5% CO2/95% air atmosphere.

Development and characterization of anti-humanadrenomedullin antibody

The adrenomedullin polyclonal antibody was developedagainst theAM1–52 peptide (Bachem) as reported previously(13) and characterized as described (Supplementary Fig.S1). All purified immunoglobulin G (IgG) of anti-adreno-medullin antibody (aAM) and preimmune serum (rabbitcontrol IgG) were affinity purified on rProtein A Sepharose

Translational RelevanceAdrenomedullin and adrenomedullin receptors were

immunohistochemically localized in the carcinomatousepithelial compartment of high-grade adenocarcinomas(Gleason score >7) of prostate cancer specimens, sug-gesting a role of the adrenomedullin system in thegrowth of human hormone–independent prostate can-cer. Adrenomedullin functions as an autocrine/para-crine growth factor to stimulate proliferation of andro-gen-independent Du145 and PC3 cells, whose effect isinhibited by a neutralizing anti-adrenomedullin anti-body causing growth cessation in vitro. The in vivo studyhighlights the significance of adrenomedullin as animportant factor to promote androgen-independentprostate cancer tumor growth and to affect the tumormicroenvironment by inducing pathologic neoangio-genesis and lymphangiogenesis. Targeting the adreno-medullin system may provide a rational basis for futuretherapeutic modalities upon androgen ablation in pros-tate cancer.

aAM Therapy Represses Hormone-Independent Prostate Cancer Tumors

www.aacrjournals.org Clin Cancer Res; 19(22) November 15, 2013 6139




Fast Flow columns (VWR) and tested for endotoxin usingthe Pyrogent plus Limulus Amebocyte Lysate Kit (Lonza).All antibody preparations used in animal studies containedless than 1.25 endotoxin U/mL.

Western blot analysisCell extracts were prepared and immunoblotted for CLR,

RAMP2, and RAMP3 as described previously (22). Immu-noblotting of phospho-CRAF (pCRAF), pMEK1/2, pERK1/2,and ERK1/2 were performed using the MAPK-phospho-ERK1/2 pathway sampler Kit (Cell Signaling Technology,Inc.). Signals were revealed using an enhanced chemilumi-nescence kit (ECL kit; Invitrogen Life Technologies Inc.).

Cyclic AMP assayDu145 cells (3 � 104 cells/mL) treated with adrenome-

dullin (10�7 to 10�9 mol/L) in the presence of I-methyl-3-isobutylxanthine (IBMX; 10�4 mol/L) were prepared tomeasure the intracellular amount of cyclic AMP (cAMP)using the cAMP enzyme immunoassay Biotrak (EIA) Systemaccording to the supplier’s protocol (GE Healthcare).

Transwell migration assaysChemoinvasion of Du145 cells (15� 103) using the filter

coated with a layer of Matrigel (0.5 mg/mL; Becton Dick-inson) in amodified Boyden chamber assay was performedas described in refs. (7, 23). Human umbilical venoussmooth muscle cells (HUVSMC; ScienCell Research Labo-ratories, Clinisciences,)were cultured in smoothmuscle cell(SMC) growthmediumM199 with 20% FBS to confluence.Confluent HUVSMCwere serumdeprived in EBM-2 growthmedium containing 0.5% FBS overnight. 1� 105 cells wereseeded in the upper chamber and allowed tomigrate towardEBM-2 supplemented with 0.5% FBS or conditioned medi-um collected from LECs over a 48-hour period in thepresence or absence of a function-blocking antibody toadrenomedullin or control IgG for 23 hours.

Cell proliferation assayThe effects of adrenomedullin (10�7, 10�8, and 10�9

mol/L), aAM (10, 30, and 70 mg/mL), and control IgG (70mg/mL) on cell proliferationwere examined at the indicatedtime points by cell counting orMTT assay (Z1 series CoulterCounter, Beckman Coulter Inc.) as described in ref. 23.

RNA preparation and quantitative RT-PCRTotal RNAwas extracted fromDu145 cells, PC3 cells, and

LECs as well as Du145 and LNCaP xenografts, reversetranscribed to cDNA, and analyzed for the expression ofadrenomedullin, CLR, RAMP2, RAMP3, GAPDH mRNAs,and 18S rRNA as described in refs. 13 and 22.

Peptide extraction and RIAProtein extracts from Du145 and LNCaP xenografts were

prepared for RIA of immunoreactive adrenomedullin (ir-AM) as previously described (22).

Animal studyAnimal work was carried out in the animal facility of the

School of Medicine according to the institutional animal

welfare guidelines. Athymic NMRI (nu/nu) nude mice(Harlan) were maintained in a sterile environment witha daily 12-hour light/12-hour dark cycle. The subcuta-neous and orthotopic models were developed for Du145cells. The subcutaneous tumors were generated by injec-tion of Du145 cells (5 � 106) in the right flank of maleathymic (NMRI; nu/nu) nude mice (n ¼ 30; Harlan).Tumors were measured with a dial-caliper, and volumeswere determined using the formula width � length �height � 0.5236 (for ellipsoid form; ref. 13). At a tumorvolume of approximately 600 � 100 mm3, animals wererandomly divided into two groups. One group (n ¼ 20)received intraperitoneal injection of the aAM (350 mgof purified IgG equivalent to 12 mg/kg) every 3 days.The amount of aAM was determined on the basis of thedata of preliminary experiments in which increasingamounts of aAM (100, 200, 350, 500, and 800 mg) wereused to determine the best concentration of aAM thatinhibits xenograft growth in vivo. As control, one group(n¼ 10) received a rabbit control IgG (350 mg equivalentto 12 mg/kg) of irrelevant specificity. Mice were sacri-ficed at the indicated time. Tumor size and generalclinical status were recorded every 3 days. For the ortho-topic Du145 xenografts, Du145 cells (1 � 106) wereimplanted orthotopically (n ¼ 20) in the dorsal prostatein nude mice. One month later and after tumor palpa-tion, mice were randomized into two groups and treatedas above.

The subcutaneous LNCaP xenograft tumors were gen-erated by injection of LNCaP cells (8 � 106) mixed at a1:1 dilution with Matrigel (BD Biosciences) as above. Ata tumor volume of approximately 500 mm3, mice (n ¼20) were randomized into two groups, a group of ani-mals (n ¼ 10) was castrated and separated into twogroups that received an intraperitoneal injection of 12mg/kg of aAM (n¼ 5) or control IgG (n¼ 5) every 3 daysfor 4 weeks. The same paradigm was applied to nonca-strated animals.

Immunohistochemical analysisThin (6 mm) sections were incubated with anti-vWF anti-

body (diluted 1:400; Dako) and anti-aSMA antibody (dilut-ed 1:80; Dako) to assess tumor vascularity, or a goat poly-clonal anti-LYVE-1 antibody (diluted1:100; R&Dsystems) toassess the lymphatic vessels, and anti-PDGFR-b antibody(diluted 1:100; eBioscience) to assess mural cells. To assessprogrammed cell death, tissue sections were evaluated usingmAb F7-26 to detect single-strand DNA (ssDNA; EurobioAbCys). Staining was carried out as detailed previously(17, 23).

Statistical analysisData are expressed as mean � SEM from at least three

independent experiments.One-wayANOVAor Fisher PLSDtest (Statview 512; Brain Power Inc.) was used for statisticalanalysis. Differenceswere considered significant at values ofP less than 0.05.

Berenguer-Daiz�e et al.

Clin Cancer Res; 19(22) November 15, 2013 Clinical Cancer Research6140




ResultsExpression of adrenomedullin, CLR, RAMP2, RAMP3,and NSE proteins in human prostate cancer specimensSerial sections of human prostate cancer specimens

(Gleason score >7; Fig. 1) were labeled with antibodiesrevealing NSE, adrenomedullin, CLR, RAMP2, and RAMP3proteins (Fig. 1). Overt NSE labeling of stromal and epi-thelial cells can be observed, suggesting a neuroendocrinedifferentiation of cancer specimen (Fig. 1). Carcinomatousepithelia displayed overt and strong labeling for adreno-medullin, CLR, RAMP2, and RAMP3 (Fig. 1). Dispersedamong the stromal collagen septa, numerous clusters oflabeled stromal cells for adrenomedullin, CLR, RAMP2,and RAMP3 can be observed (Fig. 1). Illustrating the com-plexity of its localization, the adrenomedullin protein waslocalized to either the nucleus and/or the cytoplasm indifferent biopsies (Fig. 1). The same localization for adre-nomedullin was recently reported in serial sections of lungcancer biopsies (24). Positive adrenomedullin stainingwas completely abolished by preabsorption of the antibodywith 50 mmol/L synthetic adrenomedullin peptide (Fig. 1)or CLR, RAMP2, and RAMP3 peptides (not shown). The

other human prostate cancer tissues (n ¼ 7) present thesame staining paradigm for adrenomedullin, CLR, RAMP2,and RAMP3 as shown in Fig. 1. Furthermore, the expressionof adrenomedullin was analyzed with tissue microarray inserial prostate sections from 72 patients with adenocarci-noma.More than79%(57/72) of the biopsieswere stronglypositive for adrenomedullin. Together, these data indicatethat the adrenomedullin system iswell expressed in prostatecancer tissues and might be involved in tumor cell growthin vitro and in vivo.

Exogenous adrenomedullin stimulates Du145 cellgrowth, cAMP activity, and invasion in vitro

The effects of treatmentwith adrenomedullinwere studiedin vitro on androgen-independent Du145 cells that demon-strate expression of CLR, RAMP2, and RAMP3mRNAs (Sup-plementary Fig. S2A). By Western blot analysis, Du145 cellsproduced a CLR as a distinct band of 48 kDa and multimer,presumably heterodimers CLR/RAMP2 (AM1 receptor) orCLR/RAMP3 (AM2 receptor), at 73 to 76 kDa (Supplemen-tary Fig. S2B, lane 1). RAMP2 and RAMP3 were seen as amonomer of 28 kDa andmultimer, presumably homodimer

Figure 1. Representativemicroscopic fields ofadrenomedullin (AM), CLR,RAMP2, RAMP3, and NSEimmunohistochemical analysis inprostate cancer tissues. Section ofprostate cancer tissue is stainedwith hematoxylin and eosin (H&E).Epithelial carcinomacells aswell asstromal cells showedNSE staining.Immunodetection of AM, CLR,RAMP2, and RAMP3 in serialsections of prostate cancerbiopsies. AM, CLR, and RAMP2staining of cells dispersed amongthe stroma can be observed.In a second area of the biopsy,carcinomatous epitheliumdemonstrates AM staininglocalized to either nucleusand/or the cytoplasm (�400).AM immunoreactivity is completelycanceled by the antibodypreabsorbed with 50 mmol/LAM peptide.






at 50 kDa and heterodimer at 73 to 76 kDa (SupplementaryFig. S2B, lanes 3 and 5). These data suggest that Du145 cellscould be sensitive to adrenomedullin stimulus.

We next examined whether adrenomedullin increasesintracellular cAMP, the major second messenger of adre-

nomedullin (25, 26) in cultured Du145 cells. Adrenome-dullin increased the cAMP level in a dose-dependent man-ner with a peak at 5 to 20minutes for the higher concentra-tions and then a sudden decrease at 30 minutes, probablydue to receptor desensitization (Fig. 2A). The cAMP

Figure 2. Adrenomedullin (AM) stimulates Du145 cell proliferation and invasion in vitro. A, AM induced cAMP formation in cultured Du145 cells. Cells weretreated with AM for the indicated times. Bars represent SEM of three independent experiments (��, P < 0.01; ��, P < 0.001). B, opposite effects of AM andaAM on the growth of Du145 cells (2 � 103) in vitro. Bars represent SEM of four independent experiments ( ��, P < 0.001). C, AM stimulates Du145 cellsinvasion in vitro. The bottom wells of all the chambers were filled with RPMI 1640 containing 2% FBS in the presence of control buffer (control),AM (10�7, 10�8, and 10�9 mol/L), aAM (70 mg/mL) in the presence of AM (10�7 mol/L), or bFGF (10�6 mol/L). Du145 cells (15� 103) were placed in the upperchamber and then incubated for 12 hours at 37�C. The migrated cells were stained with 4', 6'-diamidino-2-phenylindole (DAPI) and counted at �200magnification using amicroscope. Bars represent themean�SEMof three independent experiments (��,P < 0.01; ��,P < 0.001). D, the intracellular signalingpathway induced by AM in Du145 cells. Du145 cells treated with AM (10�7 mol/L) for the indicated times and immunoblotted for pCRAF, pMEK1/2, pERK1/2,and ERK1/2. The MEK inhibitor (U0126) inhibits AM-induced phosphorylation of ERK (10 mmol/L, 30 minutes). EGF was used as positive control knownto stimulate phosphorylation of CRAF, MEK1/2, and ERK1/2. Preincubation of Du145 cells with aAM or aAMR for 30 minutes inhibits AM-inducedphosphorylation of ERK1/2. Preincubation of Du145 cells with aAM without additional AM decreases pERK1/2. b-Actin was used as a loading control.






accumulation by adrenomedullin could be mediated by Gprotein, probably Gas, which is involved in many systemsof receptor-operated cAMP increase, or by other mechan-isms, such as facilitating the interaction between activatedGas and adenyl cyclase as demonstrated for other factors.The production of cAMP suggests that adrenomedullinmight be involved in the growth of Du145 cells. Accord-ingly, adrenomedullin significantly stimulates the prolifer-ation of Du145 cells in a dose-dependent manner by 8 daysof treatment (Fig. 2B). To determine whether aAM caninhibit cell growth in vitro, Du145 cells were exposed toincreasing concentrations of aAM at 10, 30, and 70 mg/mL,and the effect on proliferation was assessed by MTT assay.aAM inhibits Du145 cell proliferation in a dose-dependentmanner, reaching 15% (P < 0.05), 28% (P < 0.01), and 52%(P < 0.001), respectively, by 8 days of treatment (Fig. 2B). Incontrast, 70mg/mLof the control IgGof irrelevant specificityshowed no inhibition of cell growth (Fig. 2B).We next analyzed the effect of adrenomedullin onDu145

cells invasion. The additionof adrenomedullin (10�7, 10�8,and 10�9 mol/L) to the bottomwells increased the numberof invading cells in a dose-dependent manner after 12-hourincubation, reaching 95% � 15% (P < 0.001), 60% � 18%(P<0.001), and10%�3%(P<0.01), respectively (Fig. 2C).The induced effect of adrenomedullin on invasion wasinhibited when cells were preincubated with aAM, suggest-ing that the endogenous adrenomedullin secretedbyDu145cells might be involved in the invasion process, presumablyby autocrine/paracrine manner (Fig. 2C). The effect ofadrenomedullin on invasion was similar to that observedwith basic fibroblast growth factor (bFGF; 10�6 mol/L; Fig.2C). Interestingly, prostate hormone-independent PC3 cellsdemonstrate the expression of adrenomedullin and itsreceptors (Supplementary Fig. S3A), inhibition of cellgrowth reaching 50% upon incubation with aAM (Supple-mentary Fig. S3B), induction of migration (SupplementaryFig. S3C), and invasion (Supplementary Fig. S3C) by adre-nomedullin. Together, these data suggest that adrenome-dullin acts as an autocrine/paracrine growth factor to reg-ulate many functions of Du145 and PC3 cells in vitro.

Adrenomedullin mediates phosphorylation of MAPKExtracellular signal-regulated kinase (ERK) and Akt are

known to regulate cell proliferation and this signalingpathway was reported to function downstream of the adre-nomedullin/cAMP pathway (27, 28). Therefore, we inves-tigated the activation of different pathways and found thatadrenomedullin (10�7 mol/L) increased CRAF phosphor-ylation as early as 5 minutes and declines to reach thecontrol levels by 2-hour treatment (Fig. 2D). To investigatemitogen–activated protein kinase (MAPK) activity, wemea-sured the phosphorylation of MAP–ERK kinase (MEK1/2),ERK1 (p44MAPK), and ERK2 (p42MAPK). The levels ofpCRAF, pMEK1/2, and pERK1/2 in Du145 cells wereincreased as early as 5 minutes, respectively, after adreno-medullin (10�7 mol/L) treatment (Fig. 2D). These effectssustained to be higher than control levels for up to 1 hourand decline to control levels by 2-hour treatment (Fig. 2D).

Inhibition of MEK, an immediate upstream activator ofERK1/2, with U0126 (10 mmol/L, 30 minutes) preventedadrenomedullin’s activation of ERK1/2 (Fig. 2D). Preincu-bation of Du145 cells with aAM or aAMR inhibits thestimulus of adrenomedullin on pERK1/2, suggesting thatthe effect of adrenomedullin is specific and is one of thefactors involved in the activation of the MAPK pathway viaCLR/RAMP2 and/or CLR/RAMP3 receptors (Fig. 2D). Inter-estingly, the effects of aAM without additional adrenome-dullin demonstrate a decrease of pERK1/2 as early as 5minutes to barely detectable levels by 2-hour treatment ascompared with control cells, suggesting that adrenomedul-lin is one of the Du145 cell-derived factors involved in theactivation of the MAPK pathway in an autocrine/paracrinemanner (Fig. 2D). Dose–response studies demonstrate thatadrenomedullin (10�6 to 10�9 mol/L) induced a strongphosphorylation of CRAF, MEK1/2, and ERK1/2 by 10-minute treatment (Supplementary Fig. S4). These resultssuggest that adrenomedullin-induced cell proliferation ismediated at least in part by the cAMP/CRAF/MEK/ERKpathway.

Expression of adrenomedullin and its receptors inDu145 and LNCaP xenografts

Toassess the steady-state levels of adrenomedullinmRNAand AMR mRNAs, total RNA was prepared from subcuta-neous and orthotopic Du145 tumors and LNCaP tumorsfrom castrated and noncastrated animals. Quantification ofadrenomedullin mRNA transcripts shows equivalentexpression in subcutaneous and orthotopic Du145 xeno-grafts (Fig. 3A). In LNCaP xenografts, the data show a clearincrease of adrenomedullin mRNA by approximately 5.5�0.3-fold (n ¼ 10; mean � SEM) in castrated animals ascompared with noncastrated animals (P < 0.001; Fig. 3A) aspreviously reported (22).Quantitative PCRofCLR, RAMP2,and RAMP3 mRNAs demonstrate the expression of thesetranscripts in all xenografts analyzed (Fig. 3A). Interestingly,the expression of these transcripts is not regulated byandrogen in LNCaP xenografts in vivo, and the same findingwas observed in LNCaP cells in vitro (22).

RIA demonstrates that castration increased ir-AM levelsby 3.5 � 0.04-fold (n ¼ 10; mean � SEM), being 230 � 15pg/mg protein in castrated animals and 65� 5 pg/mg proteinin noncastrated animals (P < 0.01; Fig. 3B). The amount ofir-AM determined in both types of Du145 xenograftsshowed an average of 170 pg/mg protein (Fig. 3B). Takentogether, these data demonstrate that the adrenomedullinsystem is expressed in both xenografts and that ir-AM, thetarget of aAM, is well expressed in Du145 and LNCaPxenografts in castrated animals.

aAM inhibits growth of androgen-independentprostate cancer tumor xenografts

On the basis of our observations on the effects of adre-nomedullin on Du145 cell growth and invasion in vitro, wefurther wished to analyze the effect of adrenomedullin ontumors developed in vivo in immunodeficient mice. Wesought to determine if adrenomedullin is just a classic






growth factor involved in tumor cell proliferation in vivo, or ifadrenomedullin has a complex role to sustain tumor growthby performing a stable angiogenesis and lymphangiogenesisleading to functional blood and lymphatic vessels. To thisend, we performed a series of experiments in which Du145subcutaneous xenograft tumor–bearing mice were treated

with aAM and control IgG. In the first series of experi-ments, tumor cells were injected subcutaneously in theflanks of athymic mice (nu/nu). Once tumors reached asize of 600 � 100 mm3, mice started to receive intraper-itoneal injection of 12 mg/kg of aAM or control IgG every3 days for 5 consecutive weeks. No signs of toxicity, such

Figure 3. aAM inhibits growth of Du145 and LNCaP xenografts. A, expression of AMand its receptors (CLR, RAMP-2, and -3) in Du145 and LNCaP xenografts.Total RNA (1 mg), DNA-free, fromDu145 xenografts [subcutaneous and orthotopic (ortho)] and LNCaP xenografts fromcastrated (cast) and noncastrated (noncast) animals were reverse transcribed into cDNA and subjected to real-time quantitative reverse transcription (RT)-PCR for the estimation of relative AM,CLR, RAMP2, and RAMP3 mRNAs to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio as described in Materials and Methods(��,P < 0.001). B, extracts prepared fromDu145 xenografts (subcutaneous and orthotopic; n¼ 5 each) and LNCaP xenografts (non cast and cast; n¼ 5 each)were subjected to RIA to determine the ir-AM levels (��, P < 0.01). C, Du145 cells were subcutaneously implanted in athymic nu/nu mice and mice bearingDu145 tumors were treated with aAM or control IgG of irrelevant specificity. The sizes of Du145 xenografts were determined by measuring the volumeof the tumors. Values are mean� SEM. The asterisks indicate that the value is significantly different from the control IgG (��, P < 0.001). Orthotopic prostatetumors were established by injection of Du145 cells in the dorsal lobe of prostate. Tumor weight of aAM and control IgG–treated animals is shownand horizontal lines indicate themean tumor weight in each treatment group (��,P < 0.001). D, the LNCaP cells (8� 106) inMatrigel (v/v) were subcutaneouslyimplanted in nu/numice and 2 months later, when the primary tumors were 400 mm3 in size, animals were randomly divided into two groups. Noncastratedanimals (n ¼ 10) were randomly divided into two groups (n ¼ 5) that received aAM or control IgG by intraperitoneal administration. The castrated animals(n¼ 10) were randomly divided into two groups and treated as earlier. The asterisks indicate that the tumor volume of aAM-treated xenografts is significantlydifferent from control IgG–treated xenografts in castrated animals (��, P < 0.01; ��, P < 0.001).






as weight loss in response to aAM treatment, wereobserved. A clear regression of tumor growth wasobserved in aAM-treated tumors as compared with con-trol IgG–treated tumors (P < 0.001; Fig. 3C). At day 22,tumors in the aAM-treated mice reached a mean size ofapproximately 421 � 179 mm3, whereas the tumors inthe control group exhibited a mean size of approximately2,612 � 300 mm3. At this time of treatment, a group ofanimals was sacrificed; tumor weights were taken andtumor tissues were saved to assess vascularity. The meantumor weights in the control and the aAM-treated ani-mals were 2.5 and 0.6 g, respectively.Because the stromal environment can affect the tumor

growth, we next tested the effect of aAM on tumor growthusing the Du145 orthotopic model. Mice received intra-peritoneal injection of 12 mg/kg of aAM or control IgGthree times a week until sacrifice for analysis (Fig. 3C).Ten weeks after orthotopic tumor-cell implantation, micein the control group seemed sickly and cachectic, char-acterized by sluggishness, an unkempt appearance. At thesame time point, antibody-treated mice seemed activeand maintained normal grooming behavior. After 6weeks of treatment, animals were sacrificed and tumorburden was assessed. Nine of 10 mice treated with controlIgG developed fulminant disease (Fig. 3C). In contrast, 6of 10 aAM-treated animals showed a dramaticallydecreased response, exhibiting only small tumors upongross inspection (P < 0.001; Fig. 3C). Tumor weights weresignificantly lower in aAM-treated animals than in con-trol IgG–treated animals, further documenting tumorsuppression (P < 0.001; Fig. 3C). Pathologic examinationof tissues from control IgG and aAM-treated animalsshowed the presence of metastasis in lung, kidney, andspleen only in control orthotopic animals. No metastasiscan be observed in aAM-treated animals.

aAM represses LNCaP tumor xenograft growth in vivoonly in castrated animals

Treatment of noncastrated animals with aAM showed noinhibition of tumor growth when compared with the con-trol IgG group (Fig. 3D). Following castration, LNCaPxenografts present a hormone-independent growth patterncomparable to one observed for hormone-independentcells such as Du145 cells. To further investigate whetherthe endogenous adrenomedullin expressed upon castration(Fig. 3A) might be involved in tumor growth, intraperito-neal administration of aAM or control IgG was given tocastrated and noncastrated animals. Interestingly, thegrowth of xenografts was significantly decreased by aAMtreatment in castrated animals when compared with thecontrol IgG group, suggesting that adrenomedullin mightplay an important role in the tumor regrowth process afterandrogen ablation (Fig. 3D).

Adrenomedullin blockade depletes endothelial cellsand pericytes in tumors

Immunostaining of subcutaneous and orthotopicDu145tumors with anti-vWF antibody demonstrates that aAM-treated Du145 tumors were significantly less vascularizedthan Du145 control tumors (Fig. 4A and B). Costainingwith anti-vWF and anti-aSMA antibodies demonstrates thatboth cell types are sparce, and the vascularization is deeplydisrupted (Fig. 4A and B; inset). Quantification of vWF-stained endothelial cells and aSMA-stained pericytesdemonstrates a clear decrease of both cell types in aAM-treated tumors when compared with control IgG–treatedDu145 tumors in both xenograft models (P < 0.001; Fig. 4Cand D). In contrast, control IgG–treated Du145 tumorsshowed a well-organized vascularization. Importantly, thevascularization in normal tissues was not disrupted byaAMtreatment (Supplementary Fig. S5).

Figure 4. aAM-treated tumorsare depleted of endothelial cellsand pericytes. A and B, tumorsections from subcutaneous (A)or orthotopic (B) xenografts wereevaluated by immunofluorescencefor vWF (red) and aSMA(green). Tissue sections werecounterstained with 4',6-diamidino-2-phenylindole(DAPI; blue). Scale bar ¼ 50 mm.C and D, quantitative assessmentof cells that stained positivefor vWF in subcutaneous (C)and orthotopic (D) xenograftswas conducted through amicroscope. MBF_ImageJ 1.43U software was usedfor analysis. Values � SEM;n ¼ 6; ��, P < 0.001.






Figure 5. aAM disrupts tumor-associated lymphangiogenesis in orthotopic Du145 prostate tumor xenografts. Du145 cells (1 � 106) were implanted withorthotopic injection into dorsal prostate of athymic mice that were randomly divided into two groups and treated with aAM or control IgG. A, representativeimages of tumors from control and aAM-treated animals. Tumor sections were stained with anti-LYVE-1, or costained with anti-LYVE-1 and anti-aSMAor anti-PDGFR-b to reveal aSMA or PDGFR-b expressing cells in green and LECs in red, respectively. DAPI-stained nuclei are in blue. Pros, prostate; T,tumor tissue. B and C, quantitative assessment of cell density of cells that stained positive for LYVE-1 (B) or PDGFR-b (C) was conducted through amicroscope. MBF_ Image J 1.43U software was used for analysis. Values are mean � SEM; n ¼ 8; ��, P < 0.001. D, migration of HUVSMC towardLEC-conditioned medium (CM) � function-blocking antibody against adrenomedullin (AM). aAM and IgG control were used at 70 mg/mL, AM (10�7 mol/L),and PDGF-BB (5 ng/mL) was used as positive control. The migrated cells were stained with DAPI and counted using microscope. Bars represent themean � SEM of three independent experiments (��, P < 0.01; ��, P < 0.001).






aAM blocks the development of tumor-associatedlymphangiogenesisTo determine the effect of aAM treatment on tumor-

associated lymphangiogenesis, Du145 orthotopic tumortreated with aAM or control IgG were evaluated fortumor-associated lymphatic vessels by immunostaining forLYVE-1 (lymphatic vessel endothelial receptor 1; Fig. 5).aAM-treated tumors showedmarked disruption and reduc-tion of the lymphatic tumor vasculature compared withcontrol IgG–treated tumors (Fig. 5A). Importantly, LYVE-1–positive lymphatic vessels detected in the normal micetissue adjacent to the tumor periphery remained unaffectedby the aAM treatment (Fig. 5A). Furthermore, the smoothmuscle cells surrounding the collecting vessels in tumortissue were eradicated in aAM-treated tumors comparedwith control IgG–treated tumors (Fig. 5A). The immunos-taining for PDGFR-b, a marker of mature periendothelialsupport cells (29), revealed expression of PDGFR-b inperivascular cells that are in close contact with LECs (Fig.5A). The same finding is observed with aSMA, consistentwith the identification of the PDGFR-bþ cells in thesetumors as a class of pericyte. A very few lymphatic vesselswere found without periendothelial support cells (notshown).Quantitative evaluation of the number of lymphat-ic vessels revealed a significant reduction specifically inaAM-treated tumors (P < 0.001; Fig. 5B). No significantdifference can be observed for the number of lymphaticvessels in peritumoral tissues between control IgG andaAM-treated animals, suggesting that aAM treatment doesnot impede preexisting or non–tumor associated lymphaticvessels (Fig. 5B). Quantitative evaluation of the number ofPDGFR-b positive cells revealed an overall reduction ofmural cells of 61% � 2.5% specifically in aAM-treatedtumors compared with control IgG–treated tumors (P <0.01; Fig. 5C). Together, these data are in agreement withthe recently published work that implicates SMCs respon-sible for lymphatic vessel morphogenesis and function(30). To strengthen our findings, we therefore tested thepossibility that adrenomedullin secreted by LECs might beinvolved in the mural cells/pericytes recruitment. Adreno-

medullin receptors are expressed in cultured primaryHUVSMCs (not shown). This suggests that SMC couldtherefore respond to adrenomedullin secreted by LECs toregulate SMC recruitment during collecting vessel forma-tion. The migration assay demonstrates that LEC-condi-tioned medium promoted SMC migration in Transwellassay; this effect was significantly inhibited by a function-blocking antibody to adrenomedullin (Fig. 5D). These datastrongly suggest that adrenomedullin must be one of theLEC-derived factors responsible for SMC recruitment.

The reduction of lymphatic vessels upon aAM treatmentindicates that adrenomedullin is necessary for LECs’ sur-vival, suggesting that aAM treatment may induce LECs’death by apoptosis. The apoptotic cells, as revealed byimmunostaining of ssDNA with monoclonal antibody(mAb) F7-26 antibody (Fig. 6A; inset), were predominantlylocatedwithin the vascular lining and costainingwith LYVE-1 antibody identified them as LECs (Fig. 6B; inset). Theapoptotic index of the aAM-treated tumors reached anapproximately 6-fold increase when compared with controlIgG–treated tumors (P < 0.001; Fig. 6C). Together, thesefindings indicate that aAM treatment can (i) destroy spe-cifically tumor-associated lymphatic vessels and (ii) preventthe tumor from inducing lymphangiogenesis.

DiscussionProgression of prostate cancer toward androgen-inde-

pendent status is an oncologic challenge. The mechanismsresponsible for the tumor progression in androgen-inde-pendent manner are not well understood. Recently, wedemonstrated that adrenomedullin expression is inducedby androgenwithdrawal, suggesting that its productionmaybe important for tumor resurgence following androgenablation (22). To determine the role of the adrenomedullinsystem in androgen-independent tumor growth of prostatecancer, we used in the current study the androgen-indepen-dent Du145 and PC3 cells. Our data showed that adreno-medullin significantly increased Du145 cell proliferation,invasiveness, stimulation of cAMP, and the activation of theCRAF/MEK/ERK/MAPK pathway. We have also shown that

ssDNAA B C

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Figure 6. aAM induces LEC-associated tumor apoptosis.A, microphotographs ofimmunohistochemical-stainedtumor sections for ssDNA usinga mAb F7-26 antibody in controland aAM-treated tumors. B, todetermine cells undergoingapoptosis, tumor sections wereevaluated by immunofluorescencefor LYVE-1 (green) and ssDNA(red). DAPI-stained nuclei are blue.C, percentage of cells undergoingapoptosis was determined usingMBF_Image J 1.43U softwarefor analysis. Values are mean� SEM; n ¼ 6; ��, P < 0.001.






adrenomedullin is involved in the proliferation, migration,and invasion of PC3 cells. These data indicate that hor-mone-independent prostate cancer cells are able to respondto adrenomedullin in ways that would be expected tofurther the aggressiveness of androgen-independent pros-tate cancer. We demonstrated that aAM inhibited the basallevels of prostate cancer androgen-independent cell prolif-eration and invasion in vitro, supporting the conclusion thatadrenomedullin can act in an autocrine manner in andro-gen-independent prostate cancer. The presence of autocrineloop suggests that foci of adrenomedullin-producing cellsin a tumor could stimulate cells expressing adrenomedullinreceptors via autocrine/paracrine mechanisms.

The expression of adrenomedullin and adrenomedullinreceptors by prostate cancer cells and stromal cells suggeststhat the adrenomedullin systemmay play an important rolein situ. The role of adrenomedullin in tumor progression bystimulation of tumor cell proliferation, inhibition of apo-ptosis, and stabilization of angiogenesis has been well estab-lished (7, 12, 13, 17, 31), and all these activities may berelevant in prostate cancer. Our data demonstrate that aAMcould be efficiently delivered in vivo and significantly sup-press the growth of established Du145 xenografts. Theimmunostaining of aAM-treated tumor sections with anti-vWF antibody demonstrated that more than 84% of thevessels disappeared with a clear depletion of the endothelialcells and pericytes, suggesting strongly that the adrenome-dullin systemmust be involved in neovascularization and/orvessel stabilization in hormone-independent prostate can-cer. Because the Du145 cell proliferation is inhibited in vitrobyaAM, the inhibition of Du145 xenografts growth byaAMcould be a result of combined effects on tumor cell growth aswell as on tumor neoangiogenesis. Importantly, the physi-ologic vascularization innormal tissues that has a long actingdoubling-time (about 3 years) could not be disrupted byaAM treatment, suggesting that the adrenomedullin systemmust be highly activated in tumor neoangiogenesis in whichfar shorter doubling-time is observed (few days; ref. 32).

We have reported that in castrated animals (absence ofandrogen in vivo), intraperitoneal injection of adrenome-dullin stimulates the growth of LNCaP xenografts, suggest-ing that adrenomedullin might be involved in tumor resur-gence following androgen ablation (22). The inhibition ofLNCaP tumor growth by aAM treatment only in thecastrated animals confirms our hypothesis in agreementwith the data obtained with Du145 xenografts. These databrought strong evidence that the production of ir-AM stim-ulated by androgen ablation must participate in prostatecancer tumor growth. Interestingly, in intact animals or inthe presence of androgen, aAM could not inhibit tumorgrowth, suggesting that in noncastrated animals, LNCaPxenograft growth is adrenomedullin independent inagreement with the barely detectable levels of adrenome-dullin in xenografts developed in noncastrated animals(22). Recently, adrenomedullin was found to be over-expressed in AR-E231G prostates and shown to act asnovel effector of androgen receptor–mediated prostatetumorigenesis by promoting cell proliferation and sur-

vival (33). Collectively, these results highlight the role ofadrenomedullin as a major factor that affects the tumormicroenvironment to promote neoangiogenesis leadingthe nutrient and oxygen supply and hormone-indepen-dent prostate cancer tumor growth.

Like blood vascular angiogenesis, lymphangiogenesis hasgainedmuch attention as an important initial step in tumorpathogenesis (34–36). It has been shown that intra- and/orperitumoral lymphangiogenesis increases the risk formetastasis both in animal models and in human tumors(34). To determine whether lymphatic vessels might beimpaired by aAM treatment in prostate cancer orthotopicxenografts, we analyzed the lymphatic vessels using amurine LYVE-1 antibody. LYVE-1þ lymphatic vessels wereobserved in the control IgG–treated tumors and werecompletely devoid within the aAM-treated tumors. Impor-tantly, aAM treatment was not observed to impair preexist-ing lymphatic vessels detected in the normal tissue. Pro-longed inhibition does not affect adult lymphatics, indi-cating that activation of AM receptors (AM1 and AM2) isnecessary to induce growth of lymphatics but not requiredfor the maintenance of the lymphatics in adulthood.Accordingly, our data demonstrate that activation ofadrenomedullin receptors by adrenomedullin inducesproliferation, migration, invasion, and survival of LECs(Supplementary Fig. S6 and S7), suggesting an importantrole of adrenomedullin to build-up functional lymphaticvessels during tumor growth. Interestingly, we demon-strate that adrenomedullin secreted by LECs participate inSMCs’ recruitment in migration assay, suggesting stronglythat adrenomedullin could be involved in SMC recruit-ment during collecting-vessel formation in vivo. Together,our work provides evidence suggesting that the activationof the adrenomedullin/adrenomedullin receptors signal-ing pathway upon induction of adrenomedullin expres-sion (22, 33, 37) may not only be important for support-ing tumor cell growth and neoangiogenesis, but also forpromoting tumor lymphangiogenesis (SupplementaryFig. S8).

Adrenomedullin has been postulated to possess lym-phangiogenic properties (38, 39). Interestingly, the genet-ic loss of AM, Calcrl, or RAMP2 causes preferential reduc-tion in the proliferation of LECs of the jugular lymphaticvessels (38). It has been reported that the loss of adre-nomedullin signaling results in severely hypoplastic jug-ular lymph sacs (38). Systemic administration of adre-nomedullin stimulates both lymphangiogenesis andangiogenesis at a site of injury to mouse lymphatic vessels(39). It is well known that tumor cells enter the lymphaticvasculature by invading preexisting lymphatic vessels inthe tumor periphery or by eliciting lymphangiogenesis viagrowth factor production (40, 41). It is conceivable topropose that adrenomedullin produced by tumor cellsmay stimulate growth and dilation of the peritumorallymphatic vessels to prevent increases in tumor tissuepressure and to facilitate tumor cell entry through thelymphatic endothelium as it was previously demonstrat-ed for other lymphangiogenic growth factors (42, 43), as






reported for VEGF (44, 45) and platelet-derived growthfactor-BB (PDGF-BB; ref. 46).Many studies have brought the lymphatic system to the

forefront as an important route of tumor metastasis (47–49). aAM therapy might be expected to effectively controllymph node and systemic metastases in tumors that metas-tasize via the vasculature and the lymphatic vessels. Furthercharacterization of adrenomedullin signaling and its phar-macologic modulation via AM1 and AM2 receptors mightlead to novel therapeutic target to suppress angiogenesisand lymphangiogenesis as well as potential tumor growthin hormone-independent manner in prostate cancer anddissemination. In fact, blocking tumor-induced angiogen-esis and lymphangiogenesis is an increasingly importantstrategy in the design of antitumor drugs. The present studyand others identified the adrenomedullin system as a prom-ising target for the development of a neutralizingmAb and/or the design of a nonpeptidic modulator that could beuseful for the treatment of cancer. Efforts are underway todevelop mono-specific and/or bi-specific mAbs targetingadrenomedullin and/or adrenomedullin receptors as wellas the development of an adrenomedullin small-moleculeantagonist (13, 16, 17, 24, 50).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. Berenguer-Daiz�e, F. Boudouresque, D. Bertin,K. Mabrouk, P.-M. Martin, L’H. OuafikDevelopment of methodology: C. Berenguer-Daiz�e, F. Boudouresque, C.Bastide, Z. Benyahia, C. Delfino, N. Baeza, M. Cayol, A. El BattariAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): C. Berenguer-Daiz�e, C. Bastide, A. Tounsi, Z.Benyahia, J. Acunzo, N. Dussault, L. Daniel, M. Cayol, L’H. OuafikAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): C. Berenguer-Daiz�e, Z. Benyahia, N.Dussault, L’H. OuafikWriting, review, and/or revision of the manuscript: C. Berenguer-Daiz�e,N. Dussault, N. Baeza, L. Daniel, L’H. OuafikAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C. Berenguer-Daiz�e, C. Delfino,L’H. OuafikStudy supervision: D. Rossi, D. Bertin, L’H. Ouafik

AcknowledgmentsThe authors thank V. Gagna for her excellent secretarial assistance.

Grant SupportThis study was supported by grants from Institut National du Cancer

(INCa; grantCaPAM), Institut national de la sant�e et de la recherchemedicale(INSERM), AP-HM, ARTC Sud, and the Association pour la Recherche sur lesTumeurs de la Prostate (ARTP).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 1, 2013; revised July 29, 2013; accepted August 12, 2013;published OnlineFirst October 7, 2013.

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2013;19:6138-6150. Published OnlineFirst October 7, 2013.Clin Cancer Res Caroline Berenguer-Daizé, Françoise Boudouresque, Cyrille Bastide, et al. Independent Prostate Tumor Xenograft in Mice

−Adrenomedullin Blockade Suppresses Growth of Human Hormone

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Adrenomedullin Blockade Suppresses Growth of Human … · Cancer Therapy: Preclinical Adrenomedullin Blockade Suppresses Growth of Human Hormone–Independent Prostate Tumor Xenograft