Lactoferrin Endothelin-1 Axis Contributes to the …...Tumor and Stem Cell Biology Lactoferrin–Endothelin-1 Axis Contributes to the Development and Invasiveness of Triple-Negative

Tumor and Stem Cell Biology

Lactoferrin–Endothelin-1 Axis Contributes to theDevelopment and Invasiveness of Triple-Negative BreastCancer Phenotypes

Ngoc-Han Ha1, Vasudha S. Nair1, Divijendra Natha Sirigiri Reddy1, Prakriti Mudvari1, Kazufumi Ohshiro1,KrishnaSumanthGhanta1, SureshB. Pakala1, Da-Qiang Li1, LuisCosta2,4, Allan Lipton5, RajendraA. Badwe2,6,Suzanne Fuqua7, Margaretha Wallon3, George C. Prendergast3, and Rakesh Kumar1,2

AbstractTriple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptor-a (ER-a),

progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). However, pathwaysresponsible for downregulation of therapeutic receptors, as well as subsequent aggressiveness, remain unknown.In this study, we discovered that lactoferrin (Lf) efficiently downregulates levels of ER-a, PR, and HER-2 in aproteasome-dependent manner in breast cancer cells, and it accounts for the loss of responsiveness to ER- orHER-2–targeted therapies. Furthermore, we found that lactoferrin increases migration and invasiveness of bothnon-TNBC andTNBC cell lines.We discovered that lactoferrin directly stimulates the transcription of endothelin-1 (ET-1), a secreted proinvasive polypeptide that acts through a specific receptor, ET(A)R, leading to secretion ofthe bioactive ET-1 peptide. Interestingly, a therapeutic ET-1 receptor-antagonist blocked lactoferrin-dependentmotility and invasiveness of breast cancer cells. The physiologic significance of this newly discovered Lf–ET-1 axisin the manifestation of TNBC phenotypes is revealed by elevated plasma and tissue lactoferrin and ET-1 levels inpatients with TNBC compared with those in ERþ cases. These findings describe the first physiologically relevantpolypeptide as a functional determinant in downregulating all three therapeutic receptors in breast cancer, whichuses another secreted ET-1 system to confer invasiveness. Results presented in this article provide proof-of-principle evidence in support of the therapeutic effectiveness of ET-1 receptor antagonist to completely block thelactoferrin-inducedmotility and invasiveness of the TNBC aswell as non-TNBC cells, and thus, open a remarkableopportunity to treat TNBC by targeting the Lf–ET-1 axis using an approved developmental drug. Cancer Res;71(23); 1–11. �2011 AACR.

Introduction

Among all breast cancers, approximately 10% to 15% arecategorized as triple-negative breast cancer (TNBC; refs. 1–3).TNBC is characterized by the presence of low levels or absenceof estrogen receptor-alpha (ER-a), progesterone receptor (PR),

and human epidermal growth factor receptor (HER-2; ref. 4),and lack of effective therapies targeting these receptors leadsto poor prognosis (4, 5). While reviewing the previously pub-lished literature, we noticed evidence of an inverse correlationbetween the levels of PR or ER-a and lactoferrin (Lf; ref. 6) inendometrial adenocarcinomas or in primary breast tumors,respectively (7–10). Lactoferrin, a member of the transferrinfamily, was first discovered as an extracellular iron-bindingglycoprotein. Since then, lactoferrin has been extensively stud-ied and shown to play a major role in anti-inflammation andbactericidal events. Because lactoferrin is a hormone-respon-sive gene (11) and its levels aremodulated by a variety of signals(12), we hypothesized that elevated levels of lactoferrin may beassociated with a decreased expression of ER-a and PR and,perhaps, HER-2 and, therefore, could contribute to the devel-opment of TNBC phenotypes. Considering lactoferrin's abun-dance in exocrine secretions and its proliferative potentialon certain cell types, in this study we aimed to investigatethe role of lactoferrin in the development of TNBC, anddiscovered that lactoferrin efficiently downregulates the levelsof ER-a, PR, and HER-2 at a posttranscriptional level inmultiple breast cancer cell lines. Furthermore, we found thatlactoferrin-induced increased invasion of breast cancer cells

Authors' Affiliations: 1Department of Biochemistry andMolecular Biologyand 2Global Cancer Genomic Consortium, The George Washington Uni-versity, Washington, District of Columbia; 3Lankenau Institute for MedicalResearch, Wynnewood, Pennsylvania; 4Hospital de Santa Maria, Facul-dade de Medicina de Lisboa, Lisbon, Portugal; 5Penn State HersheyCancer Institute, Penn State College of Medicine, Hershey, Pennsylvania;6Department of Surgical Oncology, TataMemorial Hospital,Mumbai, India;and 7Lester and Sue Smith Breast Center, Baylor College of Medicine,Houston, Texas

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

V. S. Nair and D.N.S. Reddy contributed equally to this study.

Corresponding Author: Rakesh Kumar, Department of Biochemistry andMolecular Biology, The George Washington University, 2300 Eye Street,NW, Ross 530, Washington, DC 20037. E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-1143

�2011 American Association for Cancer Research.

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mechanistically mediated via transcriptional stimulation ofendothelin-1 (ET-1; ref. 13) and could be effectively blocked bytherapeutic antagonists of the ET-1 receptor. Although themechanism of loss of receptors during the development ofTNBC is currently understood poorly, our study provides aninsight into the molecules/pathways that could be responsiblefor this progression and, consequently, could contribute to thedevelopment of TNBC phenotypes.

Materials and Methods

Human patient samplesDr. Susanne Fuqua (Baylor College of Medicine, Houston,

Texas) provided human breast cancer tissue RNA samples. Dr.George C. Prendergast (Lankenau Institute for MedicalResearch, Wynewood, Pennsylvania) provided the plasmasamples along with the corresponding tissue slides. All thehuman samples were used in accordance with the InstitutionalReview Board procedures at the respective institutions.

Cell lines and cell cultureThe breast cancer cell lines MCF-7, ZR-75, MDA-MB-231,

MDA-MB-468, and SK-BR-3 and the colon cancer cell lineCaco-2 were obtained from the American Type Culture Col-lection. All cells were maintained at 37�C in 5% CO2 inDulbecco's Modified Eagle's Medium (DMEM)/F12-50/50(Mediatech, Inc.) supplemented with 10% FBS (Atlanta Biolo-gics) and 1% antibiotics (Gibco, Invitrogen). During serum-starvation, cells were incubated with serum-free DMEM sup-plemented with 1% antibiotics.

TheTNBCcell lineMDA-MB-231was selected for generatingstable clones. The cells were transfected with the followingplasmids: pcDNA 3.1 (Invitrogen; as control), ER-a, and HER-2.The stable clone cell lines (pcDNA, ER, and HER-2) weremaintained inDMEM/F12-50/50, supplementedwith 10% FBS,1% antibiotics, and 0.5 mg/mL of G418 (Sigma-Aldrich).

Protein analysisFor lactoferrin treatment, cells were maintained in serum-

free DMEM for 48 hours and then treated with 20 mg/mL oflactoferrin (Sigma-Aldrich). Protein sampleswere separated bySDS-PAGE and then transferred onto a nitrocellulose mem-brane (Bio-Rad). The primary antibodies used included: anti-ER-a (Bethyl Laboratories), anti-HER-2 (Bethyl Laboratories),anti-PR (Dako), anti-IGF-IRb (Santa Cruz Biotechnology, Inc.),anti-RXRa (Santa Cruz Biotechnology, Inc.), anti-cyclin D1(Labvision/Thermo Fisher Scientific), anti-tubulin (Sigma-Aldrich), and anti-vinculin (Sigma-Aldrich).

Immunohistochemistry and confocal microscopyDeparaffinized sections were treated with 0.3% hydrogen

peroxide in methanol and subjected to antigen retrieval byboiling the sections in antigen-unmasking solution (VectorLaboratories). The sections were then blocked with 5%skimmed milk in PBS (Mediatech Inc.) and incubated withlactoferrin antibody (Lf-Ab; 1:500) or endothelin antibody(1:100) at 4�C overnight, followed by incubation with EnVision(Dako) for 1 hour at room temperature and visualization with

liquid DABþ substrate chromogen system (Dako). Immuno-stained sections were lightly counterstained with hematoxylin,dehydrated in graded ethanol, cleared in xylene, and mountedwith the use of the permount mounting medium.

For confocal imaging, ZR-75 orMCF-7 cells were plated ontocover slips in 6-well plates, starved for 48 hours, and treatedwith 20 mg/mL of lactoferrin. After a 12-hour treatment, thecells were fixed with 4% paraformaldehyde, permeabilized in0.1% Triton X-100, and blocked in 10% normal goat serum inPBS. The cells were incubatedwith primary antibodies, washed3 times in PBS, and then incubated with goat anti-mouse orgoat anti-rabbit secondary antibodies conjugated with Alexa-488 or Alexa-555 (Molecular Probes). 40,6-Diamidino-2-pheny-lindole (DAPI) was used for nuclear staining. The slides werethen examined using a Zeiss LSM 710 confocalmicroscope andimages were acquired with the help of Zen 2009 software.Images were converted to TIFF format using Image J software.

Northern blottingFor Northern blotting, MCF-7 as well as MDA-MB-231 and

MDA-MB-468 cells were treated with 100 mg/mL lactoferrin for6 hours. A RNA sample amount of 20 mg was loaded onto aformaldehyde denaturing gel (1.5% agarose, 2.2 mol/L formal-dehyde). Subsequently, RNA was transferred to a Hybond-Nmembrane (GE Healthcare UK Limited) and then immobilizedthrough covalent linkage to themembranebyUVcross-linker.Acomplimentary RNA ET-1 probe was prepared using Ambion'sIn Vitro Transcription Kit (Ambion, Inc.) with radiolabeledUTP. Finally, themembrane was exposed to a phosphor imagerand the autoradiogram was developed using the Storm 865Imager (GE Healthcare UK Limited). For another experiment,MDA-MB-231 cells were plated and then serum-starved for 48hours. The cells were either treated with 20 mg/mL of cyclo-heximide (CHX), 100 mg/mL lactoferrin, or both. CHX wasadministered 1 hour before lactoferrin treatment. After 6 hoursof treatment with lactoferrin, RNA was isolated from the cellsusing TRIzol method, and RNA levels were detected using theNorthern blotting protocol as described above.

Wound-healing assayToanalyze the effect of lactoferrinwith orwithout tamoxifen

on cell migration, MCF-7 cells were plated with 10% fetal calfserum (FCS)–DMEM. Before treatment, each plate receivedmultiple "wounds" with a 200-mL pipette tip. Treatments werecarried out with low-serum medium supplemented with100 mg/mL lactoferrin and 100 nmol/L tamoxifen (Sigma-Aldrich). In another set, the cells were treatedwith andwithoutlactoferrin in addition to 10 nmol/L of Herceptin (Genentech,Inc.) and in combination. After 24 hours, each plate wasexamined for the amount of wound closure by measuring thephysical separation remaining between the original woundwidths using the Olympus DP2-BSW digital camera software(Olympus). Ten separate measurements were made per plate,and each experiment was carried out in triplicate.

Migration assayMCF-7 cells were serum-starved for 48 hours and then

resuspended in the presence of 0.1% BSA. Subsequently, 1 �

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105 cells/well were loaded onto the upper well of an uncoatedBoyden chamber. ET-1 and BQ123 were diluted in plainmedium before plating. The lower chamber was supplementedwith conditioned medium of NIH-3T3 fibroblasts grown inDMEM/F-12 medium with 0.1% BSA. After fixation with meth-anol and staining with 0.05% crystal violet, the number of cellsthat successfullymigrated through the filter was counted. Datawere analyzed using the Olympus DP2-BSW digital camerasoftware.

Invasion assayCells were serum-starved for 48 hours and then seeded at a

density of 1 � 105 cells/well in the upper well of the MatrigelInvasion Chambers (BD Biosciences). At the time of the cellseeding, each well was treated with 100 mg/mL of lactoferrin.For E2 treatment, cells were serum-starved in phenol red–freemedium for 48 hours. At 6 hours before trypsinizing forcollection, the cells were treated with 100 mg/mL of lactoferrin.After 24- to 48-hour incubation, the number of the invaded cellswas counted and the results were expressed as percentages ofcontrol. For MDA-MB-231 andMDA-MB-468, the invaded cellsat the bottom were fixed with 4% paraformaldehyde andstained for DAPI after 48 hours.

Microarray analysis of lactoferrin-regulated genesMDA-MB-231 and MDA-MB-468 cells were plated in tripli-

cates, serum-starved for 48 hours, and then treated with 100mg/mLof lactoferrin for 6 hours. RNAwas isolated usingTRIzolaccording to the manufacturer's instructions. cDNA sampleswere generated and hybridized onto an Affymetrix HumanExon 1.0 ST Array chip. GeneSpring GXwas used to process thedata and statistical analysis was carried out by means of anunpaired t-test. A fold change of �1.5 was used to identifydifferentially regulated genes and those with a P-value � 0.05were considered statistically significant. Heat-map analysis ofthe identified genes for individual arrays carried out using theMultiExperiment viewer version 4.4 (Dana-Farber CancerInstitute).

Luciferase assayMCF-7 as well as MDA-MB-231 andMDA-MB-468 cells were

grown to 50% confluency. ET-1 promoter, b-galactosidase, andcontrol plasmids were transfected into cells using FuGENE 6according to the manufacturer's instructions (Roche). Afterlactoferrin treatment, cell lysates were prepared using 200 mLof Tropix Lysis solution (Applied Biosystems). For luciferasepromoter activity, 10 mL of lysate was incubated with 100 mLLuciferase Assay Substrate solution (Promega) and measure-ments were taken using LUMAT LB 9507 luminometer (Bert-hold Technologies). b-Galactosidase was used as a transfectioncontrol.

Electrophoretic mobility shift assayPCR-amplified DNA fragments or oligos containing lacto-

ferrin consensus sites were 50-end labeled using g32p and PNKenzyme. End-labeled fragments were purified using sephadexG-50 spin columns (GEHealthcare UKLimited). Each fragmentwas incubated with purified holo- or apo-lactoferrin–treated

(GenWay Biotech) or lactoferrin-treated as well as untreatednuclear extracts for 15 minutes at room temperature. Thereaction samples were resolved on a 5% native PAGE for 2hours. Gels were removed and dried and exposed to phosphorimager. The final images were developed using the Storm 865Imager (GE Healthcare UK Limited).

Plasma protein detection using ELISAFor detection of lactoferrin, the AssayMax Human Lacto-

ferrin ELISA Kit (AssayPro) was used with a 1:100 dilution ofserum samples. ET-1 levels were measured using the Quanti-Glo Human Endothelin-1 Kit (R&D Systems) by following themanufacturer's manual of instructions. For ET-1 levels, the1450 Microbeta Jet Microplate Scintillation and LuminescenceCounter (Perkin Elmer Life and Analytical Sciences) were usedto measure the luminescence. A dilution of 1:5 for the serumwas used for the ET-1 ELISA assay.

RNA isolation and quantitative real-time PCRTotal RNA from human breast tumor tissue samples as well

as human breast cancer cell lines was isolated using TRIzol(Invitrogen) in accordance with themanufacturer's protocol. A2-mg RNA sample was used to synthesize cDNA using Invitro-gen's SuperScript III First-Strand Synthesis SuperMix by fol-lowing the manufacturer's instructions. The following primers(synthesized by Sigma-Aldrich) were used for target genes:lactoferrin forward: 50-ACCGCAGACATGAAACTTGT-30 andlactoferrin reverse: 50-GGGGGAGTCTCTCTTTATGC-30; ET-1forward: GCCAAGGAGCTCCAGAAACAGCAG ET-1 reverse:AGCAGGAGCAGCGCTTGGAC; actin forward: 50-TCCCTGGA-GAAGAGCTACGA-30; and actin reverse: 50-GTACTTGCGCT-CAGGAGGAG-30. Normalized expression of target geneswere calculated relative to endogenous expression of actin

(relative expression ¼ 2 �DCtð Þ).

Statistical analysisPaired Student t test was used for statistical significance of

differences in numerical data. All of the statistical tests were 2-sided. P value of less than 0.05was considered to be statisticallysignificant.

Results

Lactoferrin downregulates ER-a, PR, and HER-2receptor in human breast cancer cells

Although a large body of studies have firmly established thesignificance of the loss or reduced expression of ER-a, PR, andHER-2 in TNBC, the nature of the endogenousmolecule(s) thatmight be responsible for the downregulation of these thera-peutic receptors and, thus, progression of breast cancer cells tomore invasive phenotypes, remains unknown. As a first step toexplore this hypothesis, we examined the effect of recombinantiron-saturated lactoferrin on the levels of the 3 receptors in ER-positive ZR-75 breast cancer cells. As illustrated in Fig. 1A,lactoferrin stimulation of breast cancer cells results in a time-dependent downregulation of ER-a, PR, and HER-2 starting 6hours post treatment. Interestingly, levels of ER-a and HER-2returned to basal levels in the treated cells after 24 hours. The

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noted downregulation of these receptors by lactoferrin was notdue to a generalized toxicity as therewas no effect of lactoferrinon the levels of the insulin-like growth factor (IGF)-IRb andretinoid X receptor a (RXRa) receptors, whereas lactoferrinstimulation was accompanied by increased expression ofcyclin D1, implying an active cell-cycle progression of lacto-ferrin-treated cells (Fig. 1A). Furthermore, we did not observeany evidence of an inhibitory effect of lactoferrin on the overallprotein synthesis (Supplementary Fig. S1A). The observeddownregulation of therapeutic receptors by lactoferrin wasnot limited to ZR-75 cells because lactoferrin also downregu-lated the basal levels of ER-a, PR, and HER-2 in ER-positiveMCF-7 cells (Supplementary Fig. S1B) as well as in HER-2-positive ER-negative breast cancer SK-BR-3 or colon cancerCaco-2 cells (Supplementary Fig. S1C). Because downregula-tion of therapeutic receptors by a single dose of lactoferrin wastransient in nature, we next assessed whether noted down-regulation of receptors could be sustained by repeated lacto-ferrin dosing or by increasing the dose of lactoferrin. We foundthat replenishing the cultures with fresh or higher doses oflactoferrin results in a sustained downregulation of ER-a, PR,and HER-2 (Fig. 1B). To validate these findings, we confirmeddownregulation of all 3 receptors in ZR-75 (Fig. 1C) or MCF-7cells (Supplementary Fig. S1D) by immunocytochemical stain-ing and scanning confocalmicroscopic examination. Together,these findings indicate that persistent stimulation of breastcancer cells by lactoferrin may downregulate therapeuticreceptors, and thus, in principle, may contribute, to the devel-opment of TNBC phenotypes.

Posttranscriptional regulation of therapeutic receptorsby lactoferrin

To understand the underlying basis of lactoferrin down-regulation of therapeutic receptors, we examined the effect oflactoferrin on the levels of receptor mRNA by Northern hybrid-ization. There is no significant inhibitory effect of lactoferrintreatment on the levels of ER-a, PR, andHER-2mRNA (Fig. 2A).For subsequent studies, we decided to examine the effect oflactoferrin on ER-a and HER-2 as models of therapeuticreceptors in TNBC. In addition, there was no effect of lacto-ferrin on the half-life of the newly synthesized 35S-labeled ER-aor HER-2 (Supplementary Fig. S1E). However, cotreatment ofthe cells with a proteasomal inhibitor, MG-132, preventedlactoferrin-induced dose- and time-dependent downregula-tion of ER-a and HER-2 protein (Fig. 2B), indicating that thelactoferrin-mediated notable reduced expression of ER-a orHER-2 may use mechanisms that are sensitive to inhibitors ofproteasomal degradation of proteins. To further validate theposttranscriptional control of ER-a and HER-2 by lactoferrin,we next generated isogenic pooled clones ofMDA-MB-231 cellsstably expressing ER-a or HER-2 (Fig. 2C). We found thatlactoferrin treatment of clones is also accompanied by down-regulation of ectopically expressed ER-a and HER-2 (Fig. 2D),which indicates that lactoferrin uses posttranscriptionalmech-anism(s) to downregulate HER-2 or ER-a in breast cancer cells.

Lactoferrin promotes invasiveness of breast cancer cellsOur observation of lactoferrin stimulation of cyclin D1

expression (Fig. 1A), an endpoint for the G1 to S-phase

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Figure 1. Lactoferrin downregulatesER-a, PR, and HER-2 in humanbreast cancer cells. A, effect of asingle dose of lactoferrin on thelevels of indicated proteins in ZR-75cells. Cells were treated with20 mg/mL of lactoferrin for indicatedtimes and subjected to Westernblot analysis. B, effect of multipleand increasing doses of lactoferrinon expression levels of proteins inZR-75 cells. C, analysis of ER-a,PR, and HER-2 levels in ZR-75 cellsby confocal microscopicexamination. Cells were treatedwith 20 mg/mL of lactoferrin for 12hours, fixed, and then stained withprotein-specific antibodies. Alexa-488 (ER-a and HER-2), Alexa-555(PR), and DAPI (nucleus). Bar, 5 mm.Cont, control.

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progression, indicated that lactoferrin exposure to ZR-75 cellsmay promote cell-cycle progression. Indeed, we found thatlactoferrin treatment of ZR-75 cells leads to an acceleratedtransition into S-phase compared with untreated control cells(Supplementary Fig. S2A). As lactoferrin stimulation of breastcancer cells was accompanied by downregulation of ER-a, PR,and HER-2 receptor, characteristic of TNBC (Fig. 1A), wehypothesized that lactoferrin stimulation of ER-positive cellsmay antagonize the responsiveness of breast cancer cells totamoxifen. In support of this notion, we showed that seruminduced cell migration as well as estrogen-mediated invasionof MCF-7 cells (Fig. 3A and C, respectively) or ZR-75 cells(Supplementary Fig. S2B) could be effectively blocked bytamoxifen. However, this action of tamoxifen was compro-mised when cells were pretreated with lactoferrin, whichdownregulates ER-a and, consequently, is the target of tamox-ifen (Fig. 3A and C; Supplementary Fig. S2B). Similarly, tounderstand the implication of lactoferrin-mediated downre-gulation of HER-2 on the ability of cells to respond to HER-2–directed therapies such as Herceptin, we next showed thatlactoferrin pretreatment of HER-2–positive breast cancer cellsreduced the effectiveness of Herceptin to inhibit serum-stim-ulated cellmigration and heregulin-induced invasion inMCF-7cells (Fig. 3B and D, respectively) and in ZR-75 cells (Supple-mentary Fig. S2C). These results indicate that lactoferrinstimulation of ER- or HER-2–positive breast cancer cells isaccompanied by the loss of responses to tamoxifen or Hercep-tin, presumably due to the loss of its target receptors (Fig. 3;Supplementary Fig. S2C). However, because we did not observea complete loss of sensitivity of lactoferrin-treated cells to

tamoxifen orHerceptin,we hypothesized that lactoferrin couldpossibly be contributing to the increase in migration andinvasion (14) independent of receptor downregulation. There-fore, we presume that lactoferrin is involved in the activation ofa gene(s) that could augment migration and invasion of thesebreast cancer cells. On the basis of these results, as suspected,lactoferrin strongly promoted the migration and invasivenessof TNBC lines such as MDA-MB-231 and MDA-MB-468 asevaluated by the wound-healing or Matrigel invasion assays(Fig. 3E; Supplementary Fig. S2D), indicating the involvementof receptor-independent activation of promigration/proinva-sion gene(s).

Lactoferrin stimulates ET-1 transcriptionTo gain insight into the molecular basis of lactoferrin-

induced cell invasiveness of TNBC, we next determined thenature of lactoferrin-regulated genes usingmicroarray analysisof lactoferrin-treated or untreated MDA-MB-231 and MDA-MB-468 cells (Fig. 4A). This analysis led to the identification of 3lactoferrin-regulated genes (i.e., Et-1, Rgs-2, and Tcbp-1) in boththe cell lines (Fig. 4B). Guided by the functions of these genes inthe context of our findings in this study, we decided to focus onET-1 (15), a vasoactive peptide first isolated from vascularendothelial cells (15) that is upregulated in certain humancancers (16,17) including breast cancer (18). We found that,indeed, lactoferrin increased the steady-state levels of ET-1mRNA in MDA-MB-468, MDA-MB-231, and MCF-7 (Fig. 4C).Lactoferrin-induced increased ET-1 mRNA expression couldnot be blocked by the inclusion of a protein-synthesis inhibitor,CHX (Fig. 4D), indicating transcriptional regulation of ET-1 by

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Figure 2. Lactoferrin downregulates ER-a, PR, and HER-2 at a transcriptional level. A, Northern blot analysis of ER-a, PR, and HER-2 mRNAs in MCF-7 cellstreated with or without 100 mg/mL of lactoferrin for indicated times. B, MCF-7 cells were treated with or without lactoferrin for indicated time points or withdifferent concentrations of lactoferrin in the presence or absence of proteasomal inhibitor, MG-132. Vinculin was used as a loading control. C, MDA-MB-231stable clonesexpressingER-aorHER-2weregeneratedasdescribed in theMaterials andMethods section. Vinculinwasusedasa loading control. D, effect oflactoferrin on the levels of ER-a and HER-2 in MDA-MB-231 pooled clones stably expressing ER-a or HER-2. Cont, control; DMSO, dimethyl sulfoxide;Untrans, untransfected.






lactoferrin. Consistently, lactoferrin also stimulated the tran-scription driven by the ET-1 promoter-luc in breast cancer cells(Fig. 5A). A close scanning of the ET-1 promoter revealed thepresence of 3 lactoferrin-motifs in the ET-1 promoter. Resultsfrom the electrophoretic mobility shift assay (EMSA) usingoligonucleotides encompassing all 3 potential lactoferrin-interacting DNA regions (Supplementary Fig. S3A) in theET-1 promoter indicated the formation of specific lactofer-rin–DNA complexes with all 3 sites (Fig. 5B). On the basis of

this, next we individually mutated lactoferrin consensusmotifs 1 to 3 in the full-length ET-1 promoter-luc and foundno significant impairment in the ability of lactoferrin tostimulate the transcription of ET-1 with promoter mutantsites 1 and 2 in comparison with consensus motif number 3.This indicates that lactoferrin may preferentially use lactofer-rin consensus motif number 3 to stimulate ET-1 transcription(Fig. 5C). To identify the specific base-pair in the functionallactoferrin consensus site 3 that might be responsible for

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Figure 3. Lactoferrin promotes breast cancer cell migration and invasiveness and compromises anti-invasive activity of tamoxifen (Tam) andHerceptin (HCT).A, effect of lactoferrin on cellmigration in tamoxifen-treatedMCF-7 cells. Right, thequantification of the averagedifference inwound closure between0 and24hours; left, micrographs show representative images ofmigrated cells after 24 hours of treatment. B, effect of lactoferrin on cell migration inHerceptin-treatedMCF-7 cells. Right, quantification of the average difference in wound closure between 0 and 24 hours; left, micrographs show representative images ofmigrated cells after 24 hours of treatment. C, effect of lactoferrin on cell invasion in tamoxifen-treatedMCF-7 cells. E2was added to the upperwells to enhancetamoxifen effect. D, effect of lactoferrin on cell invasion inHerceptin-treatedMCF-7 cells. Heregulinwas added to the upperwells to enhanceHerceptin effect.E, effect of lactoferrin on the invasiveness of MDA-MB-231 and MDA-MB-468 cells. Left, micrographs that are representative images of invaded cells; right,quantification of the average number of invaded cells. Error bars indicate SD. Scale bars, 0.5 mm. �, P < 0.01. Cont, control.

Ha et al.





lactoferrin-binding, we created 3 mutant versions of the lac-toferrin consensus site 3 and tested these oligos for lactoferrin-binding using EMSA (Supplementary Fig. S3A). There was adrastic reduction in the binding of lactoferrin to oligo number3, indicating that lactoferrin may preferentially interact withthe GGCACTTGG motif in the ET-1 promoter (Fig. 5D). Fur-thermore, we observed a progressive increase in the levels ofprotein–DNA complexes that could be effectively super-shiftedby inclusion of Lf-Ab but not isogenic immunoglobulin G(IgG; Fig. 5E, lanes 6 and 7). Furthermore, only recombinantiron-saturated lactoferrin (holo-Lf), but not non-iron saturatedLf (apo-Lf), interacted with the ET-1 promoter oligo (Fig. 5F),and suchprotein–DNAcomplexes could be super-shifted by Lf-Ab (Fig. 5E, lanes 9–12). Consistent with these findings, iron-saturated holo-Lf and not apo-Lf downregulated ER-a andHER-2 in ZR-75 cells (Supplementary Fig. S3B). Taken together,these findings establish that ET-1 is a lactoferrin-induciblegene and that only the holo form of lactoferrin is responsiblefor the observed downregulation as well as increased inva-siveness in breast cancer cells. This is particularly interestingbecause a study by Duarte and colleagues (19) showed thattreatment of human breast cancer cell lines, Hs578T and T47D,with bovine apo-Lf increased apoptosis and decreased cellmigration. However, as indicated in our present data, weobserved an upregulation of ET-1, which is responsible for thelactoferrin-induced invasion andmigration, with the holo formof lactoferrin only. Consistent with these results, apo-lacto-ferrin did not bind to the ET-1 promoter and, subsequently,was unable to drive the transcription of the gene. Therefore,

we propose that the increase in invasion, as well as migration,in breast cancer cells is likely to be due to the upregulationof ET-1 after iron-saturated lactoferrin treatment.

Lf–ET-1 axis promotes TNBC phenotypeTo establish the physiologic relevance of lactoferrin stimu-

lation of ET-1, we next showed that lactoferrin-mediatedincrease in expression of ET-1 was also accompanied by asignificant accumulation of ET-1 in the conditioned media.Interestingly, the levels of baseline ET-1 in TNBC lines such asMDA-MB-231 and MDA-MB-468 were substantially higherthan that in MCF-7 cells (Fig. 6A). To assess the significanceof secreted ET-1 in the observed lactoferrin-mediated migra-tion, we determined the effect of an antagonist of the ET-1receptor [ET(A)R (20) BQ123 (21–23)], on the action of lacto-ferrin. We found that inclusion of BQ123 in the culturesabrogated the ability of lactoferrin as well as of recombinantET-1 to promote migration and invasion of MCF-7 cells in theBoyden chamber (Fig. 6B) as well as in the Matrigel invasion(Fig. 6C) assays, respectively.

To assess the significance of these observations in a phys-iologic setting, we determined the status of lactoferrin inhuman breast tumors from patients with TNBC or ERþ/PRþ

by quantitative real-time PCR (qRT-PCR). We found that thelevels of lactoferrin (Fig. 7A) as well as ET-1 (Fig. 7B) weresignificantly increased in TNBC specimens compared withthose in the control ERþ/PRþ breast tumors. Because bothlactoferrin and ET-1 are secreted polypeptides, we next deter-mined the levels of lactoferrin and ET-1 in the plasma samples

CHXLf

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Figure 4. Endothelin-1 is a transcriptional target of lactoferrin. A andB, analysis of differentially regulated genes after lactoferrin treatment inMDA-MB-231 andMDA-MB-468 cells by Affymetrix Human Exon 1.0 STmicroarrays using Gene-Spring GX 10.0.2. C, effect of lactoferrin on ET-1mRNA levels in breast cancercells. Actin was used as a loading control. D, effect of lactoferrin in the presence or absence of cycloheximide (CHX) on the induction of ET-1 mRNA in MDA-MB-231 cells. Cont, control.






from TNBC as well as from ER/PR/HER-2–positive patients.We observed an elevated level of ET-1 as well as lactoferrin inTNBC when compared with the ER/PR/HER-2–positive sam-ples (Fig. 7C). Consistent with these findings, we also foundstrong and medium matching staining of lactoferrin and ET-1in TNBC samples when compared with ER-a, PR, and HER-2–positive tissue samples as determined by immunohistochem-istry. Data in Fig. 7D illustrate representative examples ofincreased levels of lactoferrin and ET-1 staining in the tumorspecimens from patients with TNBC as compared with the ER/PR/HER-2–positive tumors, providing a proof-of-principle evi-dence of a correlative nature of lactoferrin and ET-1 in tumorsamples. Collectively, these findings revealed that lactoferrin-mediated increase in invasiveness of breast cancer cells ismediated via increased expression and secretion of ET-1, atleast in part, and these changesmight account for the generallynoted aggressive behavior of TNBC (Fig. 7E).

Discussion

Findings presented in this article show for the first timethat lactoferrin, a ubiquitous secretory protein, can down-regulate all 3 therapeutic receptors, ER-a, PR, and HER-2, inbreast cancer cells. This observation is particularly interest-ing as it raises the possibility that increased expression oflactoferrin could contribute to the development of TNBCphenotypes due to the loss or downregulation of thesereceptors. Because we have also observed an increasedexpression of lactoferrin in tumor specimens as well as inthe levels of circulating lactoferrin in plasma samples fromthe patients with TNBC as compared with those in samplesfrom ERþ patients, we hypothesize a potential contributingrole of lactoferrin in supporting the TNBC phenotype. In thiscontext, it is noteworthy that an earlier correlative report,dating back to the 1990s, has shown an inverse relationship

A

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Figure 5. Lactoferrin stimulatesendothelin-1 transcription. A, effectof lactoferrin on ET-1 promoteractivity in breast cancer cells. Cellswere treated with 100 mg/mL oflactoferrin for 36 hours. B, EMSAanalysis of lactoferrin binding to all 3lactoferrin consensus DNAsequences in the ET-1 promoterusing the nuclear extracts fromMCF-7 cells treated with or withoutlactoferrin for 6 hours. C, effect oflactoferrin on the ET-1 promoterwith wild-type ormutant lactoferrin-binding sequences in MDA-MB-468 cells. D, EMSA analysis oflactoferrin binding tooligonucleotides encompassing thewild-type lactoferrin consensusDNA sequence 3 or its 3 mutantversions using the nuclear extractsfrom MCF-7 cells treated with orwithout lactoferrin for 6 hours.E, EMSA analysis of lactoferrin-stimulated nuclear extracts fromMCF-7 cells in the presence ofantibody to lactoferrin. Antibody torabbit-IgG was used as a negativecontrol. F, effects of holo- or apo-Lfon the ET-1 promoter-luc activity inMDA-MB-468 cells. Error barsindicate SD. �,P < 0.05; ��,P < 0.01;��, P < 0.001. Cont, control.

Ha et al.





between the levels of ER-a and lactoferrin (7). In addition,there are immunocytochemical studies that show changes inthe synthesis and secretion of lactoferrin in breast cancer(24); however, in endocervical adenocarcinoma, lactoferrinhas been identified as a useful predictive marker (25).Furthermore, while our studies were being conducted,Schulz and colleagues have also noticed an increased expres-sion of lactoferrin in samples of patients with TNBC (26).Our finding that lactoferrin stimulation of both non-TNBC

andTNBC cells was accompanied by an increased invasivenessis of particular importance as it indicates the possibility thatthe loss of the 3 receptors by lactoferrin is not only associatedwith an expected insensitivity to antiestrogen or Herceptin butalso contributes to invasiveness of breast cancer cells—bothcentral features of TNBC.While exploring the molecular basis of lactoferrin-induced

invasion of breast cancer, we identified lactoferrin-regulatedgenes using a microarray approach. These and other molec-ular studies presented in this article led to the identification

of ET-1, another secreted protein, as the newest direct targetof lactoferrin. This is particularly exciting as ET-1 has beenpreviously shown to promote breast cancer progression, andincreased amounts of ET-1 participates in the process ofinvasion and metastasis, and ET-1 levels correlate well witha diminished disease-free and overall survival rate (18). ET-1exerts its biologic effects through an autocrine and/orparacrine manner through specific receptors and supportstumor cell proliferation, invasion, angiogenesis, and neovas-cularization (27–33). The relevance of these mechanisticstudies to a physiologic setting was recognized by the notedco-overexpression of lactoferrin and ET-1 in tumors as wellas elevated circulating levels in serum from TNBC as com-pared with samples from ER-, PR-, and HER-2–positivebreast tumors.

Because the ET-1 pathway is being actively targeted viaspecific pharmacologic inhibitors to its receptor and Food andDrug Administration (FDA)-approved ET-1 receptor antago-nists are moving forward in clinical trials, these studies raise a

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Figure 6. Endothelin-1 receptor antagonist blocks lactoferrin-dependent invasiveness. A, concentration of ET-1 in the conditioned media from breast cancercells treatedwith or without 100 mg/mL of lactoferrin for 24 hours asmeasured by ELISA. B, effect of ET-1 receptor inhibitor BQ123 on cell migration ofMCF-7cells treatedwith orwithout lactoferrin or ET-1, usingBoyden chambers. C, effect of ET-1 receptor inhibitor BQ123 on cell invasion ofMCF-7 cells treatedwithor without lactoferrin or ET-1 using Matrigel invasion chambers. Error bars indicate SD. �, P < 0.001.






tangible opportunity to use such ET-1 inhibitors for slowingdown the progression and invasiveness of TNBC. Resultspresented in this article provide proof-of-principle evidence

supporting therapeutic effectiveness of ET-1 receptor antago-nists to completely block the lactoferrin-induced motility andinvasiveness of the TNBC cells, and thus, open a remarkable

AR

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Yes9 ++ ++ Yes10 ++ ++

Non-TNBC 1 +++ +++ YesYes2 +++ +++ Yes3 ++ ++ Yes4 ++ ++ Yes5 ++ ++

Type Slide no. Lf staining ET-1 staining Match

n = 8(80%)

n = 2(20%)

n = 2(40%)

n = 3(60%)

P = 0.0028 P = 0.00437P = 0.000293 P = 0.000101

Figure 7. Lf–ET-1 axis promotes TNBC phenotypes. A, quantitative PCR analysis of lactoferrin mRNA levels in tumor samples from patients with or withoutTNBC. B, qRT-PCR analysis of ET-1mRNAs in TNBC or non-TNBC breast tumors. mRNA experiments were repeated 3 times, with each point in triplicate. C,concentration of lactoferrin and ET-1 in plasma samples from patients with or without TNBC by ELISA. D, immunohistochemical evaluation of lactoferrin andET-1 expression in breast tumors from the same patients used in (B). Table depicts TNBC and non-TNBC tissue samples with scoring of lactoferrin and ET-1staining intensity: þþþ (strong); þþ (medium). E, working model for lactoferrin-mediated downregulation of receptors and cell migration/invasion in TNBCcells. Error bars indicate SD.

Ha et al.





opportunity to treat TNBC by targeting the Lf–ET-1 axis usingan approved developmental drug.As expected, similar to many other new discoveries, the

present studies have not only provided several significant leadsthat can be followed, but they have also posed a number of newquestions to the scientific community such as nature of factorsthat might promote persistent elevation of lactoferrin.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

The Editor-in-Chief of Cancer Research is an author of this article. In keepingwith the AACR's Editorial Policy, the paper was peer reviewed and a member of

the AACR's Publications Committee rendered the decision concerningacceptability.

Grant Support

Work in R. Kumar's laboratory was supported by NIH grant CA090970 to R.Kumar.

The costs of publication of this article were defrayed in part by thepayment of 584 page charges. This article must therefore be hereby markedadvertisement in 585 accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 5, 2011; revised September 23, 2011; accepted September 29,2011; published OnlineFirst October 17, 2011.

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Published OnlineFirst October 17, 2011.Cancer Res Ngoc-Han Ha, Vasudha S. Nair, Divijendra Natha Sirigiri Reddy, et al. Cancer PhenotypesDevelopment and Invasiveness of Triple-Negative Breast

Endothelin-1 Axis Contributes to the−Lactoferrin

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