the canonical nf-kb pathway governs mammary tumorigenesis ... · cancer res; 70(24); 10464–73....

Tumor and Stem Cell Biology

The Canonical NF-kB Pathway Governs MammaryTumorigenesis in Transgenic Mice and TumorStem Cell Expansion

Manran Liu1,3, Toshiyuki Sakamaki4, Mathew C. Casimiro1, Nicole E. Willmarth1, Andrew A. Quong1,Xiaoming Ju1, John Ojeifo5, Xuanmao Jiao1, Wen-Shuz Yeow1, Sanjay Katiyar1, L. Andrew Shirley2,David Joyce6, Michael P. Lisanti1, Christopher Albanese7, and Richard G. Pestell1

AbstractThe role of mammary epithelial cell (MEC) NF-kB in tumor progression in vivo is unknown, as murine NF-kB

components and kinases either are required for murine survival or interfere with normal mammary glanddevelopment. As NF-kB inhibitors block both tumor-associated macrophages (TAM) and MEC NF-kB, theimportance of MEC NF-kB to tumor progression in vivo remained to be determined. Herein, an MEC-targetedinducible transgenic inhibitor of NF-kB (IkBaSR) was developed in ErbB2 mammary oncomice. Induciblesuppression of NF-kB in the adult mammary epithelium delayed the onset and number of new tumors. Withinsimilar sized breast tumors, TAM and tumor neoangiogenesis was reduced. Coculture experiments demon-strated MEC NF-kB enhanced TAM recruitment. Genome-wide expression and proteomic analysis showed thatIkBaSR inhibited tumor stem cell pathways. IkBaSR inhibited breast tumor stem cell markers in transgenictumors, reduced stem cell expansion in vitro, and repressed expression of Nanog and Sox2 in vivo and in vitro.MEC NF-kB contributes to mammary tumorigenesis. As we show that NF-kB contributes to expansion of breasttumor stem cells and heterotypic signals that enhance TAM and vasculogenesis, these processes may contributeto NF-kB–dependent mammary tumorigenesis. Cancer Res; 70(24); 10464–73. �2010 AACR.

Introduction

NF-kB proteins, which play a key role in regulating theinflammatory response (1), participate in 2 intersecting signalpathways. The canonical NF-kB pathway proteins (RelA, c-Rel,and p50) form dimers with similar DNA-binding specificity (2).The dimers are held in inactive cytoplasmic complexes withproteins of the IkB (inhibitor of NF-kB) family. IkB kinases(IKK) phosphorylate IkB bound to NF-kB, directing the IkB toubiquitin-dependent degradation (3), thereby releasing NF-kBdimers to enter the nucleus. The alternate pathway utilizes the

RelB/p52 dimer, which is regulated primarily by the IKKa andinteracts with a mostly distinct subset of target genes (4). NF-kB family members are overexpressed or activated in breastcancer cell lines, primary human breast tumors and a range ofother malignancies (5, 6). Inhibition of NF-kB in vivo enhancedboth the growth of squamous cell tumor (7, 8) and thedevelopment of hepatocellular carcinoma (9). In contrast,the inactivation of IKKb in intestinal epithelial cells reducedcolonic tumor formation in response to chemical procarcino-gens (10).

Efforts to determine the specific role of mammary epithelialcell (MEC) versus inflammatory cell NF-kB in tumorprogression have been confounded by 3 key principles. First,most NF-kB signaling proteins are essential for completingembryogenesis or postnatal mammary growth and develop-ment, precluding the use of genetically deleted mouse modelsfor exploring NF-kB function in mammary tumorigenesis invivo (11). The NF-kB proteins (RelA, p50) and the IkB proteins(IkBa) are expressed during normal murine postnatal mam-mary gland morphogenesis (12). Transplantation experi-ments, using tissue donated by mice deficient in NF-kBactivity due to IkBa overexpression, support a role for NF-kB in mammary epithelial proliferation and branching duringnormal mammary development (13). Analysis of the alternatepathway of NF-kB signaling showed that IKKa and the Rel-B/p52 complex contribute to mammary gland development andIKKa A/Amice have defective development of terminal alveolarbreast buds (14).

Authors' Affiliations: Departments of 1Cancer Biology and 2Surgery, KimmelCancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania; 3KeyLaboratory of Clinical Laboratory Diagnostics, Ministry of Education, Chongq-ing Medical University, Chongqing, China; 4Niigata University of Pharmacy andApplied Life Sciences, Niigata, Japan; 5Center to Reduce Cancer Health Dis-parities, National Cancer Institute, Rockville, Maryland; 6School of Medicine andPharmacology, The University of Western Australia, Perth, WA, Australia; and7Lombardi Comprehensive Cancer Center, Georgetown University, Washington,District of Columbia

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

Corresponding Author: Richard G. Pestell, Kimmel Cancer Center,Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA19107. Phone: 215-503-5649; Fax: 215-503-9334. E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-0732

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Second, NF-kB is expressed widely in multiple distinct celltypes. These distinct cell types (inflammatory cells, bloodvessels, adipocytes, and breast epithelial cells) may eachcontribute independently to the tumor phenotype. The roleof tumor epithelial cell NF-kB in breast tumor progression invivo in the immune-competent state is poorly understood. Theinflammatory response governing interactions between tumorcells and infiltrating immune cells in the tumor microenvir-onment can either promote or inhibit tumor development (1).Thus, dissecting the role of NF-kB in the MEC requires MECcell–specific regulation in immune-competent animals. Third,NF-kB activity may contribute differently during distinctphases of tumorigenesis. Thus, NF-kB may alter its functionto either inhibit or promote differentiation or tumorigenesisduring development or tumor progression. Temporarily con-trolled genetic regulation in the fully developed mammarygland is therefore essential for such analysis.Herein, we determined the role of NF-kB in ErbB2 mam-

mary tumorigenesis in vivo by developing an animal modelthat permits inducible suppression of NF-kB activity, targetedto the mammary gland. Transgenic mice were generated byencoding an ecdysone-regulated and stabilized IkBa protein(IkBa SuperRepressor: IkBaSR), in which serine residues 32and 36 of IkBa have been changed to alanine and are thereforeunavailable to IKKa-mediated phosphorylation and degrada-tion. The 3 cointegrated transgenes allowed temporally con-trolled suppression of NF-kB activity in the normallydeveloped mammary gland. These triple transgenics werecrossed to mammary-targeted ErbB2 to determine therequirement for NF-kB activity in ErbB2-mediated mammarytumorigenesis in vivo.Using spatial and temporal control of MEC NF-kB in vivo,

we show that MEC NF-kB promotes tumor onset, growth,angiogenesis, and the infiltration of tumor-associated macro-phages (TAM). Inhibition of NF-kB correlated with reductionsin epithelial mesenchymal transition (EMT), breast tumorstem cell expansion, and heterotypic signaling in whichMEC recruited TAM.

Materials and Methods

Cell culture, retroviral infection, and luciferasereporter assayThe 293T, NAFA, ErbB2-induced mammary tumor cell

lines and mouse embryo fibroblasts (MEF) were culturedas described (15, 16). MCF10A, MCF10A-NeuT cells werecultured in DMEM/F-12 (50/50, Cellgro; Mediatech Inc.) asrecommended by the American Type Culture Collection.Primary murine MECs were collected from transgenic miceand cultured as previously described (17). To make theMSCV-IkBaSR-IRES-GFP retroviral vector, an EcoRI frag-ment coding for IkBaSR (18) was subcloned into the MSCV-IRES-GFP vector (19) at the EcoRI site upstream of the IRESdriving expression of GFP (20). Viral supernatants wereprepared and target cells were infected as previouslydescribed (20). The expression vector plasmid pVgRXRwas obtained from Invitrogen. The ponasterone A–induciblepBPSTR1-VgEcR/RXRa-EGRE3-IkBaSR vector is an all in

one retrovirus based on the pBPSTR1 system. VgEcR andRXRa were subcloned into pBPSTR1 together with EGRE3-IkBaSR. The NF-kB–responsive reporter construct 3xRel-luc, was as previously described (21). Transfections werecarried out with Superfect transfection reagent (Qiagen) orLipotectamine 2000 Transfection reagent (Invitrogen)according to the manufacture's protocols. The relative fireflyluciferase activities were calculated by normalizing transfec-tion efficiency according to the Renilla luciferase activities.TNF-a was obtained from Sigma. The Student t test wasused for statistical analyses.

Colony formation, cellular proliferation, DNA synthesis,cell cycle, and apoptosis

Colony formation assay, [3H]thymidine (TdR) incorpora-tion, and cell proliferation analysis determined using MTTwere previously described (22). Flow cytometry used for cell-cycle analysis followed the standard protocol (22). Apoptoticcells were assessed using the Annexin V-FITC apoptosisdetection kit (Roche).

Immunohistochemistry and angiogenesisImmunohistochemical staining was carried out using the

EnVisionþ System-HRP following manufacturer's guidelines.Slides were made from paraffin-embedded murine mammarytissue from MMTV-ErbB2 and MMTV-ErbB2/IkBaSR mice(N¼ 5 per group). The following primary antibodies were used[CD31 (CM 303A; Biocare Medical; 1:50); CD34 (ab8158;Abcam; 1:50); vWF (A0082; Dako; 1:500); VEGF (sc-507; SantaCruz; 1:250); VCAM-1 (sc-8304; Santa Cruz; 1:250); CD44(#550538; BD Pharmingen; 1:50); F4/80 (ab6640; Abcam;1:700); FLAG-IkBaSR (A9594; Sigma; 1:100)]. The signal wasvisualized using a cyanine 3 (Cy-3)-labeled Tyramide ampli-fication system (Perkin Elmer Life Sciences, Inc.). Sectionswere mounted in aqueous medium containing DAPI as anuclear counterstain (Vector LABS). A negative control wasused to ensure the specificity of fluorescent immunostainingby replacing primary antibody with a nonimmune rabbit IgG.Microvascular density of the tumors was determined as pre-viously described (23).

Ponasterone A–inducible IkBaSR transgenic miceAnimal experiments were approved by the Georgetown

University and Thomas Jefferson University animal use com-mittees. The transgenic lines carrying the EGRE3-IkBaSRtransgene were crossed with animals expressing mammarygland-targeted MMTV-pVgEcR, CMV-RXRa and EGRE3-b-Galmice (15, 17) to create ponasterone A–inducible IkBaSR(PISA) mice. These lines were then crossed with MMTV-ErbB2mice (16) to create PISA/ErbB2 mice. Transgenic tumoranalysis is described in Supplemental data.

Microarray and cluster analysisMicroarrays were evaluated for normalization as previously

described (24). Arrays were normalized using robust multi-array analysis, and P ¼ 0.05 was applied as statistical criteriafor differential expressed genes. Expression profiles are dis-played using Treeview (25). Classification and clustering for

NF-kB in Tumor Stem Cell Expansion and Angiogenesis

www.aacrjournals.org Cancer Res; 70(24) December 15, 2010 10465



pathway level analysis employed ASSESS (Analysis of SampleSet Enrichment Scores; ref. 26).

Bone marrow macrophage isolation and NAFAcoculture assay

Bone marrow cells obtained from tibiae of 5- to 8-week-oldfemale FVBmice were cultured in 24-well plates (3� 105 cells/0.5 mL per well) in DMEM containing 10% FBS and 50 ng/mLof CSF-1 for 4 days (27, 28). Accordingly, almost all theadherent cells should express macrophage-specific antigensMac-1/CD11b, Moma-2, and F4/80 (28). The macrophageswere enriched and selected according to manufacturers guide-lines (Miltenyi Biotec Inc.).

In the NAFA-macrophage coculture studies, 3� 105 NAFA/GFP or NAFA/IkBSR cells were seeded on coverslips in thebottom of a 24-well plate overnight. The next day, CD11bþ F4/80þ macrophages (3 � 104 macrophages per transwell) wereplaced inside the 24-well plate (29). The cells were coculturedin phenol red–free DMEM containing 5% charcoal strippedserum and 5 ng/mL of CSF-1 for 5 days. After 5 days ofcoculture, the cells on the coverslips were then fixed with4% paraformaldehyde for 20 minutes and permeabilized usingice-cold 0.2% Triton-X in PBS for 2 minutes on ice. Afterwashing, the fixed and permeabilized cells were stained for F4/80 (CI:A3-1; Abcam) with a DAPI nuclear stain and themacrophages were counted to determine how many hadmigrated to the NAFA cells on the coverslips. Images of theF4/80-positive macrophages were captured on a confocalmicroscope (magnification �40).

Mammosphere formation and FACS analysis of stemcell surface markers

Mammosphere formation assays were conducted as pre-viously described (30). Immunostaining of cell surface mar-kers by FACS analysis for breast cancer stem cells was basedon prior publications (31).

Small interfering RNA transfectionSmall interfering RNAs (siRNAs) against NF-kB p105 (J-

003520-09-0010, NM_003998), NF-kB p65 (J-003533-08-0010,NM_021975), and nontargeting siRNA (D-001810-03) wereacquired from Dharmacon RNA Technology and were solu-bilized according to manufacturer's protocol. MCF10A-NeuTcells were transfected by the Nucleofector technology usingNucleofector Kit V and program T-024 (Amaxa Biosystems).Transfection efficiency was monitored by nonsilencing, fluor-escein-labeled siRNA from Qiagen.

Immunoprecipitation and Western blotImmunoprecipitation and Western blot analysis were con-

ducted as previously described (22). The antibodies used weredirected to p65 (Rockland Immunochemicals Inc.), p50(Upstate), E-cadherin (BD Biosciences), ZO-1 (33-9100;Zymed), N-cadherin (3B9; Zymed), fibronectin (F3648; Sigma),b-catenin (E-5; Santa Cruz), vimentin (H-84; Santa Cruz),FLAG (C-21; Santa Cruz), IkBa (C19; Santa Cruz), anti-FLAGM2 Affinity Gel (A2220; Sigma), and the loading controlguanine dissociation inhibitor (GDI; RTG Sol. LLC), vinculin

(V9131; Sigma), or b-actin (Upstate). The appropriateHRP-conjugated secondary antibodies were subsequentlyapplied, and immunodetection was conducted using theECL procedure.

RNA isolation, RT-PCR, and quantitative real-time PCRTotal RNA was isolated from NAFA cells infected with

IkBaSR-inducible system, and MMTV-ErbB2 and MMTV-ErbB2/IkBaSR mammary tumors used Trizol (17). One-stepRT-PCR for IkBaSR transgene expression was evaluated usingan mRNA Selective PCR kit (Takara Shuzo) according to themanufacturers’ instructions. Expression values were normal-ized to ribosomal protein L19 (RPL-19).

SYBR Green–based real-time PCR reactions were carriedout using QuantiTect SYBR Green PCR kit (Qiagen) andQuantiTect prevalidated Primer Assays for mouse MMP3,MMP9, PDGF-AA, ICAM1, Arcp30, and 18S rRNA as internalcontrol following manufacturer's recommendations on an ABIPrism 7900HT system (Applied Biosystems Inc.). All primersequences used in RT-PCR were listed in the Material Sup-plementary Table.

Results

Transgenic mice express IkBaSR, targeted to MECsGermline deletion of the NF-kB proteins results in embryo-

nic lethality. IkBa-deficient mice die at�9 days postnatally. Atransgenic system, in which IkBaSR expression was targetedto the mammary gland and temporally regulated by ecdysonereceptor activation, was developed to determine the role ofNF-kB in tumorigenesis. A cell line derived from tumors oftransgenic mice in which activated ErbB2 was targeted to themammary gland (NAFA) was used. In the presence of thestably integrated pVgEcR/RXRa/EGRE3-IkBaSR expressionvector, ponasterone A induced expression of the EGRE3-IkBaSR transgene in NAFA cells as determined by Westernblotting either for the FLAG epitope or for IkBaSR (Supple-mentary Fig. S1A). This led to suppression of NF-kB activity, asmeasured with the 3xRel-luc luciferase reporter construct(Supplementary Fig. S1B).

Founder lines cointegrating the EGRE3-IkBaSR transgenewere established (Supplementary Fig. S1C). Transgenic miceexpressing MMTV-VgEcR, RXRa, EGRE3-b-Gal, EGRE3-IkBaSR are known as PISA mice. The MEFs created fromPISA/ErbB2 transgenicmice were transfectedwith the NF-kB–responsive reporter, 3xRel-luc, and treated with ponasterone Ato induce IkBaSR expression (Supplementary Fig. S1D). Ponas-terone A induced IkBaSR FLAG epitope expression in theMEFs, as also demonstrated by immunohistochemical staining(Supplementary Fig. S1E). In the absence of ponasterone A,TNF-a induced NF-kB activity 15-fold at 48 hours, which wasinhibited �60% upon induction of the endogenous IkBaSRtransgene with ponasterone A (Supplementary Fig. S1F).

Endogenous NF-kB regulates epithelial cellproliferation and survival

Inducible transgenic IkBaSR expression was confirmed byWestern blot analyses of 3 separate MEC cultures (Fig. 1A).

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Inhibition of NF-kB reduced cellular growth and DNA synth-esis by approximately 35% to 50%, as measured by MTTreduction and TdR incorporation (Fig. 1A and B) but didnot significantly influence cell-cycle distribution in MEC (datanot shown).Ponasterone A induced expression of IkBaSR in NAFA cells

transfected with the pBPSTR1-VgEcR/RXRa-EGRE3-IkBaSRvector (Fig. 1C). After 5 days of IkBaSR expression, NAFAproliferation was reduced by �35% (Fig. 1C). The proportionof cells in the DNA synthetic phase of the cell cycle wasreduced by induction of IkBaSR expression via ponasterone Ain NAFA cell lines (Fig. 1D). The NAFA tumor cells formedcolonies in soft agar and induction of the IkBaSR transgenereduced colony growth 20% to 35% (Fig. 1D). IkBaSR sup-presses, and thus NF-kB maintains, cell proliferation andcolony formation of ErbB2-derived murine mammary tumorcell lines. The trend was mirrored in a PISA/ErbB2-derivedtumor cell line (Supplementary Fig. S2)

Endogenous NF-kB activity regulates initiation ofErbB2-induced mammary tumorigenesis

Mammary tumors develop spontaneously in PISA/ErbB2mice in the absence of IkBaSR transgene induction. To circum-vent the requirement for NF-kB in normal ductal development,the multigenic PISA/ErbB2 mice were allowed to develop tomaturity and then treated with ponasterone A or placeboby pellet implantation from 13 weeks, to study tumor onset(SupplementaryFig. S3A), or23weeks, tomeasuregrowth-linkedoutcomes. In the absence ofponasteroneA inductionof IkBaSR,PISA/ErbB2mice developedmultifocal mammary tumors (Sup-plementary Fig. S3B and C). In contrast, PISA/ErbB2 mice inwhich IkBaSR had been induced with ponasterone A rarelydeveloped tumors by 21 weeks. Tumors that arose in mammaryglands of IkBaSR-expressing transgenic mice were typicallyfewer in number (Supplementary Fig. S3C).

When NF-kB activity was suppressed after 23 weeks of age(Fig. 2A), IkBaSR transgene expression reduced the average

A

C D

B

Figure 1. NF-kB enhances DNA synthesis, cellular proliferation, or colony formation in MECs and NAFA cells carrying ponasterone A (Pon A)-induciblesystem. A, induction of IkBaSR expression by ponasterone A in MECs (left) and effect on proliferation (right); N � 16. B, DNA synthesis in MEC wasanalyzed by [3H]thymidine incorporation (48-hour ponasterone A treatment, N ¼ 3). C, inducible IkBaSR expression in NAFA cells carrying ponasteroneA–inducible system (pBPSTR1-VgEcR/RXRa-EGRE3-IkBaSR) determined by RT-PCR (left) and the effect on proliferation as determined by MTT assay(right). D, cell-cycle distribution was determined using FACS (N � 2), showing a decline in the proportion of S phase after IkBaSR induction viaponasterone A exposure for 48 hours (left) and reduced colony formation in soft agar after ponasterone A treatment for 2 weeks (right); 5 mmol/L ofponasterone A was used in culture. *, P < 0.05; **, P < 0.01.





number of tumors developing per mouse significantly over thefollowing 6 weeks (Fig. 2B). Fewer ponasterone A–treatedanimals had any tumors after 2 weeks and the differencewas significant at 5 weeks (Fig. 2C and D). Together, thesestudies show for the first time that in immune-competenttransgenic mice mammary, NF-kB activity governs the rate ofinitiation and number of ErbB2 tumors.

NF-kB regulates MEC gene expression of reactiveoxygen species, EMT, and stem cells

Gene expression profiles were compared between MMTV-ErbB2 animals and ponasterone A–treated PISA/ErbB2 (Sup-plementary Fig. S4). Gene targets of NF-kB were identifiedwith functions in cell survival, cell-cycle control, signaling,DNA repair, chromosomal organization, biogenesis, and func-tions in transcription, cytoskeleton formation, transport, andmetabolism (Supplementary Fig. S4A; SupplementaryTable S1). To identify functional pathways regulated by endo-genous NF-kB, genome-wide enrichment analysis was con-ducted using ASSESS. These studies identified pathwaysinhibited by IkBaSR including NF-kB signaling, reactive oxy-gen species production, EMT, cellular invasion, cellular pro-liferation, inflammation, and vasculogenesis (SupplementaryFig. S4B).

MEC NF-kB determines recruitment of TAMs andangiogenesis during ErbB2 tumorigenesis

Gene expression array analysis of the transgenic micemammary tumors (Supplementary Fig. S4) identified anumber of genes inhibited by IkBaSR that promote angio-genesis (Supplementary Table S2). As the growth of tumorsbeyond 1 to 2 mm requires the induction and maintenanceof blood supply (32), we examined the influence of IkBaSRtransgene expression on tumor vascularity in ponasteroneA- and placebo-exposed PISA mice. Tumors of transgenicmice expressing IkBaSR demonstrated a �45% reduction intumor microvessel density (Fig. 3A). Tumors were lessvascular by histomorphometric and immunohistochemicalanalyses (Fig. 3B). Integrin engagement activates mammarytumor NF-kB in vitro when assayed with 3D matrigelcultures (33). We examined candidate proangiogenic extra-cellular matrix (ECM) integrin-binding proteins to deter-mine the mechanism by which NF-kB promoted mammarytumor angiogenesis in vivo. Neither the b1a4 integrin-binding protein VCAM1 nor the avb3-binding ECMprotein vWF changed in abundance (Fig. 3C and D). Incontrast, VEGF abundance and the endothelium-expressedPECAM-1/CD31 were significantly reduced by IkBaSR exp-ression in vivo.

weeks

A

B C D

Figure 2. IkBaSR delays ErbB2-inducedmammary tumor onset. A, a group of 37 PISA/ErbB2mice was treated from 23weeks of age with ponasterone A (PonA; n ¼ 19) or placebo (n ¼ 18; left). Inducible IkBaSR expression in transgenic mice mammary tumor treated with ponasterone A and placebo, displayed byimmunoprecipitation with anti-FLAG and detection with anti-IkBa (right). B, average number of tumors per mouse in ponasterone A- or placebo-exposedgroups. C, Kaplan-Meier plots of the proportion of animals free of mammary tumor in ponasterone A- and placebo-treated groups. D, percentage of mice ineach group free of tumor at 60 days after pellet implantation. *, P < 0.05, **, P < 0.01.

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Mouse models with defective CSF-1 signaling due to muta-tion of the CSF-1 receptor suggested that TAMs contribute tomurine mammary tumor progression by enhancing angiogen-esis (34). It has been hypothesized, but never proven, thatmammary epithelial tumor–induced NF-kBmay contribute tomammary tumor TAM recruitment. To determine whetherMEC NF-kB regulates TAM recruitment, we examined theinfiltration of bone marrow macrophages assayed using F4/80immunostaining. IkBaSR expression reduced the proportionof F4/80-staining macrophages within the mammary tumorsby �50% (Fig. 4A). F4/80-expressing macrophages were iso-lated by fluorescence-activated cell sorting and migration wasassessed (Fig. 4B). The lower chamber of the Boyden chambercontained equal numbers of either cultured NAFA-IkBaSR orNAFA-GFP control cells. The number of migrating macro-phages was reduced �40% in the presence of NAFA-IkBaSR(Fig. 4C). Thus, NF-kB within MEC contributes heterotypicsignals to recruit TAM.

NF-kB in mammary tumors maintains progenitor cellexpansion

In view of the finding that NF-kB inhibition reduced EMT,we determined the function of NF-kB in regulating stem cellfeatures in transgenic mice. CD44 is a transmembrane cellsurface protein that is essential for the homing and stem cellproperties of leukemic stem cells and serves as a useful markerfor solid tumor–initiating cells in mammary tumors (35).IkBaSR expression reduced the proportion of CD44-positivecell in PISA/ErbB2 tumor by �50% (Fig. 5A).

A small number of primary breast cancer cells, tumor-initiating cell (TIC), or cancer stem cells form secondarytumors (36). TICs form nonadherent mammospheres whencultured under specific conditions in the absence of serum.Inhibition of NF-kB activity reduced mammosphere numberby greater than 60% in cell lines derived from PISA/ErbB2mice and in Met-1 cells (Fig. 5B). Cancer stem cells can beenriched by sorting for CD44þ/CD24�/low cells in human and

A

C D

B

Figure 3.Reduced vasculogenesis and bonemarrowmacrophages in PISA/ErbB2mammary tumors. Analysis of mammary tumors in PISAmice (N¼ 10), withor without ponasterone A (Pon A)–induced IkBaSR expression, to determine: (A) microvessel density; (B) histopathology by hematoxylin and eosin (H&E)staining (left), immunostaining to demarcate CD31 (middle) andCD34 (right)-positive tumor blood vessels, and (C) immunohistochemical staining for candidateproangiogenic factors; D, quantitation of immunohistochemical staining for proangiogenic factors, shown as mean � SEM (*, P < 0.05, **, P < 0.01). Allphotographs were taken using 40� objective.





murine breast cancer cells (37, 38). In mouse breast tumorcells, CD44þ/CD24� cells are enriched 50- to 100-fold in tumorgrowth potential in syngeneic transplantation and areenriched for expression of stem cell–associated genes (37).Reduction of endogenous NF-kB through IkBaSR expressionreduced the proportion of CD44þ/CD24�/low cells by 15% inthe cell lines derived from PISA/ErbB2 mice (Fig. 5C) and 75%in a second murine cell line Met1 (Fig. 5D).

The molecular circuitry governing embryonic stem (ES)cells is active in many tumors, with altered expression ofkey ES cell regulators (Oct4, Sox2, and Nanog). mRNA fromtumors of transgenic mice examined by quantitative real-timePCR (QT-PCR; Fig. 6A) for candidate ES cell regulators (Sox2,Oct4, and Nanog) showed a reduction in Sox2 (�60%) andNanog (�30%). Expression of IkBaSR reduced Nanog andSox2 promoter activity in the PISA/ErbB2-derived cell line 50%and 90%, respectively (Fig. 6B). p65/p50 induced the Sox2promoter 3-fold and the Nanog promoter was induced 6-fold.IkBaSR reduced NF-kB–dependent activity of the promoter(Fig. 6C and D).

Discussion

The current studies include the first in vivo analysis of themolecular genetic pathways regulated by endogenous MECNF-kB. The PISA mouse provided a valid model for examiningmammary tumorigenesis without disturbing antecedent phy-siologic mammary gland development or disturbing other NF-

kB–regulated functions, such as inflammatory and immuneresponses, in the tumor. This system allowed normal NF-kBactivity throughout the animal during development, providinga physiologically valid background to determine the function ofNF-kB in the normally developed adultmammary gland. Local,timed suppression of NF-kB activity in these multiply trans-genicmicewas used to examine the significance ofMECNF-kBon tumor progression and local heterotypic signals. MECIkBaSR induction delayed tumor appearance and reducedaverage tumor number. Suppressed tumor formation rate invivo was reflected in IkBaSR-regulated suppression of growthin PISA MEC lines in vitro. IkBaSR expression induced byponasterone did not reduce the relative expression of ErbB2mRNA compared with vehicle control (N ¼ 6; 8.59 vs. 8.58) bymicroarray or abundance by Western blot analysis (Supple-mentary Fig. S5). Inhibition of MEC NF-kB in vivo reduced theinfiltration of F4/80-staining macrophages and reduced tumorvascularity and features of neoangiogenesis. Reconstitutionanalysis showed that endogenous breast tumor epithelial cellNF-kB governs macrophage recruitments. Through selectivelyinactivatingMEC NF-kB in the adult mammary gland, we haveshown that MEC NF-kB governs a heterotypic signal in vivothat controls the initiation ofmammary tumor growth, recruitsTAM, and promotes neoangiogenesis.

The role of MEC NF-kB in generating angiogenesis andspecific heterotypic signals was previously unknown. IkBaSRexpression reduced angiogenesis in mammary tumors ofPISA/ErbB2 mice. Microarray analysis showed a reductionin expression of gene clusters regulating angiogenesis and a

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C

B

Figure 4.Mammary epithelial cell NF-kB determines recruitment of TAMs. Analysis of mammary tumors in PISA mice (N¼ 10), with or without ponasterone A(Pon A)–induced IkBaSR expression to determine: (A) immunohistochemical staining and quantitation for F4/80; (B and C) macrophage migration assays inresponse to secreted factors of IkBaSR-expressing mammary tumors. Schematic representation of protocol in which bone marrow macrophages weredifferentiated with G-CSF for 5 days and then assessed for migration through a transwell toward NAFA/GFP or NAFA/IkBaSR cells seeded on the bottom of aBoyden chamber. Macrophages that had migrated to the bottom were stained for F4/80 and counted (C, right) (**, P < 0.01).

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subset of secreted factors capable of regulating neovasculo-genesis. Analysis of the mechanisms by which MEC NF-kBregulated angiogenesis identified reduced secretion of VEGFand several other factors (Acrp30, Mip2, Rantes, K6, MMP-3,MMP-9) in the NAFA cell line expressing IkBaSR, correspond-ing to a reduction in gene expression within the mammarytumor expressing IkBaSR (data not shown). The reduction inangiogenesis may have contributed to the delay in onset and/or progression of the ErbB2 tumors.Herein, MEC NF-kB enhanced the infiltration of TAM.

Granulocyte colony-stimulating factor (G-CSF) is producedin advanced human and murine breast cancers (39). Breasttumor–derived CSF-1 increased hematopoietic stem cells andendothelial progenitor cell numbers in themouse (39). In priorstudies investigating the potential role of macrophages inmammary tumor progression, the polyoma middle T antigenwas expressed in the murine mammary gland and the animals

crossed withmice expressing amutation of the CSF-1 receptor(34). Tumor progression and angiogenesis were reduced in themice expressing a mutant CSF-1 receptor. Although CSF-1receptor is expressed on vascular smooth muscle cells, thesestudies were consistent with a model in which CSF-1 recruit-ment of TAM contributes to mammary tumor progression.The mechanisms by which macrophages contribute to breasttumor progression are unclear. Macrophage infiltration cor-relates with angiogenesis (34) and bonemarrow cells recruitedthrough the neuropilin-1 receptor promote neoangiogenesisformation in mice (40).

The current studies provide several lines of evidence that thecanonical NF-kB pathway contributes to self-renewal of mam-mary tumor cells in vivo. First, genome-wide analysis implicatedNF-kB function in regulating genes involved in ES cell function.Second, expression of CD44, a transmembrane receptor used asa marker of human breast tumor stem cells (35), was reduced

A

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B

D

Figure 5. NF-kB promotes breast tumor stem cell marker expression and mammosphere production. A, analysis of breast cancer stem cell marker CD44 inmammary tumors of PISAmice (N� 3), with or without IkBaSR induced by ponasterone A (Pon A). B, mammosphere assays (N� 4) were conducted on breastcancer cell derived from 3 separate PISA/ErbB2 cell lines (left) and in Met-1 cells (right), and quantitation was conducted after 10 days. Treatment was with theIKKb inhibitor (Bay11-7082, 10 mmol/L). C and D, stem cell surface markers (CD24�/low/CD44þ/þ) of breast cancer cell line PISA/ErbB2 (C) or Met-1 (D). Cellswere transduced with IkBaSR or control vector (N � 3). (**, P < 0.01).





�50% in the transgenicmouse tumors in vivo upon induction ofthe IkBaSR transgene. Third, mammosphere production, arecognized surrogate for self-renewal, using cell lines derivedfrom the transgenic mice, was inhibited by IkBaSR expression.Fourth, the generation of CD44high/CD24�/low cells, character-istic of human andmurinemammary stemcells, was reduced byIkBaSR expression (37, 38). The specific mechanism by whichNF-kB regulates self-renewal may involve transcriptionalrepression of the key regulators of ES cell identity (Sox2 andNanog). Herein, IkBaSR was shown to regulate the abundanceof Sox2 and Nanog inmammary tumors and to directly regulatetheir gene expression. Our findings with the canonical NF-kBpathway are consistent with studies of the alternate (IKKa)pathway in breast tumor self-renewal using a kinase knockinmodel (41). The canonical NF-kB and alternate pathways areoften distinct and may be mutually antagonistic. The keratino-cyte defect of IKKa�/� mice is NF-kB independent (42). IKKahas been shown to suppress NF-kB activation by acceleratingthe turnover of RelA and c-Rel, and inactivation of IKKa, incontrast with inactivation of the canonical NF-kB pathway, canlead to enhanced inflammation (43). An IKKaAA/AA knockinmouse model demonstrated a role for IKKa in normal mam-mary gland development and mammary epithelial stem cellexpansion (14, 41). Importantly, no alteration in NF-kB activitywas observed in the WT versus IKKaAA/AA mammary tumors,suggesting that the IKKa-mediated induction of mammary

stem cell expansion involves distinct mechanisms from thecurrent studies that specifically inactivated NF-kB signaling.Collectively, these studies are consistent with a role for both thecanonical and alternative NF-kB pathways in maintainingtumor stem cells.

Disclosure of Potential Conflicts of Interest

The Pennsylvania Department of Health specifically disclaims responsibilityfor any analysis, interpretations, or conclusions. There are no conflicts ofinterest associated with this study.

Acknowledgments

The authors thank Atenssa L. Cheek and Stefanie Pierpoint for the prepara-tion of the manuscript.

Grant Support

This work was supported in part by R01CA70896, R01CA75503, R01CA107382,R01CA86072 (R.G.P.), and R01CA120876 (M.P.L). The Kimmel Cancer Center wassupported by the NIH Cancer Center Core grant P30CA56036 (R.G.P.). Thisproject is funded in part from the Dr. Ralph andMarian C. FalkMedical ResearchTrust and a grant from Pennsylvania Department of Health (R.G.P.).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 03/08/2010; revised 09/28/2010; accepted 10/05/2010; publishedOnline 12/15/2010.

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2010;70:10464-10473. Cancer Res Manran Liu, Toshiyuki Sakamaki, Mathew C. Casimiro, et al. ExpansionTumorigenesis in Transgenic Mice and Tumor Stem Cell

B Pathway Governs MammaryκThe Canonical NF-

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