RAGE Mediates S100A7-Induced Breast Cancer Growth and Metastasis by Modulating the Tumor Microenvironment Mohd W

Published OnlineFirst January 8, 2015; DOI: 10.1158/0008-5472.CAN-14-2161 Cancer Microenvironment and Immunology Research

RAGE Mediates S100A7-Induced Breast Cancer Growth and Metastasis by Modulating the Tumor Microenvironment Mohd W. Nasser1, Nissar Ahmad Wani1, Dinesh K. Ahirwar1, Catherine A. Powell1, Janani Ravi1, Mohamad Elbaz1, Helong Zhao1, Laura Padilla2, Xiaoli Zhang3, Konstantin Shilo1, Michael Ostrowski4, Charles Shapiro1, William E. Carson III4,5, and Ramesh K. Ganju1

Abstract

RAGE is a multifunctional receptor implicated in diverse pro- ability to activate ERK, NF-kB, and cell migration. In an S100A7 cesses including inflammation and cancer. In this study, we report transgenic mouse model of breast cancer (mS100a7a15 mice), that RAGE expression is upregulated widely in aggressive triple- administration of either RAGE neutralizing antibody or soluble negative breast cancer (TNBC) cells, both in primary tumors and RAGE was sufficient to inhibit tumor progression and metastasis. in lymph node metastases. In evaluating the functional contribu- In this model, we found that RAGE/S100A7 conditioned tions of RAGE in breast cancer, we found that RAGE-deficient mice the tumor microenvironment by driving the recruitment of displayed a reduced propensity for breast tumor growth. In an MMP9-positive tumor-associated macrophages. Overall, our established model of lung metastasis, systemic blockade by results highlight RAGE as a candidate biomarker for TNBCs, and injection of a RAGE neutralizing antibody inhibited metastasis they reveal a functional role for RAGE/S100A7 signaling in linking development. Mechanistic investigations revealed that RAGE inflammation to aggressive breast cancer development. Cancer Res; bound to the proinflammatory ligand S100A7 and mediated its 75(6); 1–12. 2015 AACR.

Introduction RAGE was first described as a receptor for advanced glycation end products (AGE), but it has since been shown to be the receptor Receptor for advanced glycation endproducts (RAGE) is the for several other molecules involved in innate immunity, includ- signal transduction receptor that senses a variety of signaling ing high mobility group box 1 peptide (HMGB-1), amyloid-b molecules (1). The variety of ligands allows RAGE to be impli- peptide, and the S100 family of proteins (1, 2). Phorbol ester cated in a wide spectrum of pathologic conditions such as inflam- 12-O-tetradecanoylphorbol-13-acetate-induced proinflammatory mation and cancer (1, 2). Epidemiologic and molecular studies, mediators were shown to be decreased in RAGE-deficient mice including mouse models, have shown that if inflammation is (7), which suggests that RAGE expression is involved in sustaining prolonged, it promotes cancer development (3–6). It is now inflammation and cancer (1, 2, 7, 9). It has also been well- believed that most solid tumors, including those in the breast, documented that RAGE ligands bind to RAGE and activate its have an inflammatory microenvironment (4, 5). RAGE expression downstream signaling mechanisms that sustain chronic inflam- and activation have been shown to be associated with chronic matory conditions, leading to neoplastic stage (1, 12, 13). It is inflammation, which in turn enhances the malignant transfor- interesting to note that there is very low or no RAGE expression in mation of various cancers (1, 2, 7–11). However, its role in breast normal tissues but enhanced expression in chronic inflammation cancer, especially in the modulation of breast cancer microenvi- and cancer (2, 10). Although, these features of RAGE make it an ronment, is unknown. ideal candidate for therapeutic strategies against chronic inflammation, not much is known about its role in breast cancer. RAGE has been shown to bind to its ligand S100A7 in kera- 1Department of Pathology, The Ohio State Medical Center, Columbus, 2 tinocytes and leukocytes (14, 15). S100A7 has been shown to be Ohio. Biomed Division, LEITAT Technological Center, Barcelona, Spain. 3Centre for Biostatics, The Ohio State Medical Center, Colum- highly expressed in estrogen receptor (ER)a breast cancer (16, bus, Ohio. 4Comprehensive Cancer Center, The Ohio State Medical 5 17). It is believed that S100A7 mediates breast cancer growth and Center, Columbus, Ohio. Department of Surgery, The Ohio State fl fi Medical Center, Columbus, Ohio. metastasis by recruiting proin ammatory cell in ltrates (18, 19). Also, proinflammatory cytokines enhance triple-negative breast Note: Supplementary data for this article are available at Cancer Research cancer (TNBC) growth and metastasis (20). However, very little is Online (http://cancerres.aacrjournals.org/). known about mechanisms through which RAGE/S100A7 axis M.W. Nasser and N.A. Wani contributed equally to this article. modulates tumor microenvironment and enhances breast cancer Corresponding Author: Ramesh K. Ganju, Department of Pathology, The Ohio growth and metastasis. State University, 810 Biomedical Research Tower, 460 West 12th Avenue, Macrophages can be divided into subtypes M1 and M2, where Columbus, OH 43210. Phone: 614-292-5539; Fax: 614-292-7072; E-mail: M1 macrophages are associated with an anti-cancer phenotype [email protected] and M2 macrophages express a pro-cancer phenotype (21, 22). doi: 10.1158/0008-5472.CAN-14-2161 Cytokines/chemokines and growth factors modulate the tumor 2015 American Association for Cancer Research. microenvironment, which could directly/indirectly polarize

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macrophages toward the M2 tumor-associated macrophages Cell culture (M2-TAM) phenotype (22, 23). Murine macrophage cell line RAW264.7 and human breast In this investigation, we for the first time show that RAGE is carcinoma cell lines MDA-MB-231, MDA-MB-453, MCF7, T47D, expressed in a panel of aggressive breast cancer cell lines, TNBC, BT-474 were obtained from ATCC. SCP2 cells were kindly pro- and metastatic lymph node deposits. We also demonstrate that vided by Dr. Massague (28). MVT-1 cells (derived from MMTV-c- blocking of RAGE reduces tumor metastasis and that RAGE Myc; MMTV-VEGF bitransgenic mice) were obtained from Dr. ablation inhibits breast cancer growth. In addition, we show that Johnson and PyMT cells derived from MMTV-PyMT C57BL/6 RAGE mediates its tumor-promoting effects in breast cancer mice were obtained from Dr. Hai (OSU; ref. 29). MVT-1 highly through binding to S100A7. Our findings also uncovered that metastatic clone, PyMT, Met1, and 4T1 cells were cultured as the RAGE/S100A7 pathway enhanced breast cancer growth and described (18, 29). metastasis. These studies further demonstrate that RAGE neutralizing antibodies/soluble RAGE could be used to inhibit breast Chemotaxis cancer growth and metastasis, especially in S100A7-expressing Chemotactic assays were performed using Transwell chambers invasive cancers. Furthermore, these studies suggest that RAGE (Costar; 8-mm pore size) as described (18, 30). could be used as novel biomarker and therapeutic strategy against TNBCs. Mice Nude mice were obtained from Charles River. C57B/6 back- Materials and Methods ground RAGE / mice were kindly provided by Dr. Schmidt (New Patients York University, New York, NY), and TetO-mS100a7a15 mice Institutional Review Board of the Ohio State University (OSU; were kindly provided by Dr. Yuspa (NIH; Bethesda, MD). TetO- Columbus, OH) has approved protocol for the constructed TNBC mS100a7a15 mice (15) were cross-bred with MMTV-rtTA mice to tissue microarrays (TMA; n ¼ 173). The clinical and pathologic generate bitransgenic MMTV-mS100a7a15 mice. Knockout and characteristics of TNBC TMAs have been recently described (24). transgenic littermates were genotyped by PCR. Female MMTV- TMA for lymph node metastasis (BR1008) was obtained from US mS100a7a15 mice were fed with doxycycline-chow 1 g/kg (Bio- Biomax, Inc. Serv), and mice with normal diet served as controls. All mice were kept in The OSU's animal facility in compliance with the guide- Immunohistochemistry, immunofluorescence, and ELISA lines and protocols approved by the OSU Institutional Animal Samples from mammary glands and tumors were formalin- Care and Use Committee. fixed and paraffin-embedded (18). Standard IHC techniques were used according to the manufacturer's recommendations (Vector Orthotopic injection assay Laboratories) using antibodies against RAGE (Abcam, 1:400) MVT-1 or PyMT cells were injected into the mammary glands of Ki67 (Neomarkers, 1:100), CD31 (Santa Cruz 1:100), F4/80 transgenic or knockout mice. Transgenic mice injected with MVT- (AbD Serotec, 1:50), arginase 1 (Santa Cruz, 1:200), and iNOS 1 cells were either fed with doxycycline-chow 1 g/kg or normal diet (Abcam, 1:200) for 60 minutes at room temperature. Vectastain (control). Tumors were measured weekly with external calipers Elite ABC reagents (Vector Laboratories), using avidin DH:bioti- ¼ 2 0 and volume was calculated according to the formula V 0.52 a nylated horseradish peroxidase H complex with 3,3 -diamino- b, where a is the smallest superficial diameter and b is the largest benzidine (Polysciences) and Mayer's hematoxylin (Fisher Scien- superficial diameter. Orthotopically injected animals were sacri- fi ti c), were used for detection of bound antibodies. Staining of ficed and tumors were excised (18). RAGE neutralizing antibody fl TMAs was graded as previously described (25). Immuno uores- (human or murine) and soluble RAGE (human or murine) were fi fl cence was performed on paraf n-embedded tissues. Brie y, sec- purchased from R&D Systems. tions were stained with F4/80 (1:75), and MMP9 (R&D Systems, 1:150). Alexa Fluor–conjugated secondary antibodies (Life Tech- FACS analysis nologies) were used to detect primary antibody. Sections were fi mounted by VECTASHIELD mounting media containing DAPI Freshly prepared single-cell suspensions of tumor-in ltrating cells were incubated with anti-F4/80 PE or anti-CD11b APC (18). (Vector Laboratories, Inc.). Images were analyzed by confocal microscopy. Binding of RAGE with human recombinant S100A7 RAGE expression was analyzed by staining with RAGE antibody (Abcam) followed by Alexa Fluor 488 antibody. After staining, was performed as described (26). cells were analyzed by FACS Caliber using CellQuest software (BD Biosciences). Cancer patient data analysis High RAGE and S100A7 expressions were defined as overexpression of ager mRNA being greater than 0.5-fold and over- Western blot and coimmunoprecipitation expression of s100a7 being greater than 1.0-fold of SD above the Western blot analysis of cell or tumor lysates was done as mean, respectively. Association of gene expression alterations was described (30). Coimmunoprecipitation was carried out using performed on the basis of The Cancer Genome Atlas (TCGA) protein G plus A-agarose beads as described (31), with S100A7 database by Fisher exact test. Analysis of RAGE expression rabbit (Novus Biologicals) and RAGE mouse (Santa Cruz Bio- between basal and non-basal breast cancer samples was based technology) antibodies. on a subtype-specific breast cancer study (GEO accession GDS2250; ref. 27). For Kaplan–Meier survival analysis, patient Luciferase reporter assay samples with RAGE expression values greater than its median NF-kB activity was determined using NF-kB luciferase reporter were grouped as high RAGE and the other half as low RAGE. assay (Promega) per manufacturer's protocol.

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A B

4T1 cells - Met1 ER

MVT1 Murine cells SCP2 MDA-231 MDA-453 BT474 cells

T47D + cells ER + MCF7 ER

MDA-453 MCF7 T47D BT474

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3.0 80 0.6 ## 2.5 70 High 2.0 # Low 1.5 60 ( actb )

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−1.0 % Samples 20 ( ager )/Log 0.3 2

median-centered ratio −1.5

2 10 −2.0 Log 0.2 − 0 Log 2.5 Non-Basal Basal Normal IBC Basal HER2

G H 1.0 High expression 120 0.9 # Low expression 0.8 100 0.7 80 0.6 0.5 60 High n = 94 0.4 Low n = 414 % Samples 40 0.3

Survival probability 0.2 20 0.1 0 0.0 Nonmetastatic Metastatic 0.0 24 48 72 Follow-up in months

Figure 1. þ RAGE expression in breast cancer cell lines and patient samples. A, FACS analysis of RAGE in human TNBC, ERa , and highly metastatic murine mammary tumor cell lines. B, quantiﬁcation of RAGE expression obtained by FACS. C, RAGE expression in basal and non-basal type breast cancer. RAGE (ager) expression values are normalized to b-actin (actb). D, RAGE expression in normal and invasive breast cancer. E, representative photographs of RAGE expression in TNBC TMA. F, bar graph showing RAGE expression in TNBC (n ¼ 80) and HER2 (n ¼ 33) TMA samples. G, bar graph showing RAGE expression in metastatic breast cancer. We used TMA (n ¼ 100) that contained n ¼ 40 lymph node metastasis, n ¼ 50 malignant, and n ¼ 10 normal tissues. H, expression level of RAGE predicts survival differences by Kaplan–Meier analysis using R2 microarray dataset. Scale bar, 100 mm. #, P < 0.05; ##, P < 0.01. www.aacrjournals.org Cancer Res; 75(6) March 15, 2015 OF3

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Statistical analysis compared with normal control (Fig. 1D). Furthermore, we ana- To test the association between two categorical variables, c2 lyzed the expression of RAGE in breast cancer TMAs with accom- tests or Fisher exact tests were used. For continuous variables, 2- panying outcome data and other clinical information by IHC. We sample t tests were used if two groups were compared, and found that 92% of the samples showed high RAGE expression in ANOVAs were used if more than two groups were compared. TNBC tissues (Fig. 1E and F). However, RAGE was expressed only or # indicates P < 0.05; or ## indicates P < 0.01. in 29% HER2-positive TMAs (Fig. 1E and F). Using another TMA that contained metastatic and malignant patient samples, we Results found that metastatic tissue tends to have higher RAGE expression than malignant tissue (P < 0.0001; Fig. 1G). Next, we analyzed RAGE is expressed in highly metastatic breast cancer cells and open-access dataset for clinical outcome of RAGE expression. We its expression correlates with worse clinical prognosis found that high RAGE expression was associated with poor We analyzed RAGE expression in a panel of breast cancer cell prognosis (Fig. 1H). Taken together, these results show that RAGE lines. RAGE expression was higher in metastatic TNBC cell lines, þ expression is associated with highly aggressive and metastatic whereas low or no RAGE expression was observed in ERa breast breast cancers, including TNBC. cancer cell lines (MCF7, T47D, and BT474; Fig. 1A and B), which are weakly metastatic (32–34). These data suggest that RAGE is predominantly expressed in ERa and highly metastatic breast Blockade of RAGE inhibits tumor growth and metastasis in vivo cancer cell lines. To test the correlation of RAGE with ERa status, To investigate the role of RAGE in metastasis, we used the IVIS we analyzed open-access Gene Expression Omnibus (GEO) data- imaging system to analyze the metastatic potential of RAGE- sets for the expression of RAGE. In a subtype-specific breast expressing SCP2 cells. We injected highly metastatic single-cell cancer study (GEO accession GDS2250), RAGE expression is progeny clone 2 (SCP2) of MDA-MB-231 cell lines intracardially significantly enhanced in basal type (majorly TNBC) invasive into nude mice and blocked RAGE activity with RAGE neutraliz- breast cancer patient tumor samples compared with non-basal ing antibody (naRAGE). As shown in Fig. 2A and B, naRAGE þ type tumors (majorly ERa cancer; Fig. 1C). Next, we analyzed treatment significantly reduced the metastatic potential of SCP2 open-access dataset for RAGE expression. We found that high cells as compared with IgG control–treated mice. Next, we elu- RAGE expression was observed in invasive breast cancer (IBC) cidated the role of host RAGE on mammary cancer progression

ABDay 0 Day 60

IgG naRAGE IgG naRAGE 0.3 Day 0 Figure 2. Inhibition of RAGE reduces breast 0.2 cancer growth and distant metastasis. )

6 0.1 A, luciferase-positive SCP2 cells (1 105/100 mL) were injected 0 intracardially into nude mice (n ¼ 6) IgG naRAGE pretreated with mouse RAGE 60 Day 60 neutralizing (naRAGE) or IgG antibody 40 (20 mg/mouse). Representative Photon flux (10 Photon flux bioluminescent images (BLI) show 20 * comparative metastases of naRAGE- or IgG-treated mice. B, normalized photon 0 ﬂux of mice treated with naRAGE or IgG naRAGE IgG. C, RAGE expression was analyzed C D E in PyMT cells by FACS. Red, IgG; black, 6 ) 3,000 2.5

64 3 RAGE. D, PyMT (1 10 ) cells were RAGE+/+ 2,500 RAGE–/– 2.0 injected into the mammary gland 32 þ/þ / ¼ 2,000 of RAGE and RAGE mice (n 5) 1.5 and tumor volume was measured every 1,500 * * 16 1.0 week. E, after 5 weeks, tumors were

Events 1,000 excised from mice and weighed. F, 0.5 8 500 representative photograph of mice Tumor weight(g) Tumor

Tumor volume (mm volume Tumor 0 showing tumors dissected from 0 0 RAGE+/+ RAGE–/– different experimental groups. G, PyMT 12345 þ þ 100 101 102 103 104 cell line–derived tumors from RAGE / Weeks FL2H and RAGE / mice were subjected to IHC staining for CD31 and Ki67 F GHCD31 Ki67 RAGE+/+ RAGE–/– expression. H, tumor lysates (50 mg) þ þ from RAGE / and RAGE / mice were

+/+ P-ERK subjected to Western blotting using +/+ phospho-ERK (P-ERK), total ERK

RAGE (T-ERK), cyclin D1, and MMP-2 T-ERK RAGE antibodies. GAPDH served as loading Cyclin D1 control. Data represent mean SD of –/– –/– MMP2 three independent experiments. Scale bar, 100 mm. , P < 0.05. RAGE RAGE GAPDH

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Figure 3. A Co-currency of gene Odds ratio B C RAGE receptor binds to S100A7 and upregulation (>1.0 SD) Tendency toward MDA-MB-231 FACS enhances themigrationofbreast cancer 180 Gene ager s100a7 mapk1 co-currency cell lines. A, co-currency of ager (rage) Vec SA7 150 and s100a7 gene upregulation (>1.0 SD) ager P = 0.0055 P = 0.45 2.0 120 was analyzed as described (TCGA, No asso- 90 s100a7 P = 0.39 0.5 RAGE Nature, 2012; ref. 50). An irrelevant gene ciation 60 30 mapk1 (ERK gene) was used as control, Tendency toward mapk1 Expression % which showed no association with ager mutual exclusivity GAPDH 0 Vec SA7 and s100a7. B, RAGE expression was analyzed in S100A7-overexpressing DFE Vec SA7 RAGE-Fc EGFR-Fc MDA-MB-231 (SA7)cells comparedwith 0.9 vec control (Vec) as determined by 0.8 IP Western blotting. C, ﬂow cytometric 0.7 IgG SA7 TCL 0.6 analysis.D,immunoﬂuorescence. E, 0.5 IB: RAGE 0.4 S100A7 binding to RAGE as determined 0.3 by ELISA using RAGE-Fc or EGFR-Fc 0.2 (OD 450 nm) 0.1 proteins. Graph shows mean SEM of Specific binding 0.0 IB: S100A7 three independent experiments. F, one –0.1 DAPI RAGE 0 50 100 150 200 250 300 350 milligram of cell lysates from S100A7- Concentration of receptor (nmol/L) overexpressing MDA-MB-231 cells were subjected to immunoprecipitation with GH45 IgG or S100A7 and probed with anti- 1,400 40 * RAGE antibody. G and H, MDA-MB-231 ** ** 1,200 35 * cells (G) and SCP2 cells (H) were 30 1,000 pretreated with RAGE neutralizing 25 800 # antibody (naRAGE) or control IgG (10 20 mg/mL) for 30 minutes before being 15 ## 600 subjectedtorecombinantS100A7 (SA7; 10 migrated 400 migrated (×10) 50 ng/mL)-induced migration. Number of cells 5 Number of cells 200 I and J, MDA-MB-231 (I) and SCP2 (J) 0 Con SA7 SA7 + SA7 + 0 cells were pretreated with soluble IgG naRAGE Con SA7 SA7 + SA7 + RAGE (sRAGE, 50 ng/mL) for 30 IgG naRAGE minutesbefore stimulationwith S100A7 I K at the indicated times. Cell lysates (50 PBS sRAGE 3 mg) were subjected to Western blotting SA7 100 ng/mL 0 5 30 60 0 5 30 60 min using phospho-ERK (P-ERK) and total * P-ERK ERK (T-ERK). K, MDA-MB-231 cells were 2 transfected with either wild-type or T-ERK ## NF-kB plasmid for 24 hours, stimulated with S100A7 (SA7, 100 ng/mL) or IgG J 1 (10 mg) ornaRAGE (10mg) for additional * PBS sRAGE 24 hours, lysed, and analyzed for SA7 100 ng/mL 0 5 15 30 60 5 15 30 60 min luciferase activity. Renilla luciferase P-ERK Relative luciferase units 0 vector served as internal control. NF-kB + + + + Graphs represent mean SD for each SA7 (100 ng/mL) – + – + T-ERK experiment repeated three times IgG (10 µg) – + –– with similar results. , P < 0.05 and naRAGE (10 µg) – – + + , P < 0.01 versus con; #, P < 0.05 and ##, P < 0.01 versus SA7 IgG.

and development using the RAGE / model. First, we analyzed RAGE mediates its effect in breast cancer cells through S100A7 surface expression of RAGE on PyMT cells by FACS analysis. As RAGE has been shown to bind to S100A7 in various immune shown in Fig. 2C, PyMT cells expressed high RAGE expression. cells (14). When analyzing the RAGE and S100A7 expression in Next, mice were injected with PyMT cells and observed for tumor breast cancer (TCGA), we discovered that RAGE and S100A7 are growth for 35 days (Fig. 2D). Interestingly, PyMT-derived tumor often simultaneously upregulated in breast tumors among growth was significantly inhibited in RAGE / mice as compared patients of the IBC cohort (P ¼ 0.0055; Fisher exact test), whereas þ þ with RAGE / mice (P ¼ 0.0125; Fig. 2E and F). To identify the an irrelevant gene ERK (mapk1) showed no correlation with RAGE signaling molecules that are associated with breast cancer tumor or S100A7 (Fig. 3A). This co-currency of gene upregulation growth, we examined the expression of ERK, cyclin D1, and implies a functional link between RAGE and S100A7 in breast MMP2 in PyMT-derived tumor tissues obtained from RAGE / cancer. Furthermore, we observed enhanced expression of RAGE mice. We observed reduced phospho-ERK, MMP2, cyclin D1, in S100A7-overexpressing MDA-MB-231 cells by Western blot, CD31, and Ki67 expression in RAGE / mice compared with FACS, and immunofluorescent analyses (Fig. 3B–D). This sug- þ þ RAGE / mice (Fig. 2G and H). Cyclin D1 and Ki67 are markers gests that RAGE could be a receptor for S100A7 as ligand in breast for active proliferation (35, 36). These data demonstrate that cancer cells. We further verified the direct interaction between RAGE blockade significantly inhibits mammary tumor develop- human RAGE and human S100A7 protein using an ELISA-based ment and progression. binding assay, in which EGFR, an irrelevant receptor, was used as a

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negative control. As shown in Fig. 3E, the total binding was PBS doxycycline-induced group. Furthermore, we observed that dependent on RAGE-Fc concentration, whereas no binding was blocking RAGE or mS100a7a15 substantially decreased prolif- observed between EGFR-Fc and S100A7 protein. To further con- eration and angiogenesis in MVT-1–derived tumors obtained firm the association of RAGE with S100A7, we performed a from inducible MMTV-mS100a7a15 mice (Fig. 4G and H). coimmunoprecipitation assay. S100A7 coimmunoprecipitated Next, we investigated whether RAGE inhibition reduces surface with RAGE in S100A7-overexpressing MDA-MB-231 cells lung metastases in inducible MMTV-mS100a7a15 mice. We (Fig. 1F). Next, we analyzed the effect of human S100A7 on observed a significant decrease in surface lung metastases in MDA-MB-231 and SCP2 cell migration and that of its murine themicetreatedwithnaRAGE(Fig.4IandJ)orsRAGE(Fig.4K paralog mS100A7a15 on MVT-1 cell migration. S100A7 and L) in MVT-1–derived tumors obtained from inducible enhanced the migration of MDA-MB-231 and SCP2 cells, respec- MMTV-mS100a7a15 mice as compared with control groups. tively, and these effects were significantly abrogated by blocking Taken together, these results suggest that RAGE plays an impor- RAGE using neutralizing antibodies (Fig. 3G and H). We also tant role in mS100a7a15-induced breast cancer progression showed that its murine ortholog mS100a7a15 enhanced migra- and metastasis. tion of MVT-1 cells (Supplementary Fig. S1A). Next, we analyzed the S100A7-induced wound-healing capacity of MDA-MB-231 RAGE/S100A7 axis modulates the tumor microenvironment and MDA-MB-453 cell lines and observed that RAGE neutralizing Macrophages, especially M2-TAMs, have been shown to antibody significantly abrogated this effect (Supplementary Fig. enhance tumor growth and metastasis (21, 22, 38, 39). To identify S1B and S1C). In addition, we observed that S100A7 or the molecular mechanism of RAGE-mediated breast tumor mS100a7a15 significantly enhanced the invasion of SCP2 and growth and metastasis, we analyzed macrophage recruitment in þ þ MVT-1 cell lines (Supplementary Fig. S1D and S1E). To examine the PyMT-derived tumors of RAGE / and RAGE / mice. As whether activation of RAGE/S100A7 enhanced downstream sig- shown in Supplementary Fig. S2, F4/80/Arg1-positive macro- naling in breast cancer cells, we analyzed ERK activation. We phages were substantially reduced in RAGE / PyMT–derived þ þ showed that S100A7-induced ERK activation was inhibited by tumors as compared with RAGE / tumors. Next, we observed blocking S100A7 with soluble RAGE (sRAGE) in MDA-MB-231 that mS100a7a15-overexpressing mice significantly increased the þ and SCP2 cells (Fig. 3I and J). To further confirm these effects, we recruitment of F4/80 /Arg1 macrophages and RAGE blockage by used RAGE neutralizing antibody to block S100A7-induced ERK naRAGE treatment significantly reduced the recruitment of F4/ þ activation in MDA-MB-231 cells (Supplementary Fig. S1F). In 80 /Arg1 macrophages compared with IgG control, as analyzed addition, we observed that sRAGE inhibits S100A7-induced by IHC (Fig. 5A). In addition, we observed substantial decrease in MMP9 activation in SCP2 cells (Supplementary Fig. S1F). NF-kB the recruitment of M2 macrophages in MVT-1–derived tumors has also been shown to be the downstream target of RAGE (37). pretreated with sRAGE in inducible mice compared with PBS- þ Using NF-kB reporter assay, we showed that S100A7 significantly treated mice (Fig. 5B). We also observed reduced CD11b/F4/80 enhanced NF-kB activity and this effect was inhibited by naRAGE TAMs by FACS (Fig. 5C). We further showed decreased expression (Fig. 3K). Taken together, these results imply that the RAGE/ of iNOS (M1 marker) in primary tumors compared with IgG S100A7/mS100a7a15 signaling axis is necessary for breast cancer control (Fig. 5A and B). We also analyzed the infiltrations of cell migration and invasion. macrophages in the lung tissues and observed reduced expression þ of F4/80 macrophages and Arg1 expression in naRAGE- or Blockade of RAGE inhibits mammary tumor growth and sRAGE-treated MMTV-mS100a7a15–inducible mice when com- metastasis in inducible MMTV-mS100a7a15 mice pared with control mice (Supplementary Fig. S3). These studies To analyze the relevance of RAGE/mS100a7a15 in a mam- suggest that blockade of RAGE in mS100a7a15 transgenic mice mary tumor growth and metastasis model, we used an MVT-1 inhibits tumor growth and metastasis through inhibition of M2 syngeneic orthotopic model that recapitulates the stages of macrophage recruitment. human primary tumors. We injected MVT-1 cells into inducible Because macrophage recruitment to primary tumors plays an MMTV-mS100a7a15 transgenic mice and blocked RAGE with important role in promoting S100A7/mS100a7a15-induced neutralizing RAGE antibody. Mice were treated with doxycy- metastasis(18),wewantedtoknowwhetherrecombinant cline-chow (1 g/kg) 1 week before MVT-1 injection to switch on mS100a7a15 might affect macrophage activity in a RAGE- the expression of mS100a7a15. When tumors grew as large as dependent manner. We analyzed the migration of RAW264.7 100 mm3, the doxycycline-treated group was given naRAGE (20 (RAW), a macrophage cell line, in the presence of mS100a7a15 mg/mouse) or IgG (20 mg/mouse) intraperitoneally 3 times a recombinant protein. We showed that mS100a7a15 significant- week for 20 days. MMTV-mS100a7a15 mice fed with normal ly enhanced the migration of RAW and that pretreatment with chow were used as a negative control. Inducible mice treated naRAGE significantly abrogated mS100a7a15-induced migra- with naRAGE showed reduced tumor progression compared tion compared with IgG control (Fig. 6A and B). Although with the IgG-treated group (Fig. 4A–C). To determine whether S100A7 has been shown to regulate MMPs in cancer cells (17), a blocking ligand of RAGE inhibits mammary tumor progres- its role in the macrophage is not known. We analyzed MMP9 sion, we used soluble RAGE (sRAGE). Mice were fed with secretion in the presence of mS100a7a15 with or without doxycycline-chow 1 week before the injection of MVT-1 cells sRAGE treatment. mS100a7a15-induced MMP9 secretion was into the #4 mammary gland. After day 1 of doxycycline induc- enhanced in RAW cells and this effect was diminished in the tion, mice were injected intraperitoneally with murine sRAGE presence of sRAGE (Fig. 6C). Using double immunofluores- (2 mg/mouse) or PBS. MMTV-mS100a7a15 mice who received cence, we also observed enhanced recruitment of MMP9- þ normal chow served as a negative control. As shown in Fig. expressing F4/80 macrophages in the MVT-1 tumors of 4D–F, sRAGE treatment significantly reduced tumor progres- MMTV-mS100a7a15–inducible mice compared with nonindu- þ þ sion in the doxycycline-induced group as compared with the cible mice. The recruitment of MMP9 /F4/80 macrophages

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ABC1,400 1.6

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) 1.2 3

1,250 * Dox (–) Dox (+) PBS 1 1,000 Dox (+) SR # 0.8

750 PBS # 0.6 Dox (+) 500

Tumor weight(g) Tumor 0.4 250 0.2 SR Dox (+) Tumor volume (mm Tumor volume 0 0 WK1 WK2 WK3 WK4 Dox (–) Dox (+) Dox (+) Time PBS SR G H Dox (–) Dox (+) IgG Dox (+) naRAGE Dox (–) Dox (+) PBS Dox (+) SR Ki67 Ki67 CD31 CD31

I JLK Lung nodules H&E Lung nodules H&E 50 * 50 * 40 40 Dox (–) # Dox (–) # 30 30 PBS

IgG 20 Dox (+) 20 Dox (+)

10 10 Number of lung nodules SR Number of lung nodules

0 Dox (+) Dox (+) naRAGE 0 IgG SR Dox (–) PBS Dox (+) Dox (+) Dox (+) naRAGE Dox (–) Dox (+)

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A –DOX DOX (+) (IgG) DOX (+) naRAGE B Dox (–) Dox (+) PBS Dox (+) SR F4/80 F4/80 Arg-1 Arg-1 iNOS iNOS

C D 7 * DOX (+) IgG DOX (+) naRAGE –DOX 6 5 4 3 # F4/80 2

% CD11b/F4/80 % 1 0 (–) DOX DOX (+) DOX (+) IgG naRAGE CD11b

Figure 5. RAGE/mS100a7a15 regulates TAM recruitment. A, representative IHC staining of macrophage markers (F4/80þ, Arg1, and iNOS) in MVT-1 tumors derived from naRAGE or IgG-treated Dox-induced (þDox) and uninduced (Dox) MMTV-mS100a7a15 mice. B, intratumoral macrophages were highlighted with IHC for macrophage markers in MVT-1 tumors derived from sRAGE (SR) or PBS-treated Dox-induced and uninduced MMTV-mS100a7a15 mice. Scale bar, 100 mm. C and þ D, quantitative analyses of F4/80 macrophages by FACS in MVT-1 tumors derived from naRAGE or IgG-treated þDox or Dox MMTV-mS100a7a15 mice. , P < 0.05 and , P < 0.01 versus Dox (); #, P < 0.05 and ##, P < 0.01 versus Dox (þ) IgG or PBS.

was substantially decreased in the naRAGE-treated group com- Discussion pared with IgG-treated MMTV-mS100a7a15–inducible mice Emerging data have implicated importance of RAGE in the (Fig. 6D). We further analyzed the molecular mechanism of pathogenesis of various human disorders including cancers the mS100a7a15-induced migration of RAW cells. As shown (2, 10). The interactions between RAGE and its ligands trigger in Fig. 6E and F, mS100a7a15-induced ERK activation was the activation of MAPK, JAK/STAT, and NF-kB in various cancers signiﬁcantly reduced in the presence of sRAGE treatment. These (1, 2). In this work, we identiﬁed that RAGE plays a critical role in studies suggest that RAGE receptor regulates mS100a7a15- promoting breast cancer growth and metastasis. We documented induced ERK activation in macrophages. that RAGE is highly expressed in human TNBC and murine breast

Figure 4. Blockade of RAGE inhibits mammary tumor growth and metastasis in MMTV-mS100a7a15–inducible mice. A, MVT-1 (1 105) cells were injected into the mammary gland of doxycycline-treated (þDox) or untreated (Dox) MMTV-mS100a7a15 mice (n ¼ 5). Dox-treated mice (n ¼ 10) were injected intraperitoneally with RAGE neutralizing antibody (naRAGE) or control IgG (20 mg/mouse) for every alternate day and tumor volume was measured every week. B, after 28 days, the tumors were excised. C, representative photograph of tumors dissected from different experimental groups. D, MVT-1 (1 105) cells were injected into the mammary gland of þDox or Dox MMTV-mS100a7a15 mice (n ¼ 5). Dox-treated MMTV-mS100a7a15 mice (n ¼ 10) were either treated with sRAGE (SR, 2 mg/mouse) or PBS for every alternate day and tumor volume was measured every week. E, after 28 days, the tumors were excised. F, representative photograph of tumors dissected from different experimental groups. IHCs were performed for CD31 and Ki67 in tumors from Dox-induced MMTV-mS100a7a15 mice that were either treated with naRAGE or control IgG (G) or SR (H). Data represent mean SD of three independent experiments. Scale bar, 100 mm. , P < 0.05 and , P < 0.01 versus Dox (); #, P < 0.05 and ##, P < 0.01 versus Dox (þ) IgG or PBS. I, left, representative photograph of lungs dissected from naRAGE-treated groups. Right, hematoxylin and eosin (H&E) staining of metastatic deposits. J, bar graph showing the number of metastatic nodules on the lungs (18). K, left, representative photograph of lungs dissected from SR-treated groups. Right, hematoxylin and eosin (H&E) staining of metastatic deposits. L, bar graph showing the number of metastatic nodules on the lungs. Data represent mean SD per experimental group. Scale bar, 20 mm. , P < 0.05 and , P < 0.01 versus Dox (); #, P < 0.05 and ##, P < 0.01 versus Dox (þ) IgG or PBS.

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RAGE Enhances Breast Cancer Growth and Metastasis

ACB 190 64 * CSRSA15 SA15+SR IgG * RAGE 170

32 MMP9 150 # 3.5 16 130 3

Events 2.5 110 2

8 cells % Migrated 1.5 90 1 0.5 0 0 expression Relative 0 0 1 2 3 4 0 SA15 IgG 10 10 10 10 10 C SR SA15 SA15+SR IgG+ FL2H SA15 + SA15 naRAGE naRAGE

D DAPI F4/80 MMP9 Merge –Dox +Dox IgG +Dox naRAGE

EF SA15 IgG naRAGE 0.8 SA15 0 30 60 30 60 30 60 min 0.7 P-ERK 0.6 0.5 0.4 T-ERK 0.3 0.2 0.1 GAPDH expression Relative 0 0 SA15 30' SA15 60' SA15+ SA15+ SA15+ SA15+ IgG 30' IgG 60' naRAGE naRAGE 30' 60'

Figure 6. RAGE/mS100a7a15 regulates MMP9þ macrophages. A, RAGE expression was analyzed on RAW cells by FACS. B, RAW cells were subjected to mS100a7a15 (100 ng/mL)-induced migration in presence of murine RAGE neutralizing (naRAGE) or control IgG antibodies. C, RAW cells were stimulated with mS100a7a15 (100 ng/mL) in the presence or absence of sRAGE (SR, 50 ng/mL) for 24 hours. Conditioned media were analyzed for MMP activity. C, bottom, quantification. D, tumors excised from (Dox) MMTV-mS100a7a15 mice were subjected to double immunofluorescence for MMP9 (green), F4/80 (red), or DAPI (blue). E, RAW cells were pretreated with naRAGE or IgG antibodies for 1 hour, stimulated with mS100a7a15 (SA15, 100 ng/mL), and subjected to Western blotting for P-ERK. Scale bar, 63 um. F, quantification of Western blot analyses. Data represent mean SD per experimental group. or #, P < 0.05 versus con; # versus SA15 IgG.

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in S100A7-overexpressing MDA-MB-231 cells. Our bioinformat- ics data also showed that RAGE is co-overexpressed with S100A7 in human breast cancer tissue. Proinﬂammatory RAGE ligands, naRAGE S100A7/S100a7a15 such as S100B, S100A4, and S100A8/A9, have been shown to enhance RAGE expression, and continuous activation of RAGE maintains an inﬂammatory milieu at the tumor microenviron-

RAGE ment (2, 44). Our data suggest that S100A7 enhances activation of Extracellular NF-kB through RAGE activation. We also showed that blocking sRAGE RAGE or inhibiting S100A7/mS100a7a15 binding to RAGE by Intracellular soluble RAGE (sRAGE) inhibits breast cancer cell migration and Decoy ERK activation. The sRAGE acts as a decoy that prevents ligands Cytokines from interacting with the cell surface receptor. The application of sRAGE in vitro and in vivo resulted in an effective blockade of RAGE, in accordance with this decoy mechanism, in a range of animal models (11). It is well documented that S100A8/A9-RAGE axis plays a significant role in breast and colon cancer growth TAMs Tumor and metastasis by modulating its downstream targets such as ERK1/2 (P44/p42), MAPK, and NF-kB signaling pathways MMPs (7, 42, 45). In the current study, we have shown that RAGE deficiency in the host reduced breast cancer growth by decreasing recruitment of TAMs and tumor angiogenesis. We have shown previously that mS100a7a15 overexpression in mammary glands enhanced mammary tumor growth metastasis through Metastasis macrophage recruitment (18). To further analyze molecular mechanism of these effects, we blocked RAGE activation in Figure 7. MMTV-mS100a7a15–inducible model by naRAGE or sRAGE. Schematic representation of RAGE-mediated S100A7-induced signaling that regulates breast cancer growth and metastasis. Epithelial cells release Blocking RAGE reduced macrophage recruitment into the S100A7/mS100a7a15, which binds to RAGE and activates signaling cascades MVT-1–derived tumors in the MMTV-mS100a7a15–inducible that recruit TAMs to the tumor stroma. TAMs in turn enhance growth and model.Ourstudyfurtherrevealed that blocking the RAGE/ metastasis by secreting growth factor, chemokines/cytokines, and MMPs. mS100a7a15 axis inhibits M2 marker arginase expression. Blocking of RAGE/S100A7 axis by sRAGE or naRAGE may reduce breast M2-polarized TAMs are known to drive tumor progression tumor growth and metastasis, especially to lungs. by stimulating angiogenesis and metastasis (21, 38, 46). We did not observe a significant change in CD4/CD3/CD8-pos- þ cancer cell lines and weakly expressed in low metastatic ER itive T cells and other immune cells such as natural killer cells, cells. However, there are some conflicting reports regarding the as detected by FACS in MVT-1–derived tumors obtained from expression of RAGE in the MCF7 cell line (40, 41). It could be MMTV-mS100a7a15 –inducible mice treated with RAGE neu- due to the difference in the techniques that were used. We tralizing antibody or sRAGE. detected RAGE expression by FACS analysis. However, other We showed that blocking of RAGE inhibits mS100a7a15- studies detected RAGE expression by Western blot analysis. induced recruitment and MMP9 activation in macrophages. There is possibility that RAGE detected by Western blotting MMP9 has been shown to degrade the extracellular matrix and could be a truncated or soluble form of RAGE. In addition, we release growth factors to enhance angiogenesis (47, 48). Further- showed that RAGE is preferentially expressed in invasive and more, it has been shown that MMP9 induction by primary tumors metastatic tumor deposits. This is consistent with a recent in macrophages and the lung endothelium promotes metastasis, report that demonstrated that high RAGE expression was especially to lung (48, 49). observed in lymph node and distant metastases patients sam- In summary, our study shows that RAGE is highly expressed in ples (42). Moreover, we observed that RAGE expression was basal-type breast cancer, especially TNBC, and is preferentially associated with poor prognosis in breast cancer. We also char- expressed in invasive and lymph node metastasis tissues. As acterized the role of RAGE in breast cancer progression and depicted by our model (Fig. 7), elucidation of the molecular metastasis. Using a genetic approach, we showed that RAGE mechanism behind enhanced breast cancer growth and metastasis ablation significantly reduced PyMT cell–derived tumor growth shows that this is likely due to binding of RAGE to S100A7. In while blocking RAGE with neutralizing antibodies inhibits turn, the RAGE/S100A7 axis is responsible for enhanced recruit- breast cancer visceral metastasis in preclinical mouse models. ment of MMP9-positive TAMs. We have also shown that RAGE It has been shown recently that RAGE knockdown by siRNA neutralizing antibody and soluble RAGE significantly decrease significantly inhibited tumorigenic potential of MDA-MB-231 tumor growth and metastasis in an inducible mS100a7a15 trans- cell line (40). RAGE ablation in a triple transgenic model of genic mouse model. These data imply that the RAGE/S100A7 spontaneous pancreatic cancer has also been shown to delay signaling axis could be used to inhibit TNBC growth and metas- pancreatic cancer development (9, 43). tasis. Furthermore, these studies demonstrate that RAGE could be Moreover, we demonstrated that RAGE binding to S100A7 used as a novel biomarker and that neutralizing antibodies/ enhanced RAGE expression in breast cancer cells. In addition, we soluble RAGE could be used to develop novel therapeutic strat- provide evidence that S100A7 coimmunoprecipitated with RAGE egies against TNBC.

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Disclosure of Potential Conﬂicts of Interest Acknowledgments No potential conﬂicts of interest were disclosed. The authors thank Kristin Kovach for assistance with IHC. They also thank Drs. Z. Qamri, A. Sneh, D. Chakroborty, and G. Amponsah Authors' Contributions for technical assistance and S. Adamovich for critical reading of the Conception and design: M.W. Nasser, N.A. Wani, W.E. Carson III, R.K. Ganju article. Development of methodology: M.W. Nasser, N.A. Wani, H. Zhao, L. Padilla, R.K. Ganju Acquisition of data (provided animals, acquired and managed patients, Grant Support provided facilities, etc.): M.W. Nasser, N.A. Wani, D.K. Ahirwar, C.A. Powell, This work was supported by grants from NIH (CA109527 and CA153490) J. Ravi, M. Elbaz, K. Shilo, C. Shapiro, R.K. Ganju and Department of Defense to R.K. Ganju. N.A. Wani, D.K. Ahirwar, and H. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Zhao were supported by Pelotonia Fellowship from the Comprehensive Cancer computational analysis): M.W. Nasser, N.A. Wani, D.K. Ahirwar, C.A. Powell, Center, OSU. M. Elbaz is supported by Fellowship from Government of Egypt. M. Elbaz, H. Zhao, L. Padilla, X. Zhang, R.K. Ganju The costs of publication of this article were defrayed in part by the Writing, review, and/or revision of the manuscript: M.W. Nasser, D.K. Ahir- payment of page charges. This article must therefore be hereby marked war, C.A. Powell, J. Ravi, L. Padilla, X. Zhang, K. Shilo, M. Ostrowski, C. Shapiro, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate W.E. Carson III, R.K. Ganju this fact. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.W. Nasser, J. Ravi, M. Elbaz, K. Shilo, M. Ostrowski, R.K. Ganju Received July 21, 2014; revised November 4, 2014; accepted November 19, Study supervision: R.K. Ganju 2014; published OnlineFirst January 8, 2015.

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OF12 Cancer Res; 75(6) March 15, 2015 Cancer Research

RAGE Mediates S100A7-Induced Breast Cancer Growth and Metastasis by Modulating the Tumor Microenvironment

Mohd W. Nasser, Nissar Ahmad Wani, Dinesh K. Ahirwar, et al.

Cancer Res Published OnlineFirst January 8, 2015.

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