Adrenomedullin Promotes Formation of Xenografted Endometrial Tumors by Stimulation of Autocrine Growth and Angiogenesis

Adrenomedullin promotes formation of xenografted endometrial tumors by stimulation of autocrine growth and angiogenesis

Martin K Oehler1,2, Stephen Hague1,2, Margaret CP Rees2 and Roy Bicknell*,1

1Molecular Angiogenesis Laboratory, Cancer Research UK, Institute of Molecular Medicine, John Radclie Hospital, University of Oxford, Oxford OX3 9DS, UK; 2Nueld Department of Obstetrics and Gynaecology, John Radclie Hospital, University of Oxford, Oxford OX3 9DS, UK

The angiogenic peptide adrenomedullin (ADM) has been speci®city for ADM being conferred by the receptor implicated as a mediator of the increased risk of associated modifying protein 2 (RAMP2) (McLatchie endometrial hyperplasia and cancer resulting from the et al., 1998; Kamitani et al., 1999). use of tamoxifen for the treatment and prevention of Since its identi®cation (Kitamura et al., 1993) ADM breast cancer. ADM has been shown to be induced by has been shown to be a pluripotent peptide with tamoxifen in the endometrium and to be a growth factor properties ranging from inducing vasorelaxation to for endometrial endothelial cells in vitro. We have now acting as a regulator of cellular growth (Hinson et al., shown ADM to be strongly angiogenic in the mouse 2000). ADMs special role in vascular physiology was subcutaneous sponge angiogenesis assay. To examine the demonstrated by an ADM gene knockout mouse which role of ADM in tumor growth, the ADM cDNA was showed extreme hydrops fetalis and cardiovascular transfected into endometrial carcinoma cells followed by abnormalities (Caron and Smithies, 2001). We have xenografting into athymic mice. Two endometrial cancer previously shown that ADM is an endothelial cell cell lines were employed, those in which transfection and growth factor and angiogenic in the chick chorioallan- expression of ADM resulted in no eect on growth toic membrane (CAM) assay (Zhao et al., 1998). in vitro (Ishikawa cells) and those in which expression ADM is known to be a hypoxia regulated peptide of exogenous ADM stimulated in vitro growth (Cormier-Regard et al., 1998; Nakayama et al., 1998) (RL95.2 cells). A clear enhancement of tumor growth which is under hypoxia-inducible transcription factor-1 was seen with both cell lines but the eect was far (HIF-1) control similar to that of other angiogenic greater with the RL95.2 cells. We conclude that ADM is factors like vascular endothelial growth factor (VEGF) pro-tumorigenic by stimulating either angiogenesis alone (Garayoa et al., 2000; Semenza, 1999). We have shown or by stimulating angiogenesis and carcinoma cell growth that ADM when upregulated under hypoxia acts as an directly. The combined activities lead to a striking antiapoptotic factor antagonizing hypoxia induced cell increase in tumor growth. These results provide the ®rst death of endometrial cancer cells by up-regulation of direct evidence of tumorigenic activity of ADM and Bcl-2 (Oehler et al., 2001). provide further support for ADMs involvement in ADM is expressed in a variety of malignant tissues tamoxifen induced endometrial neoplasia. (Lal et al., 1999; Hata et al., 2000; Rocchi et al., 2001) Oncogene (2002) 21, 2815 ± 2821. DOI: 10.1038/sj/ and was shown to be mitogenic for human cancer cell onc/1205374 lines including lung, breast and colon lineages in vitro (Rocchi et al., 2001; Miller et al., 1996; Pio et al., Keywords: adrenomedullin; angiogenesis; tumorigen- 2000). Its role in tumorigenesis, however, is yet to be esis; tamoxifen; endometrial cancer established. Tamoxifen is currently the drug of choice for the treatment of hormone dependent breast cancer and is being evaluated as a preventative agent in women at Introduction high risk of developing the disease (Cuzick, 2000; Dalton and Kallab, 2001). A concern, however, is the Adrenomedullin (ADM) is a 52-amino-acid peptide proliferative eect of TAM on the endometrium, which which belongs to the calcitonin gene peptide super- is associated with a twofold to sevenfold increased risk family based on its slight homology with calcitonin of endometrial cancer for long-term tamoxifen users gene-related peptide (CGRP) and amylin (Wimala- (White, 1999; Bergman et al., 2000). wansa, 1997). It acts through the G protein-coupled In an attempt to understand why tamoxifen has the receptor calcitonin receptor-like receptor (CRLR), with aforementioned eect on the endometrium we initiated a study to identify tamoxifen induced gene expression in endometrial isolates. PCR dierential display was used to identify tamoxifen-induced genes. One such *Correspondence: R Bicknell; E-mail: [email protected] Received 31 July 2001; revised 22 January 2002; accepted 31 gene was ADM for which we then showed increased January 2002 expression in the endometrium of patients receiving Adrenomedullin is tumorigenic MK Oehler et al 2816 tamoxifen (Zhao et al., 1998). We subsequently showed that ADM is an autocrine regulator of endothelial cell growth in the endometrium, being both produced by endometrial endothelial cells and stimulating their growth (Nikitenko et al., 2000). Although these observations implicate an involvement of ADM in tamoxifen induced proliferative changes of the endometrium, evidence for a direct tumorigenic activity of ADM is still missing. We here report for the ®rst time that ADM is angiogenic in the mouse subcutaneous sponge angiogenesis assay and that constitutive expression of ADM enhances tumor take and endometrial carcinoma cell growth in vivo. These results provide direct evidence for the tumorigenic activity of ADM and supports ADM as a mediator of tamoxifen induced endometrial hyper- and neoplasia.

Results

Angiogenic activity of ADM in the mouse subcutaneous sponge assay We have previously shown that ADM is angiogenic in the CAM, an avian in vivo assay. Here we examined the eect of ADM in a mammalian in vivo assay. ADM was shown to be strongly angiogenic in the Figure 1 (a) Histology of control and adrenomedullin treated mouse subcutaneous sponge angiogenesis assay sponge implants at day 20 after implantation (hematoxylin/eosin (P50.05 (n=4, mean+s.e.m.) Wilcoxon matched pairs stained). (b) Vascular density of sponge implants determined by test) (Figure 1). Chalkley counting. (n=4+s.e.m.). Note the greater penetration of ®brotic tissue into and the presence of many more erythrocyte containing vessels in the sponge that received ADM Characterization of ADM expressing Ishikawa and RL95.2 clones Growth of transfectants in vitro The eect of ADM on endometrial cancer cell growth was examined by experiments with the endometrial In vitro growth of ADM transfectants and controls was carcinoma cell lines Ishikawa and RL95.2. These cell determined by MTS-assay. Constitutive expression of lines were chosen to represent major types of ADM had no eect on Ishikawa growth but markedly endometrial carcinoma cells. Ishikawa cells are estro- stimulated that of RL95.2 cells (P50.05) (Figure 4). gen receptor (ER) positive cells whereas late passage RL95.2 cells do not express the ER (Sundareshan and Growth of transfectants in vivo Hendrix, 1992; Zhang et al., 1995a; Bilimoria et al., 1996). The RL95.2 cells used in this study were of Transfected clones were examined for their ability to passage 4100. ADM under control of the CMV form tumors in vivo. Ishikawa cells are estrogen- promoter in the mammalian expression vector dependent and very weakly tumorigenic in athymic pcDNAI/Neo was transfected into Ishikawa and mice. In view of this they were co-xenografted with an RL95.2 cells by electroporation followed by selection MDA-435S carrier line and estrogen supplemented as with G418. After 16 weeks, surviving clones were previously described (Zhang et al., 1995b; Ali et al., isolated and expanded. Two Ishikawa cell clones (Ish- 2000). Figure 5 shows that while expression of ADM ADM1 and Ish-ADM2) and two RL95.2 cell clones had a comparatively small eect on Ishikawa/MDA- (RL-ADM1 and RL-ADM2) highly expressing ADM 435S tumor growth (P50.05) it had a marked eect on mRNA and peptide were identi®ed by immunohisto- growth of RL95.2 tumors (P50.01). Similarly striking chemistry of cytospins and Western analysis of was the eect of ADM expression on tumor take where conditioned media (Figure 2). The secreted ADM ADM expression greatly facilitated tumorigenicity in peptide was shown to be active by stimulation of RL95.2 cells (P50.05) (Table 1). growth of human dermal microvascular endothelial cells (HDMEC) by transfectant conditioned media Vascular density of xenografted tumors (Figure 3). Finally RL95.2 cells were shown to express CRLR and RAMP2, that together function as the Frozen sections of the xenografted tumors were ADM receptor, by RT ± PCR (data not shown). immunostained for the endothelial marker CD31 and

Oncogene Adrenomedullin is tumorigenic MK Oehler et al 2817

Figure 2 Characterization of two Ishikawa and two RL95.2 transfected clones that overexpress adrenomedullin. The cells were shown to overexpress ADM by immunohistochemistry (top) and Western analysis (bottom)

cells in athymic mice. It was shown that ADM increases both tumor take and growth rate, but that the latter varies considerably on whether the carcinoma cell line itself is or is not growth stimulated by ADM. Two ADM expressing Ishikawa clones were isolated and their growth properties compared to that of a control empty vector transfectant. Expression of ADM had no eect on in vitro growth of those cells but caused a small but statistically signi®cant enhancement of in vivo growth. Ishikawa cells are known to express ADM receptors in vitro (Oehler et al., 2001), this however, appeared not to lead to a growth eect in the Figure 3 Stimulation of human dermal microvascular endothe- presence of ADM. Despite the lack of a growth eect lial cell growth by conditioned media from control and of ADM on Ishikawa cells, ADM clearly enhances adrenomedullin transfected Ishikawa cells (*P50.05, n=5, their survival in culture especially under hypoxia mean+s.e.m.). O.D. is from the MTS assay and is proportional (Oehler et al., 2001). to the cell density (see Materials and methods) In the case of RL95.2 cells, transfectants grew faster than controls in vitro (Figure 4). Far more striking, however, was the dramatic increase in tumor growth the vessel density quantitated by Chalkley counting. seen in xenografts (Figure 5). The tumorigenic activity A signi®cantly increased vascular density of tumors of ADM was supported by the tumor take data shown overexpressing ADM could be found in both in Table 1. Thus while only 6 out of 10 implants of Ishikawa/MDA-435S (data not shown) and RL95.2 control transfectants gave tumors, 9 and 10 out of 10 tumors (P50.05 (n=3. Mean+s.e.m.) Wilcoxon of the ADM transfectant implants gave tumors. This matched pairs test) (Figure 6). eect was highly reproducible, indeed two separate experiments gave identical results. Examination of the vasculature in sections of the Discussion xenografted tumors immunostained with CD31 showed that in both Ishikawa/MDA-435S (data not shown) The purpose of this study was to examine a role for the and RL95.2 tumors (Figure 6) there was a marked angiogenic peptide ADM in tumor formation. To this increase in the vascular density in those expressing end, we have examined the eect of expression of ADM ADM compared to controls. Vascular density was on the tumorigenesis of human endometrial carcinoma quantitated by Chalkley counting.

Oncogene Adrenomedullin is tumorigenic MK Oehler et al 2818

Figure 5 Eect of ADM expression on (a) Ishikawa/MDA-435S (P50.05, n=10 mean+s.e.m.) and (b) RL95.2 (P50.01, Figure 4 Eect of ADM expression on (a) Ishikawa and (b) n=10+s.e.m.) cell growth in vivo RL95.2 cell growth in vitro. O.D. is from the MTS assay and is proportional to the cell number (see Materials and methods) Table 1 Eect of ADM-transfection on tumor take of RL95.2 endometrial carcinoma cells. The experiment was performed twice Dierent growth patterns of Ishikawa and with identical results RL95.2 cells in vivo may partly be explained by cell Clone Tumor takea line speci®c properties. Ishikawa cells are known to be RL-ADM1 10/10 (100%) ER positive estrogen dependent cells and in this sense RL-ADM2 9/10 (90%)* they are closer to endometrial epithelium than ER Control 6/10 (60%) negative late passage RL95.2 cells. ER expression is aNumber of positive mice/number of injected mice. *P50.05 well documented to inhibit in vitro and in vivo growth of endometrial cancer cells and was recently shown to inhibit VEGF induced angiogenesis (Ali et al., 2000). While xenografted RL95.2 cells readily formed tumors Of the many angiogenic polypeptides now identi®ed it in athymic mice, Ishikawa did not. For this reason it appears that those induced by hypoxia are key players was necessary to co-implant Ishikawa cells with carrier in tumor angiogenesis. ADM up-regulation under cells (in this case MDA-435S cells) and estrogen hypoxia is controlled by the hypoxia-inducible tran- supplementation to observe growth in vivo. This scription factor-1 (HIF-1) as is VEGF (Semenza, 1999). widely used strategy (Kamitani et al., 1999) permitted HIF-1 is a heterodimer composed of the oxygen- a direct comparison of the growth of transfectants in regulated HIF-1a and the constitutively expressed vivo. HIF-1b/ARNT subunits, both representing members We conclude that ADM is pro-tumorigenic in of the PAS (Per, ARNT, Sim) basic-helix-loop helix endometrial cancer cells as a result of its mitogenic family (Semenza, 1999). HIF-1 binds to hypoxia and angiogenic properties. With Ishikawa cells en- response elements (HREs) several of which are located hanced in vivo growth due to increased angiogenesis in the ADM promoter (Nakayama et al., 1998). The was seen whereas in RL95.2 cells a direct mitogenic importance of the HIF-1 response pathway in human eect of ADM on the carcinoma also occurred. This tumorigenesis is underscored by the ®nding that HIF-1 resulted in a greater enhancement of tumor growth is overexpressed in many cancers (Zhong et al., 1999) after transfection of ADM into RL95.2 cells compared and that patients with strong expression of HIF-1 show to the same experiment with Ishikawa cells. a signi®cantly shorter overall survival (Birner et al.,

Oncogene Adrenomedullin is tumorigenic MK Oehler et al 2819 al., 1993). Human ADM-(1-52) was synthesized by the ICRF- Peptide-Unit, Clare Hall, London, UK. All reagents were purchased from Sigma, Poole, UK, unless stated otherwise. The tumor cell line RL95.2 (CRL-1671) was obtained from the American Type Culture Collection, Bethesda, USA, the cell lines Ishikawa and MDA-435S from the Clare Hall Laboratories, Imperial Cancer Research Fund, London, UK and human dermal microvascular endothelial cells (HDMEC) from Clonetics/Biowhittaker, Wokingham, UK. Ishikawa cells were routinely cultured in DMEM supplemented with 10% FCS and 4 mM glutamine, RL95.2 cells in DMEM/Nutrient Mixture F-12 K Kaighn's Modi®cation ± 1 : 1 supplemented with 10% FCS and HDMEC in EGM-2MV Bullet kit medium (Clonetics/Biowhittaker, Wokingham, UK). All cells were cultured at 378C and 5% CO2.

Vector construction and stable transfection The full-length cDNA for human ADM was cloned into the XhoI/BamHI-restriction sites of pcDNAI/Neo to give plasmid pCMV-ADMneo in which expression of ADM is constitutively driven by the CMV-promoter. Ishikawa and RL95.2- cells were transfected with pCMV-ADMneo or pcDNAI/Neo (empty vector control) by electroporation at 400 V with a capacitance of 25 mF in transfection buer (272 mM sucrose, 7mM sodium phosphate pH 7.4, 1 mM MgCl2). Transfected cells were then selected for neomycin resistance by treatment with G418 (500 mg/ml) for 16 weeks. Isolated clones were Figure 6 Immunohistochemistry of xenografted tumors that are expanded for further characterization. formed on implantation of ADM-transfected RL95.2 cells and controls. (a) Vessels were visualized by staining with anti-CD31. (b)Vascular density quantitated by Chalkley counting. *P50.05 (n=3, mean+s.e.m.) Reverse transcription (RT)-polymerase chain reaction (PCR) CRLR and RAMP2 gene expression in RL95.2 cells were examined by RT ± PCR using the Reverse-iT-kit (1st strand 2000). More signi®cantly, disruption of hypoxia- synthesis kit) (Advanced Biotechnologies, Epsom, UK). For inducible transcription severely abrogates tumor growth the RT-reaction anchored oligo-dT primers included in the (Kung et al., 2000). The critical eectors by which HIF- kit were used. Primers and PCR conditions were chosen as 1 modulates tumor vascularization and subsequent previously described (Oehler et al., 2001). growth are, however, still unknown. One is VEGF and we propose that ADM may be another in that Immunoblot analysis ADM is a mitogen for both endothelial and carcinoma Preparation of samples Serum-free cell culture medium was cells as well as a survival factor for malignant cells conditioned by con¯uent cultures for 48 h. The medium was under hypoxia (Zhao et al., 1998; Nikitenko et al., 2000; concentrated (1006) with a Centricon-3-Concentrator fol- Oehler et al., 2001). ADMs role in the vasculature has lowed by a Microcon-5-Concentrator (Amicon, Beverley, been recently reviewed (Nikitenko et al., 2002). USA). Protein concentrations were quanti®ed using the BCA In contrast to a hypoxic stimulus, the mechanism by protein assay (Pierce, Rockford, USA). The samples were stored at 7808C until assay. which TAM stimulates ADM expression is not clear as the promoter lacks palindromic oestrogen response Immunoblotting For immunoblotting, samples were run on a elements (Ishimitsu et al., 1994) and it is not induced gradient 10 ± 20% tricine, SDS ± PAGE (BioRad, Hercules, by oestrogen. The ADM promoter does, however, have USA). The proteins were electrophoretically transferred onto two AP-1 binding sites at positions ± 1549 and ± 1801 a polyvinylidene di¯uoride membrane (Immobilon PVDF ± respectively (Ishimitsu et al., 1994). As TAM has been Millipore, Watford, UK). The PVDF membrane was blocked demonstrated to initiate gene transcription via AP-1 for 2 h in PBS with 0.1% Tween-20, 5% bovine serum sites (Webb et al., 1995) this could be a possible albumin and incubated in a 1 : 1000 dilution of a polyclononal mechanism of transcription. rabbit anti-human ADM antibody (Peninsula Laboratories, Overall, this study presents evidence of a tumorigenic Merseyside, UK). Binding was visualized by a horseradish activity of ADM that further supports a role for ADM peroxidase-conjugated pig anti-rabbit IgG and developed with ECL Western blotting reagents (Amersham, Little Chalfont, in tamoxifen induced endometrial hyper- and neoplasia. UK). Quanti®cation of protein expression was performed by densitometry (Fluorchem, Alpha Innotech Corp, USA).

Materials and methods Determination of in vitro cell growth Ishikawa and RL95.2 cell clones were quiescent for 24 h in their respective medium The human ADM cDNA was a gift from Dr Kitamura, First supplemented with 0.5% FCS before growth assays. Cells Department Internal Medicine, Kihara, Japan (Kitamura et were seeded at 26103 cells/well in 96-well plates. The culture

Oncogene Adrenomedullin is tumorigenic MK Oehler et al 2820 medium was changed every 48 h and cell growth measured by pellet containing 1.5 mg of 17b-estradiol (Innovative Re- MTS-assay (Promega, Madison, USA) every 24 h for 6 days. search of America, Toledo, Ohio, USA). Tumors were In the assay viable cells reduce MTS tetrazolium to a measured twice a week from ®rst appearance. When tumors coloured formazan which is measured at 490 nm with a 96- reached 1.44 cm2 animals were sacri®ced by cervical well plate reader (Dynex Technologies, Billingshurst, UK). dislocation. The tumor volume was calculated using the For cell growth experiments with HDMECs, cells were formula: Tumor volume (cm_)=(length6(width)2_)60.4 seeded at 46103 cells/well in gelatin-coated 96-well plates. (Attia and Weiss, 1966). All animal experiments were The cells were quiesced in endothelial growth medium (EGM- performed in accordance with the British Home Oce 2MV; Clonetics/Biowhittaker, Wokingham, UK) supplemen- Animals (Scienti®c Procedures) Act of 1986 under license ted with 1% FCS for 24 h. Cells were then cultured in number 70/4949. endothelial growth medium (EGM)/1% FCS with various concentrations of ADM or in EGM/1% FCS with condi- Immunohistochemistry of mouse tumor tissue samples Cryo- tioned media of ADM transfected Ishikawa clones (1 : 1). The stat sections (8 mm) of tumors were air dried for 24 h, ®xed in cell culture medium was changed after 24 h and cell growth acetone (10 min), air-dried for 40 min, and then stored at measured by MTS-assay after 48 h. 7208C until use. The mouse tumor vasculature was examined immunohistochemically. Tissue sections were Mouse subcutaneous sponge angiogenesis assay The sponge incubated with PECAM-1 (rat anti-mouse CD31) (Pharmin- implantation model was set up as described previously gen, San Diego, USA) for 1 h at a 1 : 200 dilution, rinsed in (Walsh et al., 1996) with some modi®cations. C57 black PBS for 10 min, incubated with biotinylated rabbit anti-rat mice were anesthetized using ¯uothane and received a IgG at a 1 : 300 dilution for 30 min, rinsed again in PBS for subcutaneous sterile polyether sponge disc (868 mm) (Cali- 10 min, incubated with avidin ± biotin ± peroxidase complex, gen Foam Ltd., Accrington, UK) under the dorsal skin. Rat (Dako, Ltd.) for 30 min, with a ®nal rinse in PBS. The adrenomedullin (rADM) (Peninsula Laboratories, Mersey- chromogen was developed for 10 min using diaminobenzidine side, UK) at concentrations 1078 ±10712 M in a total volume tetrahydrochloride, 0.6 mg/ml in PBS containing 3 ml/ml of of 90 ml physiological saline was injected daily through the hydrogen peroxide. All sections were lightly counterstained skin into the sponges from day 1 to 20. The control mice with haematoxylin-eosin. received physiological saline alone. At the end of the experiment, the animals were sacri®ced by cervical dislocation Statistical analysis Statistics were performed using the SPSS and the sponges rapidly excised together with overlaying skin software package. Comparisons between groups were made and underlying connective tissue. The sponges were ®xed in applying the Wilcoxon matched pairs test or the Fisher exact 10% formalin at 48C for 1 h and then immersed in 75% test. ethanol for 30 min and ®nally kept in 90% ethanol. For analysis of ®brovascular growth areas samples were embedded in paran wax and 10 mM thick sections evenly Abbreviations distributed throughout each sponge were stained with adrenomedullin, ADM; calcitonin receptor-like receptor, hematoxylin/eosin and CD31. CRLR; receptor activity modifying protein 2, RAMP-2; tamoxifen, TAM; hypoxia-inducible transcription factor-1, Assessment of sponge vascularity The vascularity of the HIF-1; vascular endothelial growth factor, VEGF; ery- explanted sponges was determined by Chalkley counting (Fox thropoietin, EPO; ER, estrogen receptor; human dermal et al., 1995). The three most vascular areas with the highest microvascular endothelial cells, HDMEC; endothelial number of discreet vessels were identi®ed by scanning at low growth medium, EGM; chick chorioallantoic membrane power (640 and 6100). Vascular density was determined assay, CAM assay; optical density, O.D. using a 25-point Chalkley eyepiece graticule at 6250 magni®cation (the graticule covers an area of 0.155 mm72 at this magni®cation). The graticule was rotated in the Acknowledgments eyepiece to where the maximum number of vessels was The authors acknowledge the excellent technical assistance overlaid by graticule dots. Individual density was then of Sandra Peak, ICRF Clare Hall Laboratories, London in obtained by taking the mean of three graticule counts. performing the xenograft and sponge assays, Helen Turley, Department of Cellular Science, University of Oxford for Xenograft experiments in BALB/c nu/nu mice BALB/c nu/nu help with immunohistochemistry and cytospins and Mr mice were anesthesized using ¯uothane and RL95.2 cells Daryl Harman, Caligen Foam Ltd, Accrington for help in injected (106 cells in 0.2 ml cell suspension) subcutaneously obtaining sponge implants. Financial support was provided into one ¯ank. In a second setting mice received two by the Imperial Cancer Research Fund, The Deutsche subcutaneous implants: Ishikawa (106 cells) and MDA-435S Forschungsgemeinschaft and The Sir Jules Thorn Chari- cells (106) in 0.2 ml cells suspension and a 60-day release table Trust.

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Oncogene