mol cancer res-2012-bohrer-1294-305.pdf

8/9/2019 Mol Cancer Res-2012-Bohrer-1294-305.pdf

1/13

2012;10:1294-1305. Published OnlineFirst August 14, 2012.Mol Cancer Res

Laura R. Bohrer and Kathryn L. Schwertfeger Tumor Cell Migration and Invasion in a Cxcr2-Dependent MannerMacrophages Promote Fibroblast Growth Factor Receptor-Driven

Updated version

10.1158/1541-7786.MCR-12-0275doi:

Access the most recent version of this article at:

Material

Supplementary http://mcr.aacrjournals.org/content/suppl/2012/08/14/1541-7786.MCR-12-0275.DC1.htmlAccess the most recent supplemental material at:

Cited Articles http://mcr.aacrjournals.org/content/10/10/1294.full.html#ref-list-1This article cites by 42 articles, 13 of which you can access for fr ee at:

Citing articles

http://mcr.aacrjournals.org/content/10/10/1294.full.html#related-urlsThis article has been cited by 2 HighWire-hosted articles. Access th e articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints [email protected]

To order reprint s of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

[email protected]

To request permission to re-use all or part of this article, contact the AACR Publications Department at

on October 1, 2014. 2012 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from

Published OnlineFirst August 14, 2012; DOI: 10.1158/1541-7786.MCR-12-0275


http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-12-0275http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-12-0275http://mcr.aacrjournals.org/content/suppl/2012/08/14/1541-7786.MCR-12-0275.DC1.htmlhttp://mcr.aacrjournals.org/content/10/10/1294.full.html#ref-list-1http://mcr.aacrjournals.org/content/10/10/1294.full.html#ref-list-1http://mcr.aacrjournals.org/content/10/10/1294.full.html#related-urlshttp://mcr.aacrjournals.org/content/10/10/1294.full.html#related-urlshttp://mcr.aacrjournals.org/cgi/alertshttp://mcr.aacrjournals.org/cgi/alertsmailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://mcr.aacrjournals.org/http://mcr.aacrjournals.org/http://mcr.aacrjournals.org/http://mcr.aacrjournals.org/http://mcr.aacrjournals.org/http://mcr.aacrjournals.org/mailto:[email protected]:[email protected]://mcr.aacrjournals.org/cgi/alertshttp://mcr.aacrjournals.org/content/10/10/1294.full.html#related-urlshttp://mcr.aacrjournals.org/content/10/10/1294.full.html#ref-list-1http://mcr.aacrjournals.org/content/suppl/2012/08/14/1541-7786.MCR-12-0275.DC1.htmlhttp://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-12-0275


2/13

Angiogenesis, Metastasis, and the Cellular Microenvironment

Macrophages Promote Fibroblast Growth Factor Receptor-Driven Tumor Cell Migration and Invasion in a

Cxcr2-Dependent MannerLaura R. Bohrer and Kathryn L. Schwertfeger

AbstractIn ltration of immune cells, specically macrophages, into the tumor microenvironment has been linked to

increased mammary tumor formation and progression. Activation of growth factor receptor signaling pathways within mammary epithelial cells, such as the broblast growth factor receptor 1 (FGFR1) pathway, inducesrecruitment of macrophages to the mammary epithelium. These macrophages promote increased epithelial cellproliferation and angiogenesis. However, the specic mechanisms by which these macrophages are regulatedby thepreneoplastic epithelial cells and the mechanisms of action of the macrophages within the developing FGFR1-driven tumor microenvironment remain unknown. In this study, we show that activation of inducible FGFR1 in

mammary glands leads to decreased activity of the TGFb

/Smad3 pathway in macrophages associated with early stage lesions. Further studies show that macrophages have increased expression of inammatory chemokines thatbind Cxcr2 following exposure to conditioned media from mammary epithelial and tumor cells in which the FGFpathway had been activated. The increase in these ligands is inhibited following activation of the TGFb pathway,suggesting that decreased TGFb signaling contributes to the upregulation of these chemokines. Using coculturestudies, we further show that macrophages are capable of promoting epithelial and tumor cell migration and invasion through activation of Cxcr2. These results indicate that macrophage-derived Cxcr2 ligands may beimportant for promoting mammary tumor formation regulated by FGFR signaling. Furthermore, these resultssuggest that targeting Cxcr2 may represent a novel therapeutic strategy for breast cancers that are associated withhigh levels of inltrating macrophages. Mol Cancer Res; 10(10); 1294 305. 2012 AACR.

IntroductionBreast tumor formation and progression involve complex

interactions between tumor cells and their surrounding environment. In ltration of immune cells into the tumor microenvironment has been linked to tumor formation and progression (1). Specically, increased numbers of tumor-associated macrophages are linked to poor prognosis inbreast cancer patients (2). Although macrophages wereinitially expected to inhibit tumor growth via their cytotoxicfunctions, it is now clear that exposure to the tumor microenvironment polarizes macrophages towards a tumor-promoting phenotype (3). Therefore, obtaining a better understanding of the mechanisms through which

macrophages regulate tumor growth and progression may result in the development of strategies that either inhibit theactivities of tumor-promoting macrophages or reprogramthe macrophages to increase their tumor cytotoxic activities.Numerous functions have been ascribed to macrophagesduring tumor progression, including promotion of tumor cell invasion, angiogenesis and immune suppression (4). Although published studies have identied speci c mechan-isms through which macrophages contribute to mammary tumor metastasis in mouse models (5, 6), less is knownregarding the mechanisms of macrophage function during mammary tumor initiation.

Macrophages associated with late stage tumors have beenshown to express high levels of TGFb (7). TGFb, which is a keyregulator of development, immune function and wound

healing, acts primarily through a core-signaling pathway involving the Smad family of transcription factors (8).During mammary gland development, TGFb is a potentinhibitor of epithelial cell proliferation and branching mor-phogenesis (9). In premalignant lesions, TGFb acts as a tumor suppressor by inhibiting proliferation and promoting apoptosis. Paradoxically, malignant tumors are associated with high levels of TGFb, which promote later stages of tumor progression and metastasis (8, 10). Pleiotropic effectsof TGFb have also been observed in macrophages,

Authors' Af liation: Department of Laboratory Medicine and Pathologyand Masonic Cancer Center, University of Minnesota, Minneapolis,Minnesota

Note: Supplementarydata forthis article are available at MolecularCancer Research Online (http://mcr.aacrjournals.org/).

Corresponding Author: Kathryn L. Schwertfeger, Department of Labora-tory Medicine and Pathology and Masonic Cancer Center, University of Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455, USA. Phone:612-626-9419; Fax: 612-626-2600; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-12-0275

2012 American Association for Cancer Research.

Molecular Cancer

Research

Mol Cancer Res; 10(10) October 20121294


http://mcr.aacrjournals.org/http://mcr.aacrjournals.org/http://mcr.aacrjournals.org/


3/13

depending on their stage ofdifferentiation. Although TGFbacts as a chemoattractant for monocytes, it inhibits phago-cytosis and production of proin ammatory mediators indifferentiated macrophages (11). TGFb expression levels are

high in macrophages associated with late stage tumors (7), which is thought to in uence many aspects of malignantprogression including invasion, angiogenesis and immunesuppression (10). However, the functional consequences of regulating the TGFb/Smad signaling pathway withinmacrophages during different stages of tumorigenesis havenot been investigated.

Inappropriate activation of growth factor signaling pathways has been strongly linked to breast cancer formation and progression. Recent studies have implicated the broblastgrowth factor (FGF) pathway in tumor growth, progressionand resistance to standard therapies (12, 13). Amplicationof the chromosomal region of 8p12 that includes the FGF receptor 1 (FGFR1) gene is associated with poor prognosis,and approximately 10% of breast cancers exhibit amplied FGFR1 (13, 14). Transgenic mice expressing an inducibleFGFR1 (iFGFR1) transgene in mammary epithelial cellsdevelop early epithelial lesions that progress to alveolar hyperplasia, ultimately resulting in mammary tumor forma-tion (15). Activation of iFGFR1 leads to alterations in themicroenvironment, including increased angiogenesis and a rapid in ammatory response characterized by inltrating macrophages (15, 16). Macrophage depletion in this modelleadsto reduced epithelialcell proliferation and angiogenesisassociated with early stage lesions showing that in anFGFR1-dependent model of mammary tumor formation,macrophages are capable of promoting the development of early stage epithelial lesions (16).

In these studies, we have further used the iFGFR1 modelto identify mechanismsthat regulate the protumor functionsof macrophages during early stage tumor formation. Weshow here that macrophages associated with iFGFR1-drivenearly stage epithelial lesions exhibit decreased activation of the TGFb/Smad3 pathway. The decrease in TGFb-associ-ated genes within macrophages correlates with increased expression of macrophage-derived chemokines that bind tothe chemokine receptor Cxcr2. Restoration of TGFb sig-naling leads to inhibition of expression of these chemokinesin macrophages.Thesestudies suggest that repressedTGFb/Smad3 signaling may be functionally important for regu-lating the protumorigenic function of macrophages in early stages of tumor formation. Furthermore, these studies show that macrophage-derived chemokines, specically Cxcr2binding chemokines, promote migration and invasion of

preneoplastic mammary epithelial cells, suggesting a poten-tial therapeutic target for early stage breast tumors.

Materials and Methods Animals

Generation of mouse mammary tumor virus (MMTV)-iFGFR1 transgenic mice has been described previously (15)and the mice were obtained from Dr. Jeffrey Rosen (Baylor College of Medicine, Houston, TX). Animal care and

procedures were approved by the Institutional Animal Careand Use Committee of the University of Minnesota and were in accordance with the procedures detailed in the Guidefor Care and Use of Laboratory Animals.

Cell sorting and RT-PCR analysisSix-week-old female MMTV-iFGFR1 transgenic mice

and nontransgenic littermates were injected intraperitone-ally (i.p.)with 1 mg/kgB/Bdimerizer (Clontech). Mice weresacri ced48 hours later and mammary glands were collected for analysis. The tissue was dissociated using 2 mg/mLcollagenase A (Roche Applied Science) for 45 minutes at37 C with rocking at 200 rpm. The solutions were vigor-ously shaken every 15 minutes and the dissociated cells werecollected by centrifuging for 5 minutes at 1,500 rpm. Thecells were washed 3 times with DMEM/F12 containing 5%FBS at1,500 rpm and 2 times at800 rpm for 5 minutes each.The cells were stained with either Cd11b-APC (Life Tech-nologies) at a dilution of 1:200 or isotype control antibody at the same concentration for 1 hour at RT. The cells were then washed, ltered through a 40mm lter and sorted using a triple laser MoFlo (Cytomation). RNA was isolated from Cd11b-positive cells sorted from 6 mice per timepointas described above and pooled into duplicate samples. RNA was extracted using the Arcturus PicoPure RNA IsolationKit (Life Technologies) and RT-PCR analysis was carried out using primers speci c for ArgI and iNOS as described below. qRT-PCR analysis was carried out for TGFb1 asdescribed below. Primer sequences are listed in Supplemen-tary Table 1. Cyclophilin was used to normalize geneexpression levels.

Immuno uorescenceMammary glandsfrom MMTV-iFGFR1 transgenicmice,

or nontransgenic littermate controls, that were treated withB/B for 48 hours as described above werexed for 2 hours in4% paraformaldehyde and embedded in paraf n. Fivemicrometers sections were used for immunouorescentstaining using the following antibodies and dilutions: ratmonoclonal F4/80 (Invitrogen), 1:50; phospho-Smad3(Cell Signaling), 1:200. Immunostaining was carried outas described previously (17) in the absence of antigenretrieval. F4/80 and phospho-Smad3 positive cells werecounted and double positive cells were calculated relativeto the total number of F4/80 positive cells. A minimum of 600 cells from a total of 3 mice per treatment group wascounted for each dataset. All statistical analyses were carried out using the unpaired Student t test to compare 2 means.

Cell cultureHC-11 cells were maintained in RPMI containing 10%

FBS (Invitrogen), 1% penicillin-streptomycin, 5 mg/mLinsulin (Sigma-Aldrich) and 10 ng/mL EGF (Invitrogen).HC-11 cells stablyexpressing the iFGFR construct (HC-11/R1) were generated as previously described (18) and wereacquired from Dr. Jeffrey Rosen. HC-11/R1 cells weremaintained in HC-11 medium with the addition of 0.7mg/mL puromycin (Sigma-Aldrich). We received SUM225

Cxcr2 Contributes to Macrophage-Induced Tumor Cell Migration

www.aacrjournals.org Mol Cancer Res; 10(10) October 2012 1295




4/13

and MCF10DCIS.com fromDr.FaribaBehbod (University of Kansas City Medical Center, Kansas City, KS) and maintained as described (19). All other cell lines wereobtained from the American Type Culture Collection

(ATCC) and maintained in the suggested media. All celllines were used for fewer than 6 months after resuscitation.HC-11 cells and HC-11/R1 cells, which are not commer-cially available, are not maintained for longer than 20passages and are tested for mycoplasma, b-casein expressionand iFGFR1 expression regularly.

Two-dimensional coculture assaysHC-11/R1 cells were grown to conuence, washed with

PBS and incubated overnight in serum-free RPMI. The cells were treated with 30 nmol/L B/B or an equal amount of ethanol as solvent control for 24 hours. Conditioned media was collected (R1-CM) and added to RAW 264.7 cells thathad been incubated overnight in serum-free DMEM. Todeterminegene expression, cells were collected in Trizolafter 2 hours of treatment with R1-CM supplemented with or without 10 ng/mL recombinant TGFb (R&D Systems,Minneapolis, MN, USA) and analyzed as described below.For signaling studies, R1-CM was added to RAW264.7 cellsfor the indicated time and lysates were collected for immu-noblotting. Then, RAW 264.7 cells were pretreated for 30minutes with 2.5 or 10 mmol/L of the MEK1 inhibitor U0126 (Cell Signaling) or solvent control (DMSO) beforeincubation with R1-CM and the inhibitor or control for anadditional 2 hours. For migration assays, RAW 264.7 cells were incubated for 24 hours with R1-CM and CM wascollected (R1/RAW-CM). Migration assays were carried outusing cell culture inserts with 8 mm pore size (BD Bios-ciences, San Jose, CA, USA). HC-11 cells were washed withPBS and incubated overnight in serum-free RPMI. Cells were plated on the top of the insert and either R1-CM or R1/RAW-CM was placed on the bottom of the insert as a chemoattractant. The CXCR2 inhibitor, SB225002 (20 or 200 nmol/L, Cayman Chemical), or the solvent control,ethanol, was added with the HC-11 cells to the top of the well. After 18 hours, cells on the bottom of the insert were

xed with 4% paraformaldehyde and stained with hema-toxylin. The number of cells migrated in 4 elds of the insert were counted. Similar experiments were done with humancell lines in which MCF-7 cells were treated with 50 ng/mLbasic broblast growth factor (bFGF, Invitrogen). After 24hours, the conditioned media (MCF-CM) was then incu-bated with the human monocyte cell line THP-1 that had been treated with 5 ng/mL PMA 24 hours and then starved

overnight. Gene expression of CXCR2 ligands in macro-phages was determined by collecting THP-1 cells treated with MCF-CM for 2 or 4 hours and analyzed as described below.For migrationassays, MCF-CM wasaddedto THP-1cells for 24 hours and the conditioned media (MCF/THP-CM) was used as a chemoattractant for MCF-7 cells in thecell culture inserts as described above. The CXCR2 inhib-itor, SB225002 (100, 200, or 400 nmol/L) or solventcontrol, ethanol, was added with MCF-7 cells in the topof the well.

Quantitative reverse transcription-PCR RNA was extracted from primary macrophages or mac-

rophage cell lines (RAW 264.7 and THP-1, ATCC) using Trizol (Invitrogen) as described in the manufacturer proto-

col. cDNA was prepared using the Quantitect reversetranscription kit (Qiagen). Quantitative RT-PCR (qRT-PCR) was carried out using SYBR green (Bio-Rad) and theBio-Rad iQ5 system. The 2 DD Ct method (20) was used todetermine the relative quanti cation of gene expressionnormalized to cyclophilin.The primers used forthesestudiesare listed in Supplementary Table 1.

qRT-PCR array RNA was isolated from RAW 264.7 cells treated with R1-

CM for 2 hours in the presence or absence of 10 ng/mLTGFb, and cDNA was made, as described above. The RT2

Pro ler PCR Array: Mouse chemokines and receptors fromSA Biosciences was carried out as described for other qRT-PCR experiments.

ELISA analysis and activity assaysR1-CM was added to starved RAW 264.7 cells in the

presence or absence of 10 ng/mL TGFb. After 2 hoursthe media was removed and serum free RPMI was added.The R1/RAW-CM was collected after an additional 24hours and ELISAs were carried out to quantify the level of Cxcl1, Cxcl5, IL-10,and IL-12followingthe manufacturer'sprotocol (R&D Systems). In addition, the RAW 264.7 werecollected in lysis buffer (10 mmol/L Tris-HCl [pH 7.4]containing 1 mmol/L pepstatin A, 1 mmol/L leupeptin, and 0.4% Triton X-100) and the supernatant was used for anarginase activity assay (BioAssay Systems). For the Smad3luciferase assay, RAW 264.7 cells were transduced with a lentivirus expressing a Smad3 reporter construct (Qiagen)and were then treated with conditioned media from B/B-treated HC-11/R1 cells for 6 hours. Luciferase activity wasmeasured usingstandard procedures (Promega).For analysisof human chemokine expression, THP-1 cells were incu-bated with MCF-CM for 2 hours and then serum freeDMEM for 24 hours. The levels of secreted CXCL1 and CXCL8 in the MCF/THP-CM were measured by ELISA following the manufacturer's protocol (R&D Systems).

Immunoblot analysisCells were lysed in RIPA buffer and equal amounts of

protein were analyzed by SDS-PAGE. Immunoblotting wasdone using the following antibodies: CXCR2 (AHR1532Z;Biosource), b-tubulin (2146; Cell Signaling), pERK1/2

(9101; Cell Signaling), and ERK1/2 (sc-94; Santa CruzBiotechnology).

Three-dimensional coculture assay Mammary glands were isolated from 8- to 12-week-old

MMTV-iFGFR1 transgenic mice as described above. Bonemarrow was collected from femurs of 6- to 10-week-old wild-type (WT) mice. Cells were differentiated into macro-phages with the addition of 20% conditioned media fromL929 cells that have a high concentration of granulocyte

Bohrer and Schwertfeger

Mol Cancer Res; 10(10) October 2012 Molecular Cancer Research1296




5/13

colony-stimulating factor (G-CSF). A total of 10,000 mam-mary epithelial cells (MECs) were plated on growth-factor reduced matrigel (BD Biosciences) in 8 well chamber slidesas described previously (21). After 2 days, 3000 bone

marrow derived macrophages (BMDM) were added to the3D culture. Also, the following treatments began at thistime: 30 nmol/L B/B and 200 nmol/L SB225002,CXCR2 inhibitor (with ( ) being ethanol, solvent control). After 10 days, cells were xed with 2% paraformaldehydeand costained for macrophages (F4/80, Invitrogen) and epithelial cells (cytokeratin 8 (ab59400- Abcam) and mounted with ProLong Gold antifade reagent with DAPI(Invitrogen). Images were taken at the confocal microscopy facility at the Masonic Cancer Center (University of Min-nesota, Minneapolis, MN).

ResultsRegulation of pro- and anti-in ammatory genes in macrophages following exposure to conditioned media from iFGFR1-activated HC-11/R1 cells

As described previously, activation of iFGFR1 in mam-mary epithelial cells promotes rapid recruitment of macro-phages to epithelial structures in vivo and in vitro (16).Furthermore, depletion of macrophages leads to delayed

formation of early hyperplastic lesions in vivo (16). There-fore, we further used this model to delineate the mechanismsthat mediate the interactions between epithelial cells and macrophages. We have previously shown that exposure of

macrophages to conditioned media from HC-11/R1 cellsfollowing activation of iFGFR1 leads to increased produc-tion of the proin ammatory cytokine IL-1b by macrophagesin vitro and that IL-1b contributes to the formation of early stage lesions in vivo (17). To extend our analysis of macro-phage response to iFGFR1 activation in epithelial cells, wefurther analyzed theexpression of various genes known to bepreferentially regulated in tumor associated macrophages,including IL-10, IL-12, Arginase I (ArgI), and TGFb. For these studies, RAW 264.7 cells were exposed to conditioned media from HC-11/R1 cells treated with either the B/Bhomodimerizer, which activates the iFGFR1, or ethanol as a solvent control. After exposure to conditioned media,RAW 264.7 cells were analyzed for expression and activity of Arg1, IL-10, IL-12, and TGFb. As shown in Fig. 1,expression levelsof IL-10 and gene expression and activity of ArgI, which are typically increased in tumor associated macrophages, were induced whereas expression levels of IL-12, which are normally reduced in tumor associated macrophages, were decreased. Interestingly, expression of

Figure 1. Exposure to conditioned media from mammary epithelial cells with activated iFGFR1 leads to regulation of in ammation-associated genes in vitro .RAW 264.7 cells were exposed to conditioned media from B/B-treated HC-11/R1 cells for 2 hours and either cells were collected for gene expression or serum free media was added for 24 hours to examine secreted protein expression or activity. A, qRT-PCR analysis of ArgI gene expression levels(leftpanel)and arginaseactivity(rightpanel).B, qRT-PCRanalysisof IL-10 geneexpressionlevels (leftpanel)and secreted proteinlevel by ELISA(rightpanel).C, qRT-PCR analysis of IL-12 gene expression levels (left panel) and secreted protein level by ELISA (right panel). D, qRT-PCR analysis of TGF b1gene expression levels (left panel) and Smad3 transcriptional activity by luciferase assay (right panel). Expression levels were normalized to cyclophilin for qRT-PCR. Error bars represent standard error of the mean (SEM). , P < 0.05; , P < 0.005; , P < 0.001.






6/13

the TGFb1 gene, which is usually induced in tumor asso-ciated macrophages, was reduced in these studies. In addi-tion, the transcriptional activity of Smad3 also decreased (Fig. 1D)suggesting an overall decrease in theTGFb/Smad3

pathway. These results, together with our previously pub-lished studies (17), show that exposure of macrophages toconditioned media from epithelial cells with activated iFGFR1 leads to regulation of a number of genes associated with macrophage polarization.

Decreased TGFb gene expression and Smad3 activity in macrophages from MMTV-iFGFR1 transgenic mice

To examine gene expression of macrophages in vivo ,macrophages were isolated from the mammary glands of WT and MMTV-iFGFR1 transgenic mice following 48hours of B/B treatment, at which time macrophage recruit-ment to epithelial structures is consistently observed (16).Following a brief enzymatic digestion, macrophages wereisolated by sorting with anti-Cd11b, which stains bothmonocytes and macrophages. Approximately 8.5% of the

cells isolated from the mammary glands were positive for theCd11b antigen compared with the isotype control antibody (Supplementary Fig. 1A). These cells were collected forbothimmunostaining and RNA extraction. To determine the

percentage of the population of sorted cells that representsmature macrophages, staining was carried out using the F4/80 antibody (Supplementary Fig. 1B). Approximately 85%of thesorted cells were positive forF4/80, suggesting that thepreparation was signicantly enriched for macrophages. Toexamine gene expression in the macrophages, RT-PCR analysis was carried out and expression levels of ArgI, a marker of protumor macrophages, and iNOS, a marker of tumor-inhibitory macrophages, were examined. As shownin Fig. 2A, ArgI expression was increased and iNOS expres-sion was decreased in macrophages isolated from MMTV-iFGFR1 transgenic mice treated with B/B, consistent with a polarization of the macrophages towards the tumor-promot-ing phenotype. Further studies using qRT-PCR revealed a decrease in expression of TGFb1 in these macrophages (Fig.2B), consistent with the results observed in vitro (Fig. 1).

Figure2. Alterationsin theTGF b geneexpression and Smad3activity in macrophages isolatedfrom mammary glands following FGFR1 activation. A, Cd11b-positive sorted cells were analyzed for expression of the indicated genes using RT-PCR. The lanes represent macrophages sorted from 3 MMTV-iFGFR1transgenic (Tg) or 3 nontransgenic WT littermates and pooled. B, expression of TGF b1 in macrophages isolated from MMTV-iFGFR1 transgenicmice or nontransgenic WT littermates treated with B/B for 48 hours. Expression levels were normalized to cyclophilin. C, nontransgenic (i, ii) andtransgenic (iii,iv) micewere treated withB/B for48 hours andmammaryglands werecollected. Sectionswere costainedwith theF4/80 andphospho-Smad3antibodies. F4/80 positive cells (red) are found in the stroma surrounding the epithelial structures (blue DAPI staining of nuclei). Phospho-Smad3(green) is detected in nuclei of F4/80 positive cells in sections from the nontransgenic (arrow, ii) but not transgenic (arrow, iv), mice. Scale bars 50 mm.D, quanti cation of the percentage of F4/80 /phospho-Smad3 cells. Error bars represent SEM. , P < 0.05; , P < 0.0001.






7/13

To determine whether decreased TGFb productioncorrelated with altered Smad3 activity in the macrophages,mammary gland sections from WT and MMTV-iFGFR1transgenic mice following 48 hours of B/B treatment were

immunostained with an antibody to phosphorylated Smad3 (pSmad3; Fig. 2C). The sections were costained with an F4/80 antibody to visualize the macrophages and the percentage of F4/80 cells positive for pSmad3 expres-sion was determined (Fig. 2D). In WT mice, approxi-mately 25% of the F4/80 cells located within the stroma surrounding the ductal structures were positive for pSmad3. Interestingly, there was a signicant decrease inthe number of cells positive for pSmad3 present in thestroma following 48 hours of B/B treatment of iFGFR1mice. This observation suggests that along with decreased expression of TGFb in the macrophages, there is alsodecreased TGFb signaling within macrophages following iFGFR1 activation in mammary epithelial cells, consistent with the in vitro studies.

Effects of TGFb on chemokine expression in macrophages

On thebasisof ourresults,we hypothesized that decreased TGFb signaling in macrophages may be linked to protu-morigenic macrophage function in early stages of tumori-genesis. Activation of the TGFb/Smad3 pathway has beenshown to inhibit the expression of inammatory cytokinesand chemokines (22, 23), suggesting that repression of thispathway leads to induction of these factors. Furthermore,recent studies showed that loss of signaling through Tgfbr2led to increased expression of chemokines in a model of pancreaticductal adenocarcinoma (24).Therefore, we useda qRT-PCR-based chemokine array to analyze chemokinegene expression in macrophages exposed to conditioned media from B/B treated HC-11/R1 cells in the absence or presence of exogenous TGFb. Several chemokines werefound to be either up- or downregulated in the macrophagesfollowing exposure to these conditions (Table 1). Thechemokines were divided into 5 clusters based on thechangein their expression patterns. Although a number of genes were either induced or repressed with B/B alone and with B/B and TGFb combined, we focused on genes in cluster 1, which were upregulated with B/B treatment and down-regulated with the addition of TGFb. Interestingly, 4 of these chemokines (Cxcl1, Cxcl2, Cxcl5, and Cxcl7) areligands for the Cxcr2 receptor. Before validating expressionlevels of these chemokines, we conrmed that Cxcr2 isexpressed in the HC-11 mammary epithelial cell line by

immunoblot analysis (Fig. 3A). We validated the resultsfrom the array using distinct primer sets to determine geneexpression and ELISAs to measure secreted proteins. Weobserved similar gene and protein expression patterns for both Cxcl1 and Cxcl5 (Fig. 3B). Induction of Cxcl2 and Cxcl7 was not detected by ELISA (data not shown). Thesedata suggest that macrophages respond to FGFR1-induced soluble factors by increasing the production of chemokinesthat bind Cxcr2, primarily Cxcl1, and Cxcl5, and that thisinduction can be inhibited by restoring TGFb signaling.

Next, we wanted to determine the signaling pathway by which the Cxcr2 ligands are regulated in the macrophages.Expression of Cxcr2 ligands can be regulated by varioussignaling pathways depending on cell type and stimulus,including the NFk B and ERK pathways (24, 25). Interest-ingly, we were unable to detect an increase in NFk B activity in macrophages exposed to conditioned media from B/B-treated HC-11/R1 cells (data not shown). Further analysisfocused on examining the ERK signaling pathway. For thesestudies, RAW 264.7 cells were exposed to conditioned media from HC-11/R1 cells treated with or without B/B.Protein lysates were collectedat different time pointsand theexpression of phosphorylated and total ERK1/2 was exam-ined. There was a rapid induction of ERK1/2 activation in

macrophages treated with B/B conditioned media in com-parison to solvent controls (Fig. 3C). Further studies weredone to determine the contribution of the ERK1/2 pathway to expression of the Cxcr2 ligands. As shown in Fig. 3D,blocking ERK activation with the MEK1 inhibitor U0126led to a decrease in gene expression of the Cxcr2 ligands.Inhibitionof Cxcl1 wasobserved at the lowestconcentrationof U0126, whereas there was a dose-dependent decrease inCxcl5 expression (Fig. 3D). These results suggest thatalthough both ligands are regulated by the ERK pathway,

Table 1. Macrophage chemokines regulated byconditioned media from FGFR1-inducedmammary epithelial cells in the presence or absence of TGF b

Fold change Fold change

Gene ID B/B CM B/B CM TGF b

Cluster 1CCL8 3.9 0.93CCR1 1.86 0.863CCR1/1 2.04 0.833CXCL1 1.44 0.81CXCL2 1.79 0.716CXCL5 3.25 1.85CXCL7 1.47 0.94

Cluster 2Bmp6 0.65 1.07CCL2 0.172 0.776CCL20 0.429 1.18Rgs3 0.425 2.02

Cluster 3CX3CL1 1.76 3.15Trem1 2.32 8.05

Cluster 4Ccbp2 0.39 0.3CCR8 0.54 0.53Ecgf1 0.42 0.55

Cluster 5CCR6 3.14 3.75TNfsf14 1.97 1.73 Xcl1 1.82 2.02






8/13

they may be regulated via slightly different mechanisms.Overall, these results indicate that the ERK pathway con-tributes to the regulation of Cxcr2 ligand expression.

Macrophages induce migration of epithelial cells, whichis blocked by Cxcr2 inhibition

To determine the effects of Cxcr2 ligand secretion frommacrophages, we developeda coculturemigrationassay(Fig.4A).In this assay,conditioned mediumfrom theHC-11/R1cells containing FGFR1-induced soluble factors was added to RAW 264.7 cells and allowed to incubate overnight. Theability of the soluble factors collected from the treated RAW

264.7 cells to promote migration of parental HC-11 cells, which do not respond to B/B treatment, was determined using a transwell assay. The ability of macrophages topromote epithelial cell migration following exposure toconditioned media from cells with an activated FGFR1re ects the ability of the stimulated macrophages to act ina tumor-promoting manner. Conditioned media from B/Btreated HC-11/R1 cells alone was able to induce migrationof HC-11 cells,suggesting that activation of FGFR1 in thesecells leads to the production of migratory factors (Fig. 4B).

However, the conditioned media isolated from the treated macrophages led to a signicant increase in migration of HC-11 cells compared with conditioned media from theHC-11/R1 cells alone (Fig. 4B). Therefore, treatment of macrophages with conditioned media following FGFR1activation leads to an increase in the ability of macrophagesto produce migratory factors. To determine whether theincrease in migrationrequired Cxcr2,we further assessed theeffects of blocking Cxcr2 activity on HC-11 cell migrationusing the Cxcr2-speci c inhibitor SB225002. As shownin Fig. 4B, the increase in migration was blocked whenSB225002 was incubated with the HC-11 cells in the

presence of the macrophage conditioned media. Theseresults show an important role for macrophage-derived Cxcr2 ligands in the promotion of mammary epithelial cellmigration.

Macrophages promote invasion of primary mammary epithelial cells in a 3D coculture assay

Primary mammary epithelialcells (MEC)formacinar-likestructures when grown in 3D culture and more closely represent characteristics of mammary epithelium in vivo

Figure3. Activation of iFGFR1 in mammary epithelial cells leads to increased expression of Cxcr2-binding chemokines inmacrophages. A, HC-11 cells weretreated with R1/RAW-CMwithor without B/B. Wholecelllysates were collectedand the expression ofCxcr2and b-tubulin(loading control)was analyzed byimmunoblotting. B, RAW 264.7 cells were treated with R1-CM in the presence or absence of 10 ng/ml TGF b for 2 hours. Expression of the givengeneswas determined by qRT-PCRand normalized tocyclophilin(left panel). Forproteinexpression, RAW264.7cells wereincubated withR1-CMfor 2 hoursfollowed by serum free media for 24 hours. The conditioned media samples were analyzed using ELISAs (middle and right panel). C, RAW 264.7cells were treated with R1-CM for the given time. Whole cell lysates were collected and the expression of pERK1/2 and ERK1/2 (loading control) wasanalyzed by immunoblotting. D, RAW 264.7 cells were pretreated with DMSO (solvent control) or U0126 for 30 minutes and then treated for anadditional 2 hours with the same conditions in the presence of B/B R1-CM. The expression of the given genes was analyzed by qRT-PCR and normalizedto cyclophilin. Error bars represent SEM. , P < 0.05; , P < 0.01; , P < 0.001.






9/13

(21). We took advantage of the ability of primary MECsisolated from MMTV-iFGFR1 transgenic mice to grow in3D culture (21) and developed a coculture model to study the interactions between MECs and macrophages. For these

studies, we isolated MECs from iFGFR1 transgenic miceand plated them in growth factor reduced Matrigel. After 2days, bone marrow derived macrophages (BMDM) that were isolated from WT mice, were added to MECs. The

structures were grown in the presence or absence of B/B and theCXCR2inhibitorSB225002.After10 days of treatment,cells were xed and stained for epithelial (cytokeratin 8) and macrophage (F4/80) markers. As shown in Fig. 5A,additionof B/B led to larger MEC structures, consistent with pre-viously published studies (21). Although coculture of MECs with BMDM did not signi cantly affect size of the structures (data not shown), a signicant increase inthe number of invasive structures was observed (Fig. 5A and B). With the addition of the CXCR2 inhibitor, macro-phages were still recruited to the MECs (Fig. 5A), but thepercentage of invasive structures was signicantly inhibited (Fig. 5A and B). These data suggest that Cxcr2 ligandssecreted from primary macrophages induce invasion of primary MECs.

Human macrophages secrete CXCR2 ligands that promote migration of human breast cancer cells

To verify the results of the mouse iFGFR1 system, weused human breast cancer cell lines. We rst examined the expression of CXCR2 in different breast cell lines:normal (MCF10A), pre-invasive (MCF10DCIS.com and SUM225), estrogen receptor positive (MCF-7 and T47D)and triple negative (MDA-MB-231, 435A, and 468)

Figure 4. Cxcr2 inhibition leads to decreased macrophage-inducedmigration of HC-11 cells. A, model of the migration coculture assay. B,R1-CM, / B/B, was exposed to RAW 264.7 cells for 24 hours and theconditioned media, R1/RAW-CM was used as the chemoattractant for atranswell migration assay. HC-11 cells were plated on top of the insertwith ethanol (solvent control) or CXCR2 inhibitor ( , 20 nmol/L and ,200nmol/L). After 18 hours, themigratedcells onthe bottom of theinsertwere stained with hematoxylin and counted. Error bars represent SEM.

, P < 0.001.

Figure 5. Macrophages induceinvasion of primary mammaryepithelial cellsgrownin 3Dculture.A,primary MECs from iFGFR1 micewere grown in Matrigel in theabsence or presence of BMDM.Every 2 to 3 days fresh media wasadded with the treatments: 30nmol/LB/B and 200nmol/LCXCR2inhibitor. After 10 days of treatment,cells were xed and immunostainedwith cytokeratin 8 (green) and F4/80(red). DAPI (blue) labeled nuclei. Arrows indicate invasive structures.Left images light microscopy. Scalebars 50 mm. B, for each treatment,

approximately 80 structures wereexamined for invasion from 3independent experiments. Error barsrepresent SEM. , P < 0.05;

, P < 0.01.






10/13

(Fig. 6A). As MCF-7 cells express CXCR2 and they havepreviously been shown to respond to FGF treatment (26), we chose to use these cells to determine the ability of bFGFstimulation to promote CXCR2 ligand induction in macro-phages. MCF-7 cells were treated with or without bFGF and

the MCF-7 conditioned media was added to the humanmonocyte cell line THP-1 that had been differentiated tomacrophages with PMA as described (27). After 2 and 4hours, THP-1 cells were collected for gene expression

analysis. In addition to the Cxcr2 ligands in mice, humancells also express CXCL8. Gene expression of all CXCR2ligands, with the exception of CXCL7 (data not shown),increased following exposure to conditioned media fromMCF-7 cells treated with bFGF (Fig.6B).Further analysisof protein expression showed signicant increases in secretionof both CXCL1 and CXCL8 (Fig. 6B), whereas the other ligands were not expressed at detectable levels by ELISA (data not shown). bFGF treatment of THP-1 cells alone did not induce expression of these chemokines, suggesting thatthese results are not due to the direct action of bFGF onmacrophages (data not shown). The MCF-7/THP-1 con-ditioned media was used as the chemoattractant in a transwell migration assay to examine the migration of MCF-7cells. Media from MCF-7 cells exposed to bFGF increased migration compared to no treatment and there was anadditional increase when the media was also exposed toTHP-1 cells (Fig. 6C). Finally, migration was inhibited witha CXCR2 inhibitor (Fig. 6C), showing that the CXCR2ligands are important for macrophage-induced breast cancer cell migration.

DiscussionRecent studies have showed that FGFR signaling con-

tributes to breast cancer growth and resistance to conven-tional therapies (12, 13). Our studies focus on understand-ing how activation of FGFR1 in tumor cells leads toprotumorigenic alterations in the tumormicroenvironment. We describe here a novel mechanism of paracrine interactionbetween tumor cells and macrophages that is driven by FGFR1 activation in the tumor cells and chemokine expres-sion in the macrophages. We have previously showed thatactivation of iFGFR1 in the HC-11/R1 cells results in theinduction of a number of secreted factors that can inuencemacrophagerecruitment (16) and cytokine production(17).The studies described here further analyze the mechanismsthrough whichmacrophages contributeto earlystage tumor-igenesis. Although carrying outcoculture studies,we found a number of genes to be regulated in an expected manner based on published studies of gene expression in tumor associated macrophages, including increased ArgI and IL-10and decreased IL-12 (28, 29). Interestingly, however,decreased expression of TGFb gene expression was consis-tently observed in the macrophages bothin vivo and in vitro ,

which is not consistent with the phenotype of macrophagesassociated with late stage tumors (30). Although levels of TGFb protein were not detectable by ELISA in the in vitro coculture model (data not shown), a decrease in Smad3activity was observed in the macrophages, consistent withthe decrease in pSmad3 observed in vivo . Together thesestudies show an overall decrease in the TGFb/Smad3 sig-naling pathway in macrophages in response to factors fromFGFR1-driven mammary tumor cells. The specic factorsand mechanisms responsible for this decrease are likely

Figure6. The migratory ability of MCF-7 cellsincreases withmacrophagesecreted CXCR2 ligands. A, whole cell lysates were collected from thedifferentcell lines andequal amountswere usedfor immunoblotanalysis.The expression of CXCR2 was determined and the membrane wasstained with Ponceau S for a loading control. B, THP-1 cells wereexposed to conditioned media from MCF-7 cells treated with 50 ng/mLbFGF or no treatment (NT). Cells were collected after 2 or 4 hours, andRNA was isolated forqRT-PCRfor the given genes thatwere normalizedto cyclophilin (upper panel). For protein expression, THP-1 cells wereincubated with MCF-CM for 2 hours and then serum free media for 24hours. The conditioned media was analyzed using ELISAs (lower panel).C, MCF-CM, / bFGF, was exposed to THP-1 cells for 24 hours, andthe conditioned media, MCF/THP-CM was used as the chemoattractantfor a transwell migration assay. MCF-7 cells were plated on top of theinsert with ethanol (solvent control) or CXCR2 inhibitor ( , 100 nmol/L; , 200nmol/L;and , 400nmol/L).After18 hours,the migratedcellson the bottom of the insert were stained with hematoxylin and counted.Error bars represent SEM. , P < 0.05; , P < 0.01; , P < 0.001.






11/13

complex and remain to be determined. Early in mammary tumor formation, TGFb is known to be growth suppressive(8), so it is possible that decreased TGFb in the microen-vironment, either from epithelial cells, inltrating macro-

phages or other stromal cells, is critical for the developmentof early stage lesions. Consistent with this, we found thatadding exogenous TGFb to the media of primary MECs in3D culture completely inhibited iFGFR1-induced acinar growth (Supplementary Fig. 2). These results suggest that inthe early stages of tumor formation, the presence of TGFbproducing macrophages, such as those associated with thepromotion of late-stage tumors, might actually result intumorinhibition.Therefore, the association of macrophages with decreased levels of TGFb expression and pathway activity, as observed in our studies, may be important for successful early-stage tumor formation.

Our results show that decreased expression of genesassociated with the TGFb pathway correlates with increased expression of in ammatory chemokines. Furthermore, res-toration of TGFb signaling with exogenous TGFb inhibitsthe expression of these chemokines. An inverse correlationbetween theTGFb pathway andexpression of inammatory chemokines hasbeen observed in other studies. Forexample,decreased TGFb responsiveness in prostatic broblastscaused an upregulation of chemokines including CXCL1,leading to the adhesion of prostate cells to the bone matrix (31). Also, loss of TGFb signaling in mammary broblastsresulted in increased production of the proinammatory chemokine Ccl2, which contributed to growth and metas-tasis of 4T1 mammary tumors (32). In addition, recentstudies using a mouse model of pancreatic ductal adenocar-cinoma also showed an increase in Cxcr2 ligands associated with loss of Tgfbr2 in the pancreas (24). Interestingly, thesestudies showed that induction of the Cxcr2 ligands wasdependent on the NFk B pathway, whereas induction inmacrophages in our model is dependent upon the ERK signaling pathway. These results possibly represent cell-typespeci c differences in CXCR2 ligand regulation. Takentogether, our results, along with published studies, show that decreased TGFb signaling is associated with increased expression of inammatory chemokines, and that thesechemokines are capable of contributing to tumor formationand progression.

Interactions between chemokines and chemokine recep-tors are known to contribute to breast cancer progression(33). Recent studies have implicated CXCR2 and itsvarious ligands in breast cancer. A number of studies havefocused on CXCL8, also known as IL-8, which binds to

both CXCR1 and CXCR2 and appears to be the primary CXCR2 binding chemokine induced in response tobFGF-treated breast cancer cells (Fig. 6B). Expression of CXCL8 has been linked to increased tumor grade and experimental studies have showed its ability to regulateangiogenesis and tumor progression (3335). CXCR2expression itself has been detected on breast cancer cells(34) and inhibition of CXCR2 decreased mammary tumor cell invasion in vitro (36). Furthermore, knockdown of CXCR2 in metastatic mammary tumor cells led to

decreased metastasis in orthotopic transplantation models(36). The CXCR2 ligand CXCL3 was identied in a screen for genes associated with basal-like breast cancersand a CXCR2 inhibitor was shown to decrease viability of

basal-like breast cancer cell lines in vitro (37). Further-more, polymorphisms in the CXCR2 gene have beenidenti ed in breast cancer patients and these polymorph-isms correlated with larger tumor size, higher tumor grade,and increased lymph node metastasis (38). However, thefunctional consequences of this polymorphism onCXCR2 expression and/or activity remain to be deter-mined. Our studies show that activation of epithelial cell-speci c CXCR2 by chemokine-producing cells within thetumor stroma may play a role in early stages of tumor-igenesis. Taken together, these studies suggest that theCXCR2 axis may represent a viable pathway to targetduring both early and late stage tumor development.

Our studies have focused on the use of mammary epi-thelial cells expressing an iFGFR1 construct. To show thatthe results were not specic to the iFGFR1 model, wevalidated our results using the MCF-7 cell line, which hasbeen previously used to study endogenous FGF signaling (26, 39,40). Similar to theresults obtained with theiFGFR1model, activation of the FGF signaling pathway in MCF-7cells also led to an increase in CXCR2 ligand gene expressionin macrophages. Interestingly, FGFR1 activity has beenshown to promote resistance of estrogen receptor positivebreast cancer cells to endocrine-based therapies (13). Although it has been reported that estrogen receptor positivecells, including MCF-7, express lower levelsof CXCR2 thanmore invasive breast cancer cell lines (34), our studies as wellas another report (41) suggest that estrogen receptor positivecells express the same or even more CXCR2 than moreinvasive breast cancer cell lines. Furthermore, our studiesshow that MCF-7 cells are capable of responding to CXCR2ligands in chemotaxis assaysand that inhibition of CXCR2 issuf cient to inhibit migration towards the stimulated THP-1 cells. These results suggest that activation of the FGFpathway may contribute to enhancement of tumor progres-sion by regulating the expression of tumor-promoting chemokines in in ltrating immune and other stromal cells.In addition, it is possible that FGF signaling in tumor cellsmay contribute to breast cancer growth and therapeuticresistance by regulating both the tumor cells and themicroenvironment.

Although some studies have showed that breast cancer cells produce CXCR2 ligands, which feed back to regulatetumor cell activity in an autocrine manner, our studies show

that cells within the stroma might also represent an impor-tant sourceof CXCR2 ligands. Similarly, studies by Halpernandcolleagues showedthat mesenchymal stem cells arealso a source of CXCR2 ligands, which are capable of promoting migration of cells in vitro and suggest that CXCR2 ligandsmay be important for homing of tumor cells to bone (42).Our results show that the production of CXCR2 ligands isenhanced in macrophages in response to soluble factorsreleased from transformed mammary epithelial cells and that these factors are then capable of promoting migration






12/13

and invasion of noninvasive cells. These results offer a novelmechanism by which macrophages associated with theinvasive edges of developing tumorsmightpromote invasionthrough the basement membrane. Together, these results

suggest that targeting CXCR2 in breast cancer patients may be effective at both early and late stages of tumor formationand progression.

In summary, our studies have focused on the effects of tumor cell-speci c FGFR1 activation on alterations withinthe tumor microenvironment. We have previously showed a rapid in ammatory response characterized by recruit-ment of macrophages to the epithelial structures (16). Wehave also showed that anti-inammatory drugs are capableof inhibiting the initiation of FGFR1-driven epitheliallesions, showing that in ammation is an important pro-moter of FGFR1-driven tumorigenesis (17). Using theiFGFR1 model, we have developed novel coculture mod-els to identify specically how FGFR1-driven tumor cellscommunicate with the microenvironment. We have iden-ti ed a paracrine mechanism in which soluble factors fromthe FGFR1-activated cells lead to increased production of CXCR2 ligands, which then feed back to promote migra-tion and invasion of the tumor cells. Importantly, these

ndings were validated using an FGF-responsive breastcancer cell line, showing that activation of endogenousFGF signaling leads to similar results as the iFGFR1model. These results suggest that in FGF-driven breastcancers, targeting CXCR2 activity, possibly in conjunc-tion with speci cally targeting FGFR activity, may lead toan effective therapeutic strategy. Further studies are

required to determine which patients might benet fromthis type of therapy.

Disclosure of Potential Con icts of InterestNo potential con icts of interest were disclosed.

Authors' ContributionsConception and design: L.R. Bohrer, K.L. Schwertfeger Development of methodology: L.R. Bohrer, K.L. Schwertfeger Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L.R. Bohrer, K.L. Schwertfeger Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): L.R. Bohrer, K.L. Schwertfeger Writing, review, and/or revision of the manuscript: L.R. Bohrer, K.L.Schwertfeger Administrative, technical, or material support (i.e., reporting or organizing data,constructing databases):Study supervision: K.L. Schwertfeger Performed the cell culture 2D and 3D coculture assays: L.R. Bohrer Performed the in vivo macrophage isolation and expression studied: K.L.Schwertfeger

Acknowledgments We would like to thank Dr. Jeff Rosen for providing reagents and advice for these

studies and Dr. Fariba Behbod for providing reagents used in this study. Also, we

would like to thank Johanna Reed and Lindsey Bade for critical reading of themanuscript. In addition, we would like to acknowledge the use of the confocalmicroscope at the Masonic Cancer Center made available through an NCRR Shared Instrumentation Grant (#1 S10 RR16851).

Grant SupportFundingfor thesestudies wasprovided bygrantsfrom theAmerican CancerSociety

(RSG-09-192-01-LIB) and NCI (1R01CA132827) to KLS.Thecostsof publicationof this article weredefrayedin part bythe payment ofpage

charges.This article musttherefore beherebymarked advertisement in accordancewith18 U.S.C. Section 1734 solely to indicate this fact.

Received May 3, 2012; revised July 5, 2012; accepted July 18, 2012;published OnlineFirst August 14, 2012.

References

1. de VisserKE, EichtenA, Coussens LM.Paradoxicalrolesofthe immunesystem during cancer development. Nat Rev Cancer 2006;6:24 37.2. Bingle L, Brown NJ, Lewis CE. The role of tumour-associated macro-

phages in tumour progression: implications for new anticancer ther-apies. J Pathol 2002;196:254 65.

3. Pollard JW. Tumour-educated macrophages promote tumour pro-gression and metastasis. Nat Rev Cancer 2004;4:71 8.

4. Qian BZ, Pollard JW. Macrophage diversity enhances tumor progres-sion and metastasis. Cell 2010;141:39 51.

5. DeNardo DG,BarretoJB, AndreuP, Vasquez L,Taw k D,Kolhatkar N,et al. CD4( ) T cells regulate pulmonary metastasis of mammarycarcinomas by enhancing protumor properties of macrophages. Can-cer Cell 2009;16:91 102.

6. LinEY, Nguyen AV, Russell RG, Pollard JW. Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy. J ExpMed 2001;193:727 40.

7. Biswas SK, Gangi L, Paul S, Schioppa T, Saccani A, Sironi M, et al. A distinct and unique transcriptional program expressed by tumor-

associated macrophages (defective NF-kappaB and enhanced IRF-3/STAT1 activation). Blood 2006;107:2112 22.8. Wake eld LM, Piek E, Bottinger EP. TGF-beta signaling in mammary

gland development and tumorigenesis. J Mammary Gland Biol Neo-plasia 2001;6:67 82.

9. Lanigan F, O'Connor D, Martin F, Gallagher WM. Molecular linksbetween mammary gland development and breast cancer. Cell MolLife Sci 2007;64:3159 84.

10. Massague J. TGFbeta in Cancer. Cell 2008;134:215 30.11. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming

growth factor-beta regulation of immune responses. Annu Rev Immu-nol 2006;24:99 146.

12. Sharpe R, Pearson A, Herrera-Abreu MT, Johnson D, Mackay A, WeltiJC, et al. FGFR signaling promotes the growth of triple-negative andbasal-like breast cancer cell lines both in vitro and in vivo . Clin Cancer Res 2011;17:5275 86.

13. Turner N, Pearson A, Sharpe R, Lambros M, Geyer F, Lopez-GarciaMA, et al. FGFR1 ampli cation drives endocrine therapy resistanceand is a therapeutic target in breast cancer. Cancer Res 2010;70:2085 94.

14. Letessier A, Sircoulomb F, Ginestier C, Cervera N, Monville F, Gelsi-Boyer V, et al. Frequency,prognosticimpact, andsubtypeassociationof 8p12, 8q24, 11q13, 12p13, 17q12, and 20q13 ampli cations inbreast cancers. BMC Cancer 2006;6:245.

15. WelmBE, FreemanKW, ChenM, ContrerasA, SpencerDM, Rosen JM.Inducible dimerization of FGFR1: development of a mouse model toanalyze progressive transformation of the mammary gland. J Cell Biol2002;157:703 14.

16. Schwertfeger KL, Xian W, Kaplan AM, Burnett SH, Cohen DA, RosenJM. A critical role for the in ammatory response in a mouse model of

preneoplastic progression. Cancer Res 2006;66:5676 85.17. Reed JR, Leon RP, Hall MK, Schwertfeger KL. Interleukin-1beta andbroblast growth factor receptor 1 cooperate to induce cyclooxygen-

ase-2 during early mammary tumourigenesis. Breast Cancer Res2009;11:R21.

18. Xian W, Schwertfeger KL, Vargo-Gogola T, Rosen JM. Pleiotropiceffects of FGFR1 on cell proliferation, survival, and migration in a 3Dmammary epithelial cell model. J Cell Biol 2005;171:663 73.

19. Behbod F, Kittrell FS, LaMarca H, Edwards D, Kerbawy S, HeestandJC, et al. An intraductal human-in-mouse transplantation modelmimics the subtypes of ductal carcinoma in situ. Breast Cancer Res2009;11:R66.






13/13

20. Livak KJ, Schmittgen TD. Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods 2001;25:402 8.

21. XianW, Schwertfeger KL,Rosen JM.Distinctrolesof broblastgrowthfactor receptor 1 and 2 in regulating cell survival and epithelial-mes-enchymal transition. Mol Endocrinol 2007;21:987 1000.

22. Maggio-Price L, Treuting P, Bielefeldt-Ohmann H, Seamons A, Driv-dahl R, Zeng W, et al. Bacterial infection of Smad3/Rag2 double-nullmice with transforming growth factor-beta dysregulation as a modelfor studying in ammation-associated colon cancer. Am J Pathol2009;174:317 29.

23. Werner F, Jain MK, Feinberg MW, Sibinga NE, Pellacani A, WieselP, et al. Transforming growth factor-beta 1 inhibition of macro-phage activation is mediated via Smad3. J Biol Chem 2000;275:36653 8.

24. IjichiH,ChytilA, GorskaAE,AakreME,BierieB, Tada M,et al.InhibitingCxcr2 disrupts tumor-stromal interactions and improves survival in amouse model of pancreatic ductal adenocarcinoma. J Clin Invest2011;121:4106 17.

25. Issa R, Xie S, Lee KY, Stanbridge RD, Bhasvar P, Sukkar MB, et al.GRO-alpha regulation in airway smooth muscle by IL-1beta and TNF-alpha: roleofNF-kappaBandMAP kinases.Am J PhysiolLungCellMolPhysiol 2006;291:L66 74.

26. Bade LK, Goldberg JE, Dehut HA, Hall MK, Schwertfeger KL. Mam-mary tumorigenesis induced by broblast growth factor receptor 1requires activation of the epidermal growth factor receptor. J Cell Sci2011;124:3106 17.

27. Tjiu JW, Chen JS, Shun CT, Lin SJ, Liao YH, Chu CY, et al. Tumor-associatedmacrophage-induced invasionand angiogenesis of humanbasal cell carcinoma cells by cyclooxygenase-2 induction. J InvestDermatol 2009;129:1016 25.

28. Biswas SK,Sica A, Lewis CE.Plasticity ofmacrophage function duringtumor progression: regulation by distinct molecular mechanisms.J Immunol 2008;180:2011 7.

29. Mantovani A, Schioppa T, Porta C, Allavena P, Sica A. Role of tumor-associated macrophages in tumor progression and invasion. Cancer Metastasis Rev 2006;25:315 22.

30. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophagepolarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 2002;23:549 55.

31. Li X, SterlingJA, FanKH,VessellaRL, Shyr Y,HaywardSW, etal. LossofTGF-beta responsivenessin prostatestromal cellsalterschemokine

levels andfacilitatesthe development ofmixed osteoblastic/osteolyticbone lesions. Mol Cancer Res 2012;10:494 503.

32. Hembruff SL, Jokar I, Yang L, Cheng N. Loss of transforming growthfactor-beta signaling in mammary broblasts enhances CCL2 secre-tion to promote mammary tumor progression through macrophage-dependent and -independent mechanisms. Neoplasia 2010;12:425 33.

33. Ali S, Lazennec G. Chemokines: novel targets for breast cancer metastasis. Cancer Metastasis Rev 2007;26:401 20.

34. Freund A, Chauveau C, Brouillet JP, Lucas A, Lacroix M, Licznar A,et al. IL-8 expression and its possible relationship with estrogen-receptor-negative status of breast cancer cells. Oncogene 2003;22:256 65.

35. Lin Y, Huang R, Chen L, Li S, Shi Q, Jordan C, et al. Identi cation of interleukin-8 as estrogen receptor-regulated factor involved in breastcancer invasion and angiogenesis by protein arrays. Int J Cancer 2004;109:507 15.

36. Nannuru KC, Sharma B, Varney ML, Singh RK. Role of chemokinereceptor CXCR2 expression in mammary tumor growth, angiogenesisand metastasis. J Carcinog 2010;10:40.

37. Marotta LL, Almendro V, Marusyk A, Shipitsin M, Schemme J, Walker SR, et al. The JAK2/STAT3 signaling pathway is required for growth of CD44CD24 stem cell-like breast cancer cells in human tumors. J Clin

Invest 2011;121:2723

35.38. Snoussi K, Mahfoudh W, Bouaouina N, Fekih M, Khairi H, Helal AN,et al. Combined effects of IL-8 and CXCR2 gene polymorphisms onbreast cancer susceptibility and aggressiveness. BMC Cancer 2010;10:283.

39. LiuJF, CrepinM, LiuJM, Barritault D, LedouxD. FGF-2 andTPA inducematrix metalloproteinase-9 secretion in MCF-7 cells through PKCactivation of the Ras/ERK pathway. Biochem Biophys Res Commun2002;293:1174 82.

40. PondAC,HerschkowitzJI, SchwertfegerKL, WelmB, Zhang Y, YorkB,et al. Fibroblast growth factor receptor signaling dramatically accel-erates tumorigenesis and enhances oncoprotein translation in themouse mammary tumor virus-Wnt-1 mouse model of breast cancer.Cancer Res 2010;70:4868 79.

41. Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, et al.Involvement of chemokine receptors in breast cancer metastasis.Nature 2001;410:50 6.

42. Halpern JL, Kilbarger A, Lynch CC. Mesenchymal stem cells promote

mammary cancer cell migration in vitro via the CXCR2 receptor.Cancer Lett 2011;308:91 9.




mol cancer res-2012-bohrer-1294-305.pdf

Documents