Research Angiogenin Regulation by Estradiol in Breast Tissue

Published OnlineFirst May 25, 2010; DOI: 10.1158/1078-0432.CCR-10-0501

Cancer Therapy: Preclinical Clinical
Cancer
Research

Angiogenin Regulation by Estradiol in Breast Tissue:Tamoxifen Inhibits Angiogenin Nuclear Translocationand Antiangiogenin Therapy Reduces Breast CancerGrowth In vivo

Ulrika W. Nilsson, Annelie Abrahamsson, and Charlotta Dabrosin

Abstract

Authors' AExperiment

Corresponköping Uni10-103-85-

doi: 10.115

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Purpose: Angiogenin, a 14.2-kDa polypeptide member of the RNase A superfamily, has potent angio-genic effects. Nuclear accumulation of angiogenin is essential for its angiogenic activity. Increased angio-genin expression has been associated with the transition of normal breast tissue into invasive breastcarcinoma. In this article, we investigated whether estradiol (E2) affected angiogenin in breast tissue.Experimental Design: We used microdialysis for sampling of extracellular angiogenin in vivo. In vitro

cultures of whole normal breast tissue, breast cancer cells, and endothelial cells were used.Results:We show that extracellular angiogenin correlated significantly with E2 in normal human breast

tissue in vivo and that exposure of normal breast tissue biopsies to E2 stimulated angiogenin secretion. Inbreast cancer patients, the in vivo angiogenin levels were significantly higher in tumors compared with theadjacent normal breast tissue. In estrogen receptor–positive breast cancer cells, E2 increased and tamox-ifen decreased angiogenin secretion. Moreover, E2-induced angiogenin derived from cancer cells signifi-cantly increased endothelial cell proliferation. Tamoxifen reversed this increase as well as inhibitednuclear translocation of angiogenin. In vivo, in experimental breast cancer, tamoxifen decreased angiogen-in levels and decreased angiogenesis. Additionally, treating tumor-bearing mice with an antiangiogeninantibody resulted in tumor stasis, suggesting a role for angiogenin in estrogen-dependent breast cancergrowth.Conclusion: Our results suggest previously unknown mechanisms by which estrogen and antiestrogen

regulate angiogenesis in normal human breast tissue and breast cancer. This may be important for estrogen-driven breast cancer progression and a molecular target for therapeutic interventions. Clin Cancer Res; 16(14);

3659–69. ©2010 AACR.

Estrogen is a crucial regulator of normal breast physiol-ogy and a major risk factor for breast cancer (1–3). Themajority of breast cancers have a maintained estrogen de-pendency and antiestrogen therapy is a corner stone inmedical breast cancer therapy. However, almost 50% ofestrogen receptor (ER)–positive breast cancer cases fail tobenefit from endocrine therapy. There is a need to eluci-date which specific targets are involved in estrogen-driventumor progression to optimize therapeutic options.Angiogenesis is a key event in tumor development and

progression (4). Sex steroids are crucial for the control ofangiogenesis in the female reproductive tract. How ste-

ffiliation: Division of Oncology, Department of Clinical andal Medicine, Linköping University, Linköping, Sweden

ding Author: Charlotta Dabrosin, Division of Oncology, Lin-versity Hospital, SE-581 85 Linköping, Sweden. Phone: 46-95; Fax: 46-10-103-30-90; E-mail: [email protected].

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roids regulate angiogenesis in normal breast tissue andbreast cancer is yet under investigation.Angiogenin is a basic heparin-binding protein of 14 kDa

and one of the most potent angiogenic factors when exam-ined in various experimental models of angiogenesis (5).Angiogenin binds to endothelial cells (6, 7), undergoesnuclear translocation, a process essential for its angiogenicactivity (8–11), and thereby induces a wide variety ofresponses including cell proliferation, activation of cell-associated proteases, and cell migration and invasion.Moreover, nuclear angiogenin in endothelial cells is neces-sary for angiogenesis induced by other angiogenic factorsincluding vascular endothelial growth factor (VEGF), acidicand basic fibroblast growth factors, and epidermal growthfactor (8). Antagonists to angiogenin have been shown toinhibit the growth and angiogenesis of human tumor cellsin athymic mice (12–14). Also, the aminoglycoside antibi-otic neomycin and its nontoxic derivative neamine havebeen shown to abolish nuclear translocation of angiogeninand thereby inhibit its angiogenic activity (15, 16). Inbreast cancer, increased angiogenin expression has been

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Translational Relevance

Estrogen is a crucial regulator of normal breast phys-iology and a major risk factor for breast cancer. There isa need to elucidate which specific targets are involvedin estrogen-driven tumor progression to optimize ther-apeutic options and develop preventive strategies.

Angiogenesis plays a key role in tumor progression.Angiogenin is one of the most potent proangiogenicfactors independent of hypoxia inducible factor andvascular endothelial growth factor.

Here we show, in a translationalmanner, from single-cell level via experimental breast cancer to normal hu-man breast tissue ex vivo and in vivo to breast cancerpatients in vivo, that the expression of angiogenin is es-trogen dependent and that antiangiogenin therapy in-hibits breast cancer growth. Thus, angiogenin is apotential powerful therapeutic target in breast cancer es-pecially in light of the very high extracellular levels(10,000 pg/mL) of angiogenin detected in breast can-cer patients.

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associated with the transition from normal to invasivebreast carcinoma and with ER expression (17).There is increasing evidence suggesting that sex steroids

may be important in the regulation of angiogenesis inbreast tissue by affecting the balance between proangio-genic and antiangiogenic proteins (18). We have previ-ously shown that estradiol (E2) acts proangiogenicallyin normal breast tissue and breast cancer by increasingthe extracellular expression of VEGF and its receptor(19–25), increasing interleukin-8, and by decreasing solu-ble VEGF receptor-1 and endostatin (22, 26–28). More-over, tamoxifen, the most widely used medical therapyagainst ER-positive breast cancer, may tip the balance toinhibiting angiogenesis by downregulating extracellularlevels of VEGF while at the same time increasing solubleVEGF receptor-1 expression and the generation of endo-statin (21, 22, 26).Recently, the importance of angiogenin as a potent reg-

ulator of tumor angiogenesis independent of hypoxiainducible factor 1 and VEGF has been highlighted (29).Whether sex steroids regulate angiogenin in breast tissueis unknown. In this article, we show that E2 increasesextracellular angiogenin in normal human breast tissuein vivo and in breast cancer cells in culture. We also showthat tamoxifen decreases tumor angiogenesis both bydownregulating extracellular angiogenin in vivo and by in-hibiting the nuclear translocation of angiogenin in endo-thelial cells. Treating tumor-bearing mice with an antibodyagainst angiogenin resulted in tumor stasis, emphasizingthe biological significance of angiogenin in estrogen-dependent tumor growth. Moreover, in a novel fashion,extracellular angiogenin was sampled in vivo from breastcancer patients, and this revealed twice as high levels in

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the cancerous tissue compared with the adjacent normalbreast tissue.

Materials and Methods

SubjectsA total of 24 women were investigated with microdialy-

sis for angiogenin sampling in breast tissue in vivo. Four-teen of the women were healthy volunteers: eight werepremenopausal (ages 22-25 years) and six were postmen-opausal (ages 52-55 years). All had been off sex steroid–containing medication for more than 3 months. All pre-menopausal women had a history of regular menstrual cy-cles (cycle length, 28-35 days). Four of the premenopausalwomen were investigated in the luteal phase of the men-strual cycle whereas four were investigated in the follicularphase. Ten breast cancer patients (ages 51-86 years, post-menopausal without ongoing hormonal treatment) werealso investigated preoperatively with microdialysis. The lo-cal ethical committee approved the study and all womengave their informed consents.

Microdialysis of healthy volunteers and breastcancer patientsMepivacaine (5 mg/mL) was administrated intracutane-

ously before insertion of the microdialysis catheters. Inhealthy volunteers, the microdialysis catheter was placedin the upper lateral quadrant of the left breast and directedtoward the nipple as previously described (24, 30–33).The breast cancer patients were investigated on the day be-fore or the same day as surgery. In these women, one mi-crodialysis catheter was inserted intratumorally in thecenter of the tumor and one catheter in the adjacent nor-mal breast tissue. The microdialysis catheter (CMA/Micro-dialysis AB), which consists of a tubular dialysis membrane(10mm long × 0.52mm in diameter, 100,000 atomic masscutoff) glued to the end of a double-lumen tube (100 mmlong × 0.8 mm in diameter), was connected to a micro-infusion pump (CMA 107, CMA/Microdialysis AB) andperfused with 154 mmol/L NaCl and 40 g/L dextran-70at a perfusion rate of 0.5 μL/min. The solution enteredthe catheter through the outer tube and left it throughthe inner tube, from which it was collected and stored at−70°C for subsequent analysis.

Breast tissue biopsiesBiopsies from human breast tissue were obtained from

premenopausal women with no ongoing hormonal treat-ment and who were undergoing routine reduction mam-moplasty as previously described (34). Tissue biopsiescontaining epithelium, stroma including endothelialcells, and adipose tissue were produced by using an8-mm biopsy punch (Kai Europe GmbH) and placed ina 12-well plate (Costar). Serum-free medium was usedconsisting of a 1:1 mixture of nutrient mixture F-12 (LifeTechnologies) and DMEM without phenol red (LifeTechnologies) supplemented with transferrin (10 μg/mL;Sigma), insulin (1 μg/mL; Sigma), and bovine serum

Clinical Cancer Research



Estradiol, Tamoxifen, and Angiogenin in the Breast


albumin (0.2 mg/mL; Sigma) with or without physiologiclevels of estradiol (E2) (17β-E2; 10

−9 mol/L; Sigma) or acombination of 10−9 mol/L E2 and 10−8 mol/L progester-one (P4; Sigma) (E2 + P4). The control biopsies wereincubated in medium supplemented with the vehicle,equivalent to the hormone-treated groups (0.001% etha-nol). The biopsies were treated for 7 days at 37°C in ahumidified atmosphere containing 5% CO2 and the me-dium was changed everyday. The medium was collectedand stored at −70°C and the biopsies were formalin fixedand embedded in paraffin for subsequent analyses.

Cells and cultureER- and progesterone receptor (PgR)–positive (MCF-7

and ZR-75-1) and ER- and PgR-negative (MDA-MB-231)breast cancer cell lines were obtained from the AmericanType Culture Collection. MCF-7 and MDA-MB-231 cellswere cultured in DMEM supplemented with 2 mmol/Lglutamine, 50 IU/mL penicillin-G, 50 μg/mL streptomy-cin, and 10% fetal bovine serum, whereas ZR-75-1 cellswere cultured in DMEM/F12 without phenol red (1:1)supplemented with 50 IU/mL penicillin-G, 50 μg/mLstreptomycin, and 5% fetal bovine serum. Human umbil-ical vascular endothelial cells (HUVEC) were isolated fromfresh umbilical cords obtained from female donors. HU-VEC were isolated by collagenase digestion at 37°Cfor 15 to 20 minutes as previously described (35), andgrowth medium consisted of Medium-199 without phe-nol red supplemented with 1% nonessential amino acids,1.6 mmol/L glutamine, 4 IU/mL penicillin G, 4 μg/mLstreptomycin, 20 mmol/L HEPES, 0.02 mg/mL endothelialcell growth factor (Roche Diagnostics), and 16% fetalbovine serum. HUVEC used for the experiments were frompassages 2 to 3. All cells were cultured at 37°C in a humid-ified atmosphere of 5% CO2. Cell culture medium andadditives were obtained from Invitrogen if not otherwisestated. In all in vitro experiments, hormones were added inserum-free medium consisting of DMEM/F-12 without phe-nol red (1:1) supplemented with transferrin (10 μg/mL; Sig-ma), insulin (1 μg/mL; Sigma), and bovine serum albumin(0.2 mg/mL; Sigma). This hormone medium was changedevery 24 hours.

Hormone treatment of cells in cultureCells were trypsinized (0.05% trypsin and 0.02%

EDTA) and seeded into Petri dishes (Costar) at 20 ×103/cm2. Cells were then incubated in complete growthmedium for 24 hours and were then treated with orwithout E2 (17β-E2; Sigma), tamoxifen (Tam; Sigma),or fulvestrant (ICI 182,780; Faslodex, Tocris) in serum-free medium for 7 days. For HUVEC, 3 days was chosenfor the duration of the treatment due to shorter survivaltime without the supplements of the complete growthmedium. The control group was incubated in mediumsupplemented with the vehicle (ethanol) in a con-centration equivalent to the hormone-treated groups(0.001%). Samples were stored at −70°C until subsequentanalyses.

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MCF-7 tumor explants in nude miceFemale athymic nude mice, BALB/c nu/nu (Taconic

M&B), ages 6 to 8 weeks, were housed in a pathogen-freeisolation facility with a 12-h light/12-h dark cycle and fedwith rodent chow and water ad libitum. The study was ap-proved by the Linköping University animal ethics researchboard. Mice were anesthetized with i.p. injections of keta-mine/xylazine (Apoteket) and ovariectomized, then 3-mmpellets containing 17β-E2 (0.18 mg/60-day release; Inno-vative Research of America) were implanted s.c. The pelletsprovide a continuous release of E2 at serum concentrationsof 150 to 250 pmol/L, which is in the range of physiologiclevels seen in mice during the estrous cycle (20). One weekafter surgery, MCF-7 cells (5 × 106 in 200 μL of PBS) wereinjected s.c. on the right hind side flank. The tumors weremeasured twice weekly and the surface area was calculated(length/2 × width/2) × π. At a tumor size of ∼30 mm2, themice were divided into two subgroups such that the meanbody weight and tumor size in each group were the same.One group continued with the E2 treatment only, whereastamoxifen (1 mg every 2 days s.c.) was added to the E2treatment in the other group. In another set of animals,tumors were established as described above, and at a tu-mor size of ∼15 mm2, one group received 60 μg of anantiangiogenin antibody (goat anti-human; R&D Systems)s.c. everyday and the other group was treated with 60 μg ofcontrol antibody s.c. everyday (goat IgG; R&D Systems).

Microdialysis for in vivo sampling of angiogeninTumor-bearing mice were anesthetized i.p. with keta-

mine/xylazine and thereafter kept anesthetized by repeateds.c. injections of ketamine/xylazine. A heating lamp main-tained body temperature. Microdialysis probes (CMA/20;0.5-mm diameter; 10-mm PES membrane length; 100-kDa cutoff; CMA Microdialysis) were inserted into tumortissue, sutured to the skin, connected to a CMA/102 mi-crodialysis pump (CMA Microdialysis), and perfused at0.6 μL/min with saline containing 154 mmol/L NaCland 40 mg/mL dextran 70 (Pharmalink). After a 30-minequilibration period, the outgoing perfusates (microdialy-sates) were collected on ice and stored at −70°C for sub-sequent analysis. At the end of the experiments, the micewere euthanized and tumors were excised. Tumors weremeasured, weighed, formalin fixed, and subsequently em-bedded in paraffin for immunohistochemical analysis.

Quantification of angiogenin by ELISAMicrodialysates and conditioned media from hormone-

treated cells were analyzed using a commercial quantita-tive immunoassay kit (human angiogenin Quantikine,R&D Systems). The analyzed angiogenin in conditionedmedia was normalized to the total protein content and ex-pressed as picograms per milligram of protein or to tissueweight and expressed as picograms per milligram of tissue.Total protein content was determined by the Bio-Rad Pro-tein Assay using bovine serum albumin as standard (Bio-Rad Laboratories). E2 and P4 were analyzed using ELISAkits (DRG Instruments GmbH).

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Real-time PCRmRNA was extracted from cells using TurboCapture

8 mRNA kit (Qiagen) according to the manufacturer's in-structions. cDNA was synthesized using the High CapacitycDNA Reverse Transcription Kit (Applied Biosystems). Therelative levels of angiogenin were determined by quantita-tive reverse transcription-PCR with TaqMan Gene Expres-sion Fast Master Mix in Applied Biosystems 7900HT FastReal-Time PCR System and normalized to β-actin. Angio-genin RNA expression levels were determined by quantita-tive reverse transcription-PCR using predesigned primersand probe of the TaqMan Gene Expression Assay for an-giogenin (Applied Biosystems) and the Applied Biosys-tems 7900HT Fast Real-Time PCR System. Angiogeningene expression was normalized to β-actin (Applied Bio-systems) expression. A standard curve for angiogenin wasrun on each plate, using serial diluted cDNA. All sampleswere run as triplicates.

HUVEC proliferation assayHUVEC were seeded (4,000 per well) in gelatin-coated

96-multiwell plates (Costar). After 24 hours of incubationin complete growth medium, the cells were washed andserum-free medium consisting of DMEM/F-12 (1:1) with-out phenol red, supplemented with 10 μg/mL transferrin(Sigma), 1 μg/mL insulin (Sigma), and 0.2 mg/mL bovineserum albumin (Sigma), was added to minimize the avail-ability of growth factors. Thereafter, the serum-free medi-um was replaced by conditioned media collected fromMCF-7 cells and ZR-75-1 cells (treated for 7 days with orwithout E2, or a combination of E2 and tamoxifen) incombination with 0.1 μg/mL recombinant human angio-genin (R&D Systems), 50 μg/mL goat anti-human angio-genin neutralizing antibody (R&D Systems), or 50 μg/mLnormal goat IgG antibody (R&D Systems), or by serum-free hormone medium with or without 1 nmol/L E2 ora combination of 1 nmol/L E2 and 1 μmol/L tamoxifen(E2 + Tam). The conditioned medium and serum-freemedium with supplements were changed every 24 hours.After a total of 48 hours of treatment, HUVEC prolifera-tion was determined using the CellTiter 96 AQueous OneSolution Cell Proliferation Assay (Promega). Absorbancewas recorded after 2 hours of incubation with CellTiter96 AQueous One Solution Reagent.

Nuclear translocation of angiogenin in HUVECHUVEC were seeded at a density of 5 × 103 cells/cm2 on

gelatin-coated coverslips (19-mm diameter; VWR) placedin 12-well plates (Costar). The cells were incubated incomplete growth medium for 48 hours, washed thricewith serum-free medium, and incubated with 1 μg/mL re-combinant human angiogenin (R&D Systems) at 37°C for1 hour. Test compounds were added 5 hours before theaddition of angiogenin. After incubation with angiogenin,the cells were washed thrice with PBS and fixed with 100%methanol at −20°C for 10 minutes, blocked with PBS/5%bovine serum albumin, and incubated with 20 μg/mL ofgoat anti-human angiogenin antibody (R&D Systems) for



1 hour. After incubating with primary antibody, the cellswere washed thrice and incubated with Alexa 488–labeledrabbit anti-goat IgG (Invitrogen) at 1:500 dilution for1 hour. The cells were mounted with SlowFade Gold anti-fade reagent with 4′,6-diamidino-2-phenylindole (Invitro-gen) and examined under an Olympus BX41 and IX51light/fluorescent microscope (×40/0.75); images were cap-tured using an Olympus DP70 charge-coupled device cam-era (Solna). Negative controls included omission ofprimary antibody and cells incubated in the absence of an-giogenin. The experiment was repeated thrice.

Quantification of tumor microvessel areaFormalin-fixed, paraffin-embedded tumors were cut in-

to 3-μm sections, deparaffinized, and stained with anti–von Willebrand factor (rabbit anti-human von Willebrand;dilution 1:1000; with Envision detection, DakoCytoma-tion). Sections were counterstained with Mayer's hematox-ylin. Negative controls did not show staining. In a blindedmanner, hotspot fields (×200) were examined in nine tu-mors and six normal breast biopsies for each group. Theimages were digitally analyzed and the percentage of areathat stained positively for von Willebrand factor was quan-tified using ImageJ software version 1.39u (NIH).

Statistical analysisStatistical analysis was done using GraphPad Prism

software. One-way ANOVA with Bonferroni post hoc testand paired or unpaired t test were used where appropri-ate. All statistical tests were two-sided. Results are ex-pressed as mean ± SEM. Statistical significance wasassumed at P < 0.05.

Results

E2 exposure increased extracellular angiogenin levelsand angiogenesis in normal breast tissues inhealthy volunteersThere were no subsequent complications after the mi-

crodialysis experiments. Significant positive correlationsbetween local breast angiogenin levels and local breast tis-sue E2 and plasma E2 levels were detected. A correlationwas observed between breast angiogenin and local E2(r = 0.83, P = 0.0003; n = 14; Fig. 1A) and between angio-genin and plasma E2 (r = 0.9, P < 0.0001; n = 14; Fig. 1B).There was no correlation between plasma P4 and local ex-tracellular breast angiogenin. To investigate if the positivecorrelations found in the microdialysis samples were E2dependent, we set up an experiment using tissue biopsiesof normal breasts. We have previously shown that thesebreast biopsies maintain tissue morphology includingendothelial cells and epithelial cells, which proliferateas shown by the proliferation marker Ki67, without devel-oping necrosis during 7 days of culture (34). Exposureof breast biopsies to E2 increased the secretion of angio-genin into the culture media [2,732 ± 398 pg/mg tissuein the control group versus 8,904 ± 1,479 pg/mg tissuein the E2-exposed group (P < 0.01) and control versus






9,660 ± 1,871 pg/mg in the E2 + P4–exposed biopsies (P <0.01); n = 6 in each group; Fig. 1C]. Next, we investigatedif the breast biopsies exhibited increased microvessel areaafter exposure to the hormones. Fig. 1D shows that bothE2 alone– and E2 + P4–exposed biopsies showed increasedmicrovessel area compared with control treated tissue:1.02 ± 0.13% in the control tissue compared with 5.1 ±0.9% in the E2 tissues (P < 0.0001) and 5.3 ± 0.7% inthe E2 + P4 tissues (P < 0.0001).

Significantly higher levels of angiogenin in breastcancer versus normal breast tissue in patientsTen breast cancer patients were subjected to microdialy-

sis before surgery. One microdialysis catheter was insertedinto the center of the tumor and one catheter into the nor-mal adjacent breast tissue. Routine clinicopathologic datasuch as content of estrogen and P4 receptors (ER and PgR),



tumor histology and grade, and stage of the excised tu-mors were determined. All 10 tumors expressed ER and5 tumors coexpressed ER and PgR. There were significantlyhigher levels of angiogenin in cancerous tissue comparedwith normal breast tissue (9,020 ± 946 pg/mL in cancertissue compared with 5,074 ± 816 pg/mL in normal breasttissue; n = 10; P < 0.0001; Fig. 2). This difference may rep-resent an absolute difference between the tissues, but otherfactors such as epithelial density may have also influencedthe results.

Altered extracellular levels of angiogenin in culturedER-positive breast cancer cells after exposure to E2and tamoxifenTo explore if E2 affected the secretion of angiogenin

from ER-positive breast cancer cells, the cells were culturedwith and without E2. As shown in Fig. 3A, E2 exposure of

Fig. 1. Estradiol increases extracellular angiogenin and angiogenesis in normal human breast tissue. A and B, healthy women were investigated withmicrodialysis for sampling of extracellular proteins in vivo in normal breast tissue. Blood samples were drawn at the time of microdialysis. Eightpremenopausal women with high E2 levels and six postmenopausal women with low E2 levels were included. A, there was a significant positive correlationbetween extracellular breast angiogenin and extracellular local breast E2 (r = 0.83, P = 0.0003, n = 14). B, there was a significant correlation betweenextracellular breast angiogenin and plasma E2 (r = 0.9, P < 0.0001, n = 14). C and D, normal breast tissue biopsies from women undergoing reductionmammoplasty were cultured in the presence of E2 (10−9 mol/L), a combination of E2 and P4 (E2 + P4; 10−9 and 10−8 mol/L, respectively), or serum-freemedium alone supplemented with hormone solvent (control) for 7 d. C, angiogenin released into the culture media. **, P < 0.001, compared with controlbiopsies. Columns, mean; bars, SE. D, microvessel staining and quantification of normal breast tissue biopsies as described in Materials and Methods.Representative tissue sections from the different treatment groups. ***, P < 0.0001, compared with control biopsies. Columns, mean; bars, SE.




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MCF-7 cells significantly increased secreted angiogenin ina dose-dependent manner. Addition of 0.1 μmol/L ful-vestrant (ICI 182,780 or Faslodex), which is a pure ERantagonist by downregulating the ER, abolished theE2-stimulated angiogenin secretion completely (P <0.0001; Fig. 3A). As shown in Fig. 3B, E2 also induced



a 3-fold induction of the mRNA levels of angiogenin(P < 0.01). Moreover, exposure of the ER-negative MDA-MB-231 cells to E2 did not induce an increased secretionof angiogenin (Fig. 3C).In our next experiment, we examined how tamoxifen af-

fected angiogenin secretion in the absence or presence ofE2 in ER-positive breast cancer cells. As Fig. 3D shows, wefound that adding tamoxifen to the E2 treatment of breastcancer cells in culture significantly lowered the angiogeninlevels compared to treatment of cells with E2 alone. Theeffect of tamoxifen increased over time, and after 7 daysof culture, a potent inhibition of the estrogen effects onangiogenin secretion was detected (P < 0.0001; Fig. 3D).Similar results were repeated using another ER-positive

breast cancer cell line, ZR-75-1. After 7 days of culture, thelevels of angiogenin were 306 ± 14 pg/mg protein in con-trol cells compared with 402 ± 17 pg/mg protein in E2-exposed cells (P < 0.001) and 226 pg/mg protein in theE2 + Tam–exposed group (P < 0.0001, compared with E2alone; n = 5 in each group).

Increased proliferation of HUVEC after exposure toconditioned medium from E2-treated cancer cellsAngiogenin interacts with endothelial cells to induce

neovascularization. Therefore, we first set up an experiment

Fig. 3. Estradiol increases angiogenin in estrogen-dependent breast cancer cells. A, human ER-positive MCF-7 breast cancer cells were cultured withouthormones (control) or in the presence of increased concentrations of E2 and with the addition of the pure antiestrogen fulvestrant (Faslodex; 0.1 μmol/L)for 7 d. Cell culture supernatants were analyzed for human angiogenin using quantitative ELISA. B, MCF-7 cells were cultured in 1 nmol/L E2 for 7 dand fold changes in mRNA levels of angiogenin were determined by TaqMan quantitative PCR as described in Materials and Methods. C, humanER-negative MDA-MB-231 were cultured in 1 nmol/L E2 for 7 d and cell culture supernatants were analyzed for human angiogenin using quantitative ELISA.***, P < 0.0001, compared with control cells; ###, P < 0.0001, compared with 1 nmol/L E2; †††, P < 0.0001, compared with 10 nmol/L E2. Columns, mean;bars, SE. D, MCF-7 cells were cultured for 3, 5, and 7 d in the presence or absence of 1 nmol/L E2, 1 μmol/L tamoxifen (Tam), or a combination ofE2 and tamoxifen (E2 + Tam). Cell culture supernatants were analyzed for human angiogenin using quantitative ELISA. ***, P < 0.0001, compared with controlcells; **, P < 0.001, compared with control cells; †††, P < 0.0001, compared with E2; ††, P < 0.001, compared with E2. Columns, mean; bars, SE.

Fig. 2. Angiogenin in vivo levels in breast cancer patients. Tenpostmenopausal breast cancer patients were subjected to microdialysisin the breast cancer and adjacent normal breast tissues before surgery.***, P < 0.0001. Columns, mean; bars, SE.






where HUVEC were treated with E2 and/or tamoxifen di-rectly to confirm the ability of E2 to induce proliferation inour model. Indeed, we found that E2 significantly inducedproliferation of HUVEC, whereas tamoxifen completelyinhibited this induction [0.3 ± 0.02 in the control cellsversus 0.44 ± 0.03 in the E2-exposed cells (P < 0.05) and0.28 ± 0.02 in the E2 + Tam–treated cells (P < 0.01, versusE2-exposed cells; n = 6 in each group)]. In a tumor, pro-teins secreted by the tumor cells affect the endothelialcells. We therefore performed a second set of experimentsin which we treated HUVEC with cell culture medium col-lected from hormone-exposed breast cancer cells. Usingthis approach, we found that HUVEC exposed to condi-tioned medium from E2-stimulated MCF-7 cells proliferat-ed to a significantly higher extent compared with controlcells incubated with medium from non–hormone-treatedMCF-7 cells (P < 0.05; Fig. 4A). In addition, this increase inproliferation was completely reversed by the exposure ofHUVEC to conditioned medium from E2 + Tam–treatedMCF-7 cells (P < 0.01, compared with E2 only; Fig. 4A).Similar results were found when exposing HUVEC to con-ditioned medium from hormone-stimulated ZR-75-1 cells.Cell culture medium originating from E2-treated ZR-75-1cells induced proliferation to a significantly larger degreethan did control medium [0.46 ± 0.02 in E2 medium ver-sus 0.31 ± 0.01 in the control group (P < 0.05; n = 6 ineach group)]. This increase in proliferation by E2 culturemedium was inhibited by the exposure of HUVEC to con-ditioned medium from E2 + Tam–exposed ZR-75-1 cells(0.32 ± 0.01; P < 0.001 versus E2 medium; n = 6 in eachgroup). To explore if the increase in proliferation inducedby E2 was dependent on the increased angiogenin levels in



E2-exposed cancer cells, we first treated HUVEC with re-combinant angiogenin (1 μg/mL) alone or with an angio-genin neutralizing antibody (50 μg/mL). The proliferationof HUVEC treated with recombinant angiogenin signifi-cantly decreased after treatment with the antibody (P <0.05; Fig. 4B). Conditioned medium from E2-stimulatedMCF-7 cells in combination with the antiangiogenin anti-body significantly inhibited the increase in proliferation ofHUVEC detected after treatment with E2-exposed MCF-7cell culture medium (P < 0.05; Fig. 4B). This suggests thatangiogenin present in E2-treated MCF-7 medium affectedthe proliferation of HUVEC. The complete block of E2-induced proliferation by antiangiogenin may be explainedby several mechanisms: the method is not sensitiveenough for fine-tuned differences of HUVEC proliferation;the actions of VEGF and angiogenin are dependent oneach other (i.e., by blocking one of them, the action ofboth is inhibited); the levels of other E2-induced proangio-genic proteins are very low in this cell line.Again, experiments using the ZR-75-1 cancer cell line

confirmed these results. Incubating HUVEC with condi-tioned medium from E2-treated ZR-75-1 cells in combina-tion with an antiangiogenin antibody inhibited theproliferation increase detected with the E2-treated ZR-75-1 medium [0.47 ± 0.02 in the ZR-75-1 E2 group comparedwith 0.36 ± 0.03 in the ZR-75-1 E2 + angiogenin antibodygroup (P < 0.05; n = 6 in each group)].

Tamoxifen inhibited the nuclear translocation ofangiogenin in HUVECThe nuclear accumulation of angiogenin in HUVEC

is essential for its angiogenic activity (9). We used

Fig. 4. Conditioned medium from E2-treated MCF-7 breast cancer cells increases the proliferation of HUVEC. HUVEC were isolated from fresh femaleumbilical cords of female donors. Cells from passages 2 to 3 were seeded into 96-well plates and cultured for 48 h in conditioned medium collectedfrom hormone-treated MCF-7 cells. Proliferation was assessed by MTS assay. A, proliferation of cultured HUVEC after exposure to conditionedmedium from non–hormone-treated MCF-7 cells (MCF-7 control), E2-treated MCF-7 cells (MCF-7 E2), or E2 + Tam–treated MCF-7 cells (MCF-7 E2 + Tam).**, P < 0.001, compared with control cells; ##, P < 0.001, compared with E2 cells. Columns, mean; bars, SE. B, proliferation of HUVEC after exposure torecombinant human angiogenin with or without an antihuman angiogenin neutralizing antibody (Ab) or after exposure to conditioned medium fromE2-treated MCF-7 cells with or without an antihuman angiogenin-neutralizing antibody. *, P < 0.05, compared with vehicle and MCF-7 control; #, P < 0.05,compared with angiogenin alone; †, P < 0.05, compared with MCF-7 E2 cells. Columns, mean; bars, SE.




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immunofluorescence to monitor the nuclear translocationof angiogenin in HUVEC. Incubation with the antibodywithout recombinant angiogenin resulted in very weakto absent immunofluorescence (data not shown). Asshown in Fig. 5A and B, after 1 hour of incubation inthe absence of hormones, the majority of cell-associatedangiogenin accumulated in the nucleus. In the presenceof 1 μmol/L tamoxifen, the amount of nuclear angiogeninwas decreased (Fig. 5C and D). Instead, staining was ob-served in the cytoplasm. No difference in staining was ob-served with incubation of cells with E2 only comparedwith controls cells incubated with recombinant angio-genin only.

Tamoxifen decreased extracellular angiogenin levelsand angiogenesis in solid MCF-7 tumors in nude miceThe bioactive site for angiogenin is the extracellular

space and no sampling or quantification of angiogeninin this compartment has previously been done. In thisstudy, we used microdialysis to sample extracellular fluidin situ of MCF-7 tumors. These tumors require estrogen forgrowth in nude mice; therefore, a nontreated controlgroup is not possible to achieve in vivo. Because angiogen-in expression is regulated by hypoxia (17, 36), all experi-ments were done on size-matched tumors, and H&E



staining confirmed that tumors did not contain any ne-crotic areas. As Fig. 6A shows, there were significantly low-er levels of extracellular angiogenin in microdialysatesfrom tumors treated with tamoxifen in combination withE2 compared with the levels in tumors treated with E2 only(P < 0.001), confirming our in vitro results in cell culture.These tamoxifen-treated tumors also exhibited decreasedvessel area compared with E2-treated tumors (P < 0.001;Fig. 6B). Finally, antiangiogenin treatment resulted in tu-mor stasis, whereas control treated tumors continued togrow (Fig. 6C). This suggests that angiogenin is involvedin estrogen-dependent growth of breast cancer.

Discussion

Here we show for the first time that E2 increases theextracellular levels of angiogenin in normal human breasttissue in vivo. Our in vivo results were confirmed ex vivo byculture of normal human breast biopsies in the presenceof E2 alone or together with P4. P4 had neither a counter-acting nor an additive effect on the angiogenin levelscompared with E2. In breast cancer patients, the extracel-lular angiogenin levels in cancer tissues were twice as highcompared with the normal adjacent breast tissue. We fur-ther show that E2 increased and tamoxifen decreased the

h. 10, 2019. © 2010 American A

Fig. 5. Tamoxifen inhibits thenuclear accumulation ofangiogenin in cultured HUVEC.HUVEC were isolated from freshfemale umbilical cords of femaledonors. Cells from passages 2 to 3were cultured on coverslips andincubated with 1 μg/mLrecombinant human angiogeninat 37°C for 1 h in the absence(A and B) or presence (C and D) of1 μmol/L tamoxifen. Tamoxifenwas added 5 h before the additionof angiogenin. Angiogenin wasvisualized with a goat anti-humanangiogenin antibody and Alexa488–labeled rabbit anti-goat IgG.Images are representative of threeindependent experiments.


ssociation for Cancer




secretion of angiogenin from human breast cancer cells inculture. In vivo, in solid human breast cancers in nudemice, tamoxifen therapy decreased the extracellular levelsof angiogenin and this was associated with decreased an-giogenesis. E2-induced angiogenin derived from cancercells significantly increased endothelial cell proliferationin vitro. Exposure to tamoxifen of HUVEC in culture sig-nificantly decreased the nuclear accumulation of angio-genin. Additionally, treating tumor-bearing mice with anantibody against angiogenin resulted in tumor stasis, sug-gesting that angiogenin is one mechanism involved inestrogen-dependent breast cancer growth.Angiogenesis is a prerequisite for the growth and metas-

tasis of solid tumors, and angiogenin has been shown tobe one important regulator of this process. Inhibition ofangiogenin may prevent and inhibit the growth of humantumors cells inoculated in nude mice (12–14, 37). More-over, increased tissue levels of angiogenin have been de-tected in cancer patients with aggressive disease withpoor prognosis as recently summarized (38). In breastcancer patients, the results have been heterogeneous, onereason being that different sites, serum or tissue, of angio-genin quantification have been used and no correlation be-tween tissue and plasma levels of angiogenin has beenfound (39, 40). It has also been suggested that angiogenincirculates inactive in plasma and that only the extravascular-tissue angiogenin promotes angiogenesis (41). Tissue ho-



mogenate represents both cell-associated and free extra-cellular levels in the tissue. Clearly, a technique thatquantifies the extracellular angiogenin in vivo directly fromthe target tissue would more accurately represent the spe-cific bioactive soluble tissue angiogenin. Microdialysis, atechnique that mimics a blood vessel within a tissue, al-lows direct in situ sampling of extracellular proteins aswe have described previously (42). In this study, noveldata on extracellular angiogenin levels in human breasttissue were achieved by using microdialysis in both nor-mal and cancerous breast tissues. The results clearly showthat E2 is a potent regulator of angiogenin in normalbreast tissue, as the microdialysis data were confirmed us-ing breast tissue biopsies in culture. In human breast can-cers before surgery, the extracellular angiogenin levels wereclose to 10,000 pg/mL, compared with VEGF levels of∼10 pg/mL as we have previously reported (23). Althoughsome differences in the concentrations of the two proteinsmay be attributed to different relative recovery [i.e., theability of the protein to pass over the microdialysis mem-brane (8% for VEGF and 22% for angiogenin)], it cannotfully explain the magnitude of the difference. Previousdata have shown equivalent induction of blood vesselgrowth in vitro at identical concentrations of VEGF and an-giogenin (8). Clinical data have shown a beneficial effectof anti-VEGF (bevacizumab) therapy in breast cancer pa-tients (43). The much higher levels of angiogenin in breast

Fig. 6. Tamoxifen decreases the in vivo levelsof angiogenin and angiogenesis andantiangiogenin therapy decreases the growth ofMCF-7 tumors in nude mice. A and B, micewere oophorectomized and supplemented witha physiologic level of E2. MCF-7 cells wereinjected s.c. and tumors were established onthe right hind flank. At similar tumor sizes, onegroup of mice continued with E2 treatment only,whereas in the other group tamoxifen wasadded to the E2 treatment (E2 + Tam) asdescribed in Materials and Methods.A, microdialysis was done after 2 wk oftreatment on size-matched tumors asdescribed in Materials and Methods.Microdialysates were analyzed for humanangiogenin using quantitative ELISA.**, P < 0.001. Columns, mean; bars, SE.B, the microvessel area of tumors wasquantified as described in Materials andMethods. ***, P < 0.0001. Columns, mean;bars, SE. C, size-matched MCF-7 tumors ofoophorectomized mice supplemented with E2,as described above, were treated with dailyinjections of an antiangiogenin antibody (filledsquares) or a control IgG (filled circles) andtumor size was followed as described inMaterials and Methods. **, P < 0.001.




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cancers suggests that blocking angiogenin would be apowerful molecular approach in inhibiting angiogenesis,and ultimately tumor growth, in breast cancer patients.In our experimental breast cancer model, we show that

tamoxifen reduced the extracellular tumor levels of angio-genin compared with tumors grown in estrogen-treatedmice and that tumors with low levels of angiogenin levelsexhibited significantly decreased angiogenesis. Our in vivodata confirmed the in vitro cell culture results, using twoseparate estrogen-dependent breast cancer cell lines, whereE2 induced a dose-dependent significant increase of extra-cellular angiogenin protein levels, which was abolished onaddition of the pure ER antagonist fulvestrant. Moreover,E2 induced a 3-fold increase of angiogenin mRNA levels.Adding tamoxifen to a physiologic dose of E2 antagonizedthe E2-induced increase of angiogenin, which further sup-ports an estrogen-dependent angiogenin regulation inbreast cancer cells.In the process of tumor angiogenesis, cancer cell–

derived angiogenin acts on endothelial cells in a paracrinemanner. Here we also show that released proteins from E2-exposed cancer cells increased the proliferation of HUVEC.This increase was inhibited by the addition of an antibodyagainst angiogenin, suggesting that the proliferation was,at least in part, mediated by angiogenin. We also showthat inhibition of angiogenin by antibody therapy in vivoresults in tumor stasis. This strongly suggests thatangiogenin is one of the mechanisms behind estrogen-dependent breast cancer growth. Thus, a combination ofantiangiogenin therapy with ER targeting compoundsmay enhance the therapeutic response rate in ER-positivebreast cancer.Nuclear accumulation is essential for the angiogenic ac-

tivity of angiogenin, and inhibition of this nuclear trans-location abolishes the angiogenic activity of the protein(9, 16). The process of nuclear translocation of angiogen-in is, to date, largely unknown. The first step of internal-izing extracellular angiogenin into the cell requiresreceptor-mediated endocytosis (9). Further import to thenucleus may involve passive diffusion of angiogeninthrough the nuclear pore and/or specific proteins involved



in nuclear localization signals (44). Here, we show thattamoxifen decreased the important nuclear accumulationof angiogenin in endothelial cells, whereas E2 exerted noadditionally effects on the nuclear accumulation of angio-genin compared with control cells treated with recombi-nant angiogenin only. This may suggest that an alreadyefficient mechanism for the angiogenin translocation tothe nucleus is difficult to enhance, but can be inhibited.Hence, tamoxifen exerts two potent direct antiangiogenicmechanisms: decreased secretion of angiogenin fromcancer cells and inhibition of the nuclear accumulationof angiogenin in endothelial cells.In summary, this study shows for the first time that an-

giogenin is regulated by E2 in normal human breast tissueand that breast cancers in humans express higher extracel-lular levels of angiogenin compared with normal breasttissue. Tamoxifen counteracted E2-induced angiogenin se-cretion in breast cancer cells, inhibited the nuclear accu-mulation of angiogenin in endothelial cells, and reducedendothelial cell proliferation. Taken together, these resultssuggest previously unknown mechanisms by which E2may tip the balance to favor angiogenesis and for potentantiangiogenic actions of tamoxifen in breast cancer. Thismay be important for the understanding of estrogen-dependent breast carcinogenesis as well as for therapeuticinterventions of breast cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

Swedish Research Council grant 60294601 (C. Dabrosin) and SwedishCancer Society grants 060036 (C. Dabrosin), 070012 (C. Dabrosin), and070049 (C. Dabrosin).

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 toindicate this fact.

Received 02/25/2010; revised 04/26/2010; accepted 05/14/2010;published OnlineFirst 05/25/2010.

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2010;16:3659-3669. Published OnlineFirst May 25, 2010.Clin Cancer Res Ulrika W. Nilsson, Annelie Abrahamsson and Charlotta Dabrosin vivo

InAntiangiogenin Therapy Reduces Breast Cancer Growth Tamoxifen Inhibits Angiogenin Nuclear Translocation and Angiogenin Regulation by Estradiol in Breast Tissue:

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