S100A9 Is a Novel Ligand of EMMPRIN That Promotes Melanoma Metastasis

Published OnlineFirst November 7, 2012; DOI: 10.1158/0008-5472.CAN-11-3843

Cancer Molecular and Cellular Pathobiology Research

Toshihiko Hibino1, Masakiyo Sakaguchi2, Shoko Miyamoto1, Mami Yamamoto1,3, Akira Motoyama1, Junichi Hosoi1, Tadashi Shimokata3, Tomonobu Ito3, Ryoji Tsuboi3, and Nam-ho Huh2

Abstract The calcium-binding proteins S100A8 and S100A9 can dimerize to form calprotectin, the release of which during tissue damage has been implicated in inflammation and metastasis. However, receptor(s) mediating the physiologic and pathophysiologic effects of this damage-associated "danger signal" are uncertain. In this study, searching for candidate calprotectin receptors by affinity isolation-mass spectrometry, we identified the cell surface glycoprotein EMMPRIN/BASIGIN (CD147/BSG). EMMPRIN specifically bound to S100A9 but not S100A8. Induction of cytokines and matrix metalloproteases (MMP) by S100A9 was markedly downregulated in melanoma cells by attenuation of EMMPRIN. We found that EMMPRIN signaling used the TNF receptor– associated factor TRAF2 distinct from the known S100-binding signaling pathway mediated by RAGE (AGER). S100A9 strongly promoted migration when EMMPRIN was highly expressed, independent of RAGE, whereas EMMPRIN blockade suppressed migration by S100A9. Immunohistologic analysis of melanomas revealed that EMMPRIN was expressed at both the invasive edge of lesions and the adjacent epidermis, where S100A9 was also strongly expressed. In epidermal-specific transgenic mice, tail vein–injected melanoma accumulated in skin expressing S100A9 but not S100A8. Together, our results establish EMMPRIN as a receptor for S100A9 and suggest the therapeutic use in targeting S100A9–EMMPRIN interactions. Cancer Res; 73(1); 172–83. 2012 AACR.

Introduction Regarding receptors, RAGE (receptor for AGE, advanced fl There is a well-established link between chronic inflam- glycation end products) is known to be a general and in am- – mation and cancer development (1–3). A number of mole- mation-related S100 protein receptor (9 11). However, RAGE is cules have been identified as contributing to this process; a multiligand receptor of the immunoglobulin superfamily of these include TNF-a, interleukin (IL)-1, IL-6, chemokines, cell surface proteins that acts as pattern recognition receptors. matrix metalloproteases (MMP), angiogenic factors, and Ligands of RAGE include AGE, S100/calgranulins, high mobil- anti-apoptotic proteins. We have shown that many of these ity group box-1 (HMGB1), amyloid beta peptide, and beta-sheet fi molecules are upregulated in cultured keratinocytes stimu- brils. In addition, S100A9, S100A11, S100A12, S100A13, and lated with S100A8/A9 (4). Furthermore, S100A8/A9-induced S100P were also thought to interact with RAGE and transduce cytokines and chemokines, in turn, stimulate keratinocytes signals (12, 13). to synthesize and secrete S100A8/A9, suggesting a positive Although this variety of ligands implicates RAGE in a wide feedback mechanism between S100A8/A9 and these pro- spectrum of pathophysiologic conditions, it would be inap- inflammatory factors. In addition to the inflammatory propriate to postulate that all the S100-mediated reactions are processes, recent investigations have revealed that S100 totally dependent on their interaction with RAGE. Indeed, proteins play important roles in malignancy, especially in some S100 proteins seem to signal by engaging not only RAGE cancer cell metastasis (5–8). but also non-RAGE receptors. Other putative S100-binding proteins include CD36 (14), heparan sulfate proteoglycan (15), and carboxylated glycans (16). In the present study, we tried to identify the S100A8/A9 Authors' Affiliations: 1Shiseido Innovative Science Research & Develop- ment Center, Yokohama; 2Department of Cell Biology, Okayama University receptor involved in the positive feedback mechanism of Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, inflammation. We used Liquid chromatography coupled Okayama; and 3Department of Dermatology, Tokyo Medical University, Tokyo, Japan with electrospray tandem mass spectrometry (LC/MS- MS)-based shotgun proteomics and succeeded in identifying Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). the extracellular matrix metalloprotease inducer (EMM- PRIN) as a novel receptor for S100A9. Interestingly, EMM- Corresponding Author: Toshihiko Hibino, Shiseido Innovative Science Research & Development Center, 2-12-1 Fukuura, Kanazawaku, Yoko- PRIN is a well-known cell surface molecule that is associated hama 236-8643, Japan. Phone: 81-45-788-7289; Fax: 81-45-788-7277; with cancer cell malignancy (17, 18). We explored the E-mail: [email protected] S100A9–EMMPRIN relationship in the context of melanoma doi: 10.1158/0008-5472.CAN-11-3843 cell metastasis in vitro and in vivo. Our data provide new 2012 American Association for Cancer Research. and important insights into the molecular mechanism of

172 Cancer Res; 73(1) January 1, 2013

Identiﬁcation of a Novel Receptor of S100A9

S100A9-EMMPRIN–mediated inflammation and cancer cell Melanoma cell lines metastasis. SK-MEL-2 (HTB-68), SK-MEL-3 (HTB-69), SK-MEL-5 (HTB- 70), MeWo (HTB-65), and A2058 (CRL-11147) were purchased Materials and Methods from American Type Culture Collection in 2002, and after expanded culture, 1 106 cells/tube were kept in liquid Liquid chromatography coupled with electrospray nitrogen B16-BL6 cells (1), a highly invasive variant of a tandem mass spectrometry mouse melanoma B16 cell line, was obtained from Dr. Isaiah Proteins were identified via a shotgun-type protein identi- J. Fidler (M. D. Anderson Cancer Center, Houston, TX). All cell fication approach described previously (19, 20). Briefly a lab- lines were used from these stocks or within 3 passages. made nano-flow liquid chromatography-mass spectrometry Melanoma cells were cultured with Dulbecco's Modified platform consisting of a Nanospace SI-2 high-performance Eagle's Medium (DMEM) supplemented with 10% fetal calf liquid chromatograph (Shiseido Co.), a Yates lab-designed serum. nano-electrospray ionization source, and an LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific, Inc) were Establishment of stable clones used to obtain tandem mass spectra of tryptic peptides. The For isolation of clones stably expressing dnEMMPRIN, B16- resulting tandem mass spectrometry spectra were analyzed BL6 cells were subjected to conventional transfection method using the SEQUEST algorithm against a nonredundant human with the plasmids (pDNR-CMVi-dnEmmp and pMSCV-puro; protein database (NCBI, Feb2007) for putative protein Clontech TAKARA) using the FuGENE-HD reagent (Promega identifications. Biosciences) and then selected with puromycin.

Preparation of expression vectors Microarray analysis We prepared various expression vectors for soluble (sol) Whole human genome (4 44K) Oligo Microarray Kit EMMPRIN, dominant-negative (dn) EMMPRIN, cytoplasmic (Agilent) was used. We prepared the target RNA from SK- domains of N-terminal (RAGE-cyt; ref. 13) and C-terminal MEL-2 and primary keratinocytes in the presence or absence of (EMMPRIN-cyt), EMMPRIN variants (Mut-Traf and Mut-C- recombinant S100A8, S100A9, or S100A8/A9 (10 mg/mL each) ter), TIRAP, MyD88, TRAF2, TRAF6, dnTRAF2, dnTRAF6, for 3 or 24 hours as described. cDNAs from total RNA (1.65 mg S100A8, and S100A9. Details of constructions are provided in starting material) isolated from cell culture harvests were used the Supplementary Methods. for generation of Cy3-labeled cRNA by in vitro transcription. The cRNA was fragmented before hybridization, and a hybrid- Coimmunoprecipitation analysis ization cocktail was prepared. Microarrays were hybridized, Transient transfection of each plasmid to the HEK293 cells scanned and image analysis was conducted as described by the was conducted using FuGENE-HD (Promega Biosciences). manufacturer. After 48 hours, conditioned medium was subjected to coimmunoprecipitation experiments, using anti-HA tag Real-time quantitative reverse transcription PCR (clone HA-7) agarose (Sigma-Aldrich) and anti-Myc tag Real-time PCR was carried out with an ABI 7900HT Real (clone 1G4) Agaroses (MBL). The tag-Agarose beads were time PCR system (Applied Biosystems) using the Sybr Green mixed with conditioned mediums and incubated for 3 hours PCR mix (Invitrogen). Samples were cycled at 95Cfor10 at 4 C. The precipitated proteins were subjected to SDS- minutes, 40 cycles of: 95C for 15 seconds, 60Cfor1minute. PAGE and detected by subsequent Western blotting using Primer sets used are listed in the Supplementary Materials mouse anti-HA tag (clone 6E2; Cell Signaling Technol- and Methods. ogy) and mouse anti-Myc tag (clone 9B11; Cell Signaling Technology) antibodies. GTP-bound form of cdc42 was Melanoma tissues also determined using a Rac/cdc42 Activation Assay Kit Human skin specimens were obtained under informed (Millipore). consent from the patients with melanoma. A total of 20 melanoma tissues were analyzed immunohistochemically. The S100-EMMPRIN binding assay study was approved by the Institutional Review Board of Tokyo Recombinant EMMPRIN was immobilized on a 96-well Medical University (Tokyo, Japan) and the Shiseido Committee ELISA plate by incubation at 4C overnight. After washing on Human Ethics. with PBS containing 0.1% Tween20 (PBS-T), the plate was incubated with PBS containing 5% bovine serum albumin Knockdown of EMMPRIN by siRNA silencing (BSA) at 37C for 1 hour for blocking. Various concentrations Recombinant S100A8 and S100A9 were prepared as pre- of recombinant S100A9 were added to each well, and the viously described (4). SK-MEL-2 cells were plated at 50% to plate was incubated at 37C for 1 hour. After washing with 60% conﬂuency and transfected with 50 nmol/L siRNA PBS-T, anti-S100A9 antibody (1:2,000 in PBS) was added, and against EMMPRIN or nonspeciﬁc control (Santa Cruz) using bound S100A9 was detected with horseradish peroxidase Lipofectamine RNAi MAX (Invitrogen). After 24 hours post- (HRP)-conjugated anti-rabbit antibody (1:2,000 in PBS). Color transfection, 10 mg/mL recombinant S100A8, S100A9, or was developed using TMB Peroxydase EIA Substrate Kit (Bio- S100A8/A9 were added and incubated for an additional 3 Rad Laboratories) and read at 630 nm. or 24 hours.

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 173

Hibino et al.

In vitro migration assay Immunohistochemical procedures Migration of human melanoma cells was assayed using Primary antibodies used are listed in the Supplementary a 96-well Disposable Chemotaxis System (8-mm pore size; Materials and Methods. HRP-conjugated, goat anti-rabbit IgG Funakoshi). The lower wells of the chamber were loaded or anti-mouse IgG (Nichirei) were used as secondary antibo- with DMEM containing recombinant S100A8, S100A9, or dies and reacted with 3,30-diaminobenzidine (DAB). For double S100A8/A9 (10 mg/mL). Melanoma cells (10,000 cells per detection, Alexa Fluor 555 or 488 (Molecular Probes Inc.) was 25 mL in DMEM) were placed in the upper wells. After used. 40,6-diamidino-2-phenylindole (DAPI; Molecular Probes) incubation for 48 hours at 37C, cells on the lower surface was used to visualize nuclei. of the filter were stained with CellTracker Red CMTPX (5 mm, Molecular Probes), and fluorescence intensity Statistical analysis was measured and compared with that of the medium Data are expressed as the mean SD. We used simple control. pairwise comparison with the Student t test (2-tailed distri- bution with 2-sample equal variance). P < 0.05 was considered Generation of S100A8 and S100A9 transgenic mice significant. Human S100A8 and S100A9 were PCR-cloned from keratinocyte cDNA. Each S100 cDNA was fused to the involucrin Results promoter (ref. 21; a generous gift from Dr. Taichman) and EMMPRIN is identified as an S100A8/A9-binding protein transgenic (Tg) mice were generated using BDF1 mice. To We carried out affinity purification-mass spectrometry obtain the hairless phenotype, the involucrin-S100 Tg mice using keratinocyte membrane fractions, as S100A8/A9 is were crossed with HR-1 mice. S100A8 and S100A9 Tg mice were strongly upregulated in the epidermis in inflammatory skin used for metastasis assays after 3 passages. disorders (22). We analyzed interactions between 2 kinds of membrane extracts (from proliferating and differentiated In vivo metastatic assay cells) and glutathione S-transferase (GST)-fused S100 proteins. The SK-MEL-2 and SK-MEL-5 cell lines were injected into Overlapping proteins interacting with GST were removed and the tail vein of Tg mice (1 106 cells in 0.25 mL/mouse) and finally we identified total of 13 proteins from the analyses littermate wild mice. Six mice each were injected per mela- (Table 1). S100A9 showed the highest probability and GST noma cell line. The control group without melanoma cells were followed next in each sample, indicating high fidelity of these injected serum-free DMEM. After 4 weeks of injection, skin was analyses. Interestingly, we found 2 membrane proteins, leucine excised and stained for the melanoma markers. Metastases zipper-EF-hand transmembrane protein 1 (LETM1) and basi- were examined using LSM5 PASCAL (Zeiss). gin. As LETM1 serves as an anchor protein for complex

Table 1. Summary of LC/MC-MC analysis of S100A8/A9-interacting proteins

Protein ID Probabilitya

Keratinocyte Keratinocyte (Differentiated phase) (Growth phase)

Description S100A9 S100A8/A9 S100A9 S100A8/A9 S100 calcium-binding protein A9 [Homsapiens] 1.00E-302 2.00E-10 1.00E-30 2.26E-11 Glutathione S-Transferase (E.C.2.5.1.18) 4.00E-13 7.66E-14 4.11E-14. 4.03E-13 Xeroderma Pigmentosum Group E Complementing protein 7.29E-13 N.D. N.D. N.D. Glutathione transferase [Homo sapiens] 1.21E-12 2.84E-11 6.98E-11 9.91E-13 Tubulin alpha 6 [Homo sapiens] 1.27E-08 6.63E-09 N.D. N.D. Acyl-CoA synthetase long-chain family 5.94E-08 N.D. N.D. N.D. Leucine zipper-EF-hand containing transmembrane 1.07E-07 N.D. N.D. N.D. B Chain B, Human Glutathione S-Transferase 2.93E-07 8.76E-07 N.D. N.D. Eukaryotic translation elongation factor 1 beta 2 1.19E-05 N.D. N.D. N.D. Basigin isoform 1 [Homo sapiens] 1.62E-05 N.D. 3.16E-05 N.D. Ribosomal protein P2 [Homo sapiens] 2.71E-05 N.D. N.D. N.D. Myosin, light polypeptide 6, alkali, smooth muscle 3.86E-04 N.D. N.D. N.D. S100 calcium-binding protein A8 [Homo sapiens] N.D. 8.19E-05 N.D. 3.25E-07

NOTE: Bold indicates notable molecules including S100A8, S100A9, and basigin (EMMPRIN). Abbreviation: N.D., not deﬁned. aStringent search criteria: Sf score > 0.85; peptide probability < 0.001; number of top matches: 1; precursor mass tolerance: < 5 ppm; minimum number of peptides to identify proteins: 1; enzyme speciﬁcity: half-tryptic or fully tryptic (peptides only).

174 Cancer Res; 73(1) January 1, 2013 Cancer Research

Identiﬁcation of a Novel Receptor of S100A9

formation with the mitochondrial ribosome (23), a cell mem- S100A9–EMMPRIN interaction is the key to inducing proin- brane protein, basigin, was selected as a candidate molecule. flammatory factors and MMPs both in keratinocytes and in Basigin is also known as EMMPRIN or CD147 (24). EMMPRIN melanoma cells. was found to be an S100A9 interactor in extracts from both proliferating and differentiated cells. Signal transduction pathways are distinct between EMMPRIN and RAGE S100A9 specifically binds with EMMPRIN As RAGE is a well-known receptor of multiple S100 proteins Extracellular domain of EMMPRIN (soluble form), S100A8, (12), we compared signaling pathways for EMMPRIN and and S100A9 were expressed in HEK293 cells and immunopre- RAGE. First, we identified adaptor molecules for signal trans- cipitated with appropriate antibodies. We found that S100A9 duction. A motif search for EMMPRIN (http://elm.eu.org/) but not S100A8 was coimmunoprecipitated with EMMPRIN, indicated a TRAF-binding site in its cytoplasmic domain suggesting that EMMPRIN binds to S100A9 (Fig. 1A). In addi- (Supplementary Fig. S2). Furthermore, our previous work tion, when S100A8 and S100A9 were coexpressed and pulled (13) suggested that RAGE required MyD88 and TIRAP for the down with EMMPRIN, both proteins were detected in the signal transduction. Thus, we include MyD88, TIRAP, TRAF2, precipitated fraction. Thus, it is likely that the S100A8/A9 and TRAF6 as candidate molecules. Each cytoplasmic domain heterodimer (calprotectin) is also capable to bind with EMM- of EMMPRIN or RAGE tagged with hemagglutinin (HA) was PRIN. Binding of S100A9 was further confirmed with expressed in HEK293 cells. Coexpression analysis indicated EMMPRIN-immobilized ELISA assays. S100A9 bound with that cytoplasmic domain of EMMPRIN only bound with immobilized soluble form of EMMPRIN in a dose-dependent TRAF2, whereas that of RAGE trapped MyD88, TIRAP, and manner (Fig. 1B). We next examined EMMPRIN expression in 5 TRAF6, suggesting different signal transduction pathways melanoma cell lines and primary human keratinocytes. The between EMMPRIN and RAGE (Fig. 2A). Mutation of TRAF- highest expression was seen in SK-MEL-2, which was 3.3 times binding motif in EMMPRIN (Supplementary Fig. S2) abolished higher than in SK-MEL-5 (Fig. 1C). The gene expression profile the binding ability with TRAF2 (Fig. 2B). We confirmed that of SK-MEL-2 cells was investigated after stimulation with inherent TRAF2 was recruited by the stimulation with exter- S100A8, S100A9, or S100A8/A9 (Table 2). We used 10 mg/mL nally added S100A9 (Fig. 2C). We next examined whether of each S100 protein, as this concentration showed the highest EMMPRIN and RAGE were able to form heterodimer each stimulation of cytokine induction in keratinocytes (Supple- other. Figure 2D clearly showed that they were unable to make mentary Fig. S1A). Only 16 genes were upregulated more than up heterodimers but present as homodimers. Overexpression 2-fold at 3 hours in S100-stimulated SK-MEL-2 cells, including of dominant-negative TRAF2 in SK-MEL-2 cells showed sig- CXCL1, CXCL2, CXCL3, IL8, TNF-a, BIRC3 (cIAP2), and EFNA1 nificant reduction of S100A9-induced cdc42 activation (Fig. (ephrinA1). Components of NF-kB as well as associated factors 2E), further supporting the involvement of TRAF2 in S100A9– were also induced by these S100 proteins. Interestingly, most EMMPRIN signaling pathway. of these factors are related to inflammation and cell survival. Our previous study showed that similar genes were also Immunohistochemical analysis shows that EMMPRIN upregulated in keratinocytes stimulated with S100A8/A9 (4), and S100A9 are differentially expressed indicating common and essential features of S100A9 function. We next examined the localization of S100A9 and EMM- As we already knew that MMP10, MMP1, MMP2, and MMP9 PRIN. These proteins were hardly detectable in normal human were induced at 24 hours after S100A8/A9 stimulation in skin (Fig. 3A) or in benign pigmented nevi (Fig. 3B). In contrast, keratinocytes (see Supplementary Table S1), we tested expres- heavy staining of S100A9 was always observed in the epidermis sion of these MMPs in SK-MEL-2, a high EMMPRIN expresser adjacent to HMB-45–positive melanoma-burdened skin (Fig. MMP1 was always upregulated after stimulation with S100A9, 3C; Supplementary Fig. S3A). Interestingly, EMMPRIN was S100A8, or S100A8/A9 (Fig. 1D and E), although induction of detected at the invasive edge of melanoma lesions (Fig. 3C MMP9 and MMP2 was inconsistent in SK-MEL-2 (data not and D) but was absent in the mass of melanoma tissue shown). (Supplementary Fig. S3B). In the epidermis, EMMPRIN was To elucidate the possible involvement of EMMPRIN in these also present in the upper layer of melanoma tissue (Fig. 3c and changes, we used 2 approaches, siRNA-mediated knockdown D). Proximity ligation assay (PLA) using anti-S100A9 antibody of EMMPRIN (Supplementary Fig. S1B) and treatment with a and anti-EMMPRIN antibody indicated a positive reaction at soluble form of EMMPRIN. Under conditions of EMMPRIN the invasive edge of melanoma cells as well as the upper mRNA knockdown, induction of cytokines as well as MMPs epidermal tissue exposed to the melanoma (Supplementary was significantly suppressed compared with the control (Fig. Fig. S4). These results suggest colocalization of these molecules 1D). Induction by S100A8 and S100A8/A9 was also suppressed. and possible interaction in vivo. For melanoma cell metastasis, As S100A8 and S100A9 are thought to make a stable hetero- it is necessary to degrade the basement membrane at the dimer in vivo (25, 26), these results may, at least in part, be dermal–epidermal junction. MMP2 and MMP9 are thought to dependent on binding with EMMPRIN via S100A9. Further- play an important role in the degradation of basement mem- more, addition of soluble EMMPRIN to the medium down- brane components (27). Loss of the basement membrane regulated the induction of cytokines and MMPs in SK-MEL-2 structure was obvious at the invasive edge of melanoma as (Fig. 1E; Supplementary Fig. S1C). Collectively these results verified by the absence of type VII collagen (28; Fig. 3E and F). suggest that S100A9 is a novel ligand of EMMPRIN and that Immunohistochemical data indicate that melanoma cells and

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 175

Hibino et al.

AB Transfection

0.35

0.3 0.25 0.2 None None S100A9 S100A8/A9 S100A8 0.15 0.1 Cell lysate S100A9 WB: HA 0.05 S100A8 0

Absorbance at 630 nm at 630 Absorbance 0 0.63 1.25 2.5 5 10

Tubulin WB: Tubulin Amount of S100A9 (μg/mL) C Culture S100A9 WB: HA medium S100A8 2.5 2 Bound IP: myc 1.5 proteins EMMPRIN WB: myc 1 0.5 S100A9 IP: myc 0

S100A8 WB: HA EMMPRIN expression

D E TNF-α 4 TNF-α * 5 ** ** ** *** * 4 3 3 2 2 1

1 Fold increase Fold increase 0 0 5 IL-8 IL-8 * ** * * ** 5 * 4 4 3 3 2 2 1

Fold increase 1 Fold increase 0 0

2 MMP1 MMP1 * ** * * 12 ** 1.5 10 ** 8 1 6 0.5 4 Fold increase 2 Fold increase 0 0 A9 A9 Control A9 A9 Control (1 nmol/L) (10 nmol/L) (1 nmol/L) (10 nmol/L) +sol.EMMPRIN

Figure 1. S100A9 specifically binds with EMMPRIN. A, immunoprecipitation study showing binding of S100A9 with EMMPRIN in HEK293 cells. S100A8 and S100A9 were expressed with HA-tag. Soluble form of EMMPRIN was expressed with both HA-tag and Myc-tag. Pull-down with anti-HA antibody showed sufficient expression of these proteins (lanes 1–3). When S100A8 or S100A9 was expressed together with EMMPRIN and pulled down with anti-Myc antibody, only S100A9 was seen to bind with EMMPRIN as verified with anti-HA antibody. Coexpression of S100A8 and S100A9 resulted in the detection of both proteins. B, ELISA assay showing S100A9 binding with extracellular domain of EMMPRIN in a dose-dependent manner. C, EMMPRIN mRNA expression in 5 melanoma cell lines and normal human keratinocytes. D, effect of EMMPRIN knockdown on the expression of S100-induced TNF-a, IL-8, and MMP1. S100 proteins were added to the SK-MEL-2 culture, and total RNA samples were obtained 3 hours after incubation. E, effect of soluble EMMPRIN (sol. EMMPRIN) on the expression of S100-induced TNF-a, IL-8, and MMP1 in SK-MEL-2. , P < 0.05; , P < 0.01; , P < 0.001.

176 Cancer Res; 73(1) January 1, 2013 Cancer Research

Identiﬁcation of a Novel Receptor of S100A9

Table 2. Summary of microarray analysis for S100-stimulated SK-MEL-2 at 3 hours

Factor of increase

Gene symbol þS100A8 þS100A9 þS100A8/A9 CXCL3 7.30 0.81 14.77 0.56 12.57 0.78 IL-8 6.20 0.68 14.36 2.12 10.69 0.47 CCL2 5.35 0.85 13.62 0.62 10.03 0.35 BIRC3 5.14 0.69 12.74 0.97 10.41 0.15 ZC3H12A 4.08 0.43 7.81 0.37 6.82 0.22 CXCL2 4.03 0.49 7.37 0.52 6.55 0.15 TNF 3.12 0.26 5.92 0.29 4.86 0.39 NFKBIA 2.83 0.38 4.94 0.24 4.88 0.24 C10orf10 2.26 1.0 4.58 0.21 4.10 0.21 CXCL1 3.36 0.17 4.28 0.18 4.21 0.07 RELB 2.18 0.37 4.07 0.15 3.78 0.12 EFNA1 1.91 0.24 4.04 0.29 3.38 0.16 TIFA 1.84 0.42 3.74 0.25 3.17 0.07 CEBPD 2.07 0.24 2.79 0.07 2.68 0.04 IER3 1.94 0.32 2.78 0.18 2.69 0.13 BCL3 1.96 0.24 2.74 0.05 2.59 0.09

NOTE: Expressed as mean values SD from n ¼ 3 microarray analysis. keratinocytes respond to each other strongly at the invasive of RAGE in human melanoma cell lines. Quantitative PCR edge, where S100A9–EMMPRIN interaction takes place. (qPCR) analysis showed it was 1.5 times higher in SK-MEL-2 than in SK-MEL-5 (Fig. 4G). Collectively, these data suggest Chemotactic activity of S100A9 is dependent on that S100A9-induced cell migration is strongly dependent on EMMPRIN expression EMMPRIN. Some S100 family members are known to function as Suppression of migration activity caused by knockdown of chemoattractants to neutrophils (29) and cancer cells (30). EMMPRIN or blocking of signal transduction may be due to We analyzed the chemotactic activity of S100A8 and S100A9 on cell aggregation (Supplementary Fig. S5A). dnEMMPRIN melanoma cell lines. S100A9 showed a strong effect on SK- B16showedaverycompactcellshapeandformeddense MEL-2 migration (Fig. 4A). S100A8 and S100A8/A9 also showed cell aggregates, although control B16 or vector-transfected chemotactic activity on SK-MEL-2 to some extent. Knockdown cells grew with a rather uniform spread and an elongated cell of EMMPRIN markedly suppressed the chemotactic activity of shape. S100A9, as well as S100A8 and S100A8/A9 on SK-MEL-2. Quantitative analysis further supported the above results (Fig. Overexpression of S100A9 induced melanoma cell 4B). Either S100A9 or S100A8 had little effect on the migration migration in vivo of SK-MEL-3 and SK-MEL-5, low EMMPRIN expressers (Fig. Finally, we tested whether S100A8 or S100A9 contributed to 4C). These results indicate that chemotactic activity of S100A9 the metastatic ability of cancer cells. For this purpose, we is closely related to the expression level of EMMPRIN (Fig. 1C). generated involucrin promoter–driven S100A8 and S100A9 Tg In addition, we established a clone of B16-BL6 melanoma mice (Fig. 5A). We chose the involucrin promoter, as these cells that constitutively expresses the dominant-negative (dn) molecules were expressed in the upper epidermis of inflam- form of EMMPRIN (dnEMMPRIN B16) with 1,400 times higher matory skin. We used SK-MEL-2 and SK-MEL-5 for the metas- than intact EMMPRIN. This clone lacks the cytoplasmic tasis assay as high and low EMMPRIN expressers, respectively. domain of EMMPRIN and thus is unable to transduce SK-MEL-2 or SK-MEL-5 cells were injected into the tail vein of S100A9 signals. S100A9 showed significant chemotactic activ- wild-type and Tg mice, and their presence was examined in the ity on control B16 melanoma cells (Fig. 4D). On the other hand, skin after 4 weeks. Skin specimens were scanned with a S100A9 did not show any effect on the migrating activity of confocal microscope in an area 1 2cm2 or immunostained dnEMMPRIN B16. Quantitative analysis clearly showed strong with HMB-45 and MART-1 for the detection of melanoma cells. effect of S100A9 on B16 cell migration (Fig. 4E). To assess the In SK-MEL-2–injected S100A8 Tg mice, we found very few involvement of RAGE in melanoma metastasis, we examined HMB-45–positive cells (n ¼ 6; Fig. 5B). However, considerable expression of RAGE in these cell lines. Expression of RAGE was numbers of positive cells were always detected in the skin of 40-fold higher in dnEMMPRIN B16 than in B16 control, SK-MEL-2–injected S100A9 Tg mice (n ¼ 6; Fig. 5C). Presence although that of mouse EMMPRIN is rather suppressed in of melanin in the skin of both types of mice was confirmed with dnEMMPRIN B16 (Fig. 4F). We also checked expression levels Fontana–Masson staining (Fig. 5D). Immunohistochemical

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 177

Hibino et al.

RAGE-cyt A EMMPRIN-cyt Figure 2. Signal transduction pathways are distinct between EMMPRIN and RAGE. A, identiﬁcation of adaptor molecules Control MyD88 TIRAP TRAF2 TRAF2 TRAF6 Control MyD88 TIRAP TRAF2 TRAF2 TRAF6 TRAF6 for signal transduction. HA-tagged TRAF6 TRAF2 cytoplasmic domain of EMMPRIN TRAF2 Cell lysate (EMMPRIN-cyt) or RAGE (RAGE- MyD88 MyD88 cyt: S391E, phospho-mimic form) TIRAP TIRAP as well as each adaptor molecule with Myc-tag were coexpressed in EMMPRIN-cyt RAGE-cyt HEK293 cells. Interacting TRAF6 TRAF6 molecules in cell lysate were TRAF2 TRAF2 immunoprecipitated with anti-HA IP: anti-HA antibody and analyzed with MyD88 MyD88 Western blot using anti-Myc TIRAP TIRAP antibody. B, TRAF2 binds with cytoplasmic domain of EMMPRIN. Mutation was introduced into the +HA-EMMPRIN cytoplasmic domain of EMMPRIN B D in the TRAF-binding motif (Mut

Traf) and the C-terminal end (Mut

ter. ter.

- C-ter). They were coexpressed Transfection with EMMPRIN-cyt in HEK293 Wt Mut Traf Mut C Control Control

+EMMPRIN (myc) cells and binding with inherent +RAGE (myc) TRAF2 was analyzed with Western Transfection Cell lysate TRAF2 RAGE blotting. C, recruitment of TRAF2 to the cytoplasmic domain of EMMPRIN EMMPRIN. Full-length EMMPRIN EMMPRIN was expressed in HEK293 cells, (cyt) EMMPRIN and interaction with inherent IP: anti-HA TRAF2 was investigated in the TRAF2 IP: anti-HA RAGE presence of 1 or 10 nmol/L GST and S100A9. D, EMMPRIN forms homodimer. Dimer formation EMMPRIN between EMMPRIN and RAGE was analyzed after coexpression of HA-tagged and Myc-tagged +S100A9 EMMPRIN and RAGE. C EMMPRIN (transfected) E Immunoprecipitation was carried out using anti-HA antibody and GST S100 A9 Transfection Stimulation veriﬁed with anti-Myc antibody. E, 1 10 1 10 nmol/L TRAF2 but not TRAF6 mediates None None Control dnTRAF2 dnTRAF6 dnTRAF6 Cell lysate dnTRAF2 signal transduction in S100A9/ TRAF2 dnTRAF6 EMMPRIN system. Dominant- negative form of TRAF2 (dn Tubulin TRAF2) or TRAF6 (dn TRAF6) was EMMPRIN Cell lysate overexpressed in SK-MEL-2 cells, IP: anti-HA cdc42 (Active) and effect of cdc42 activation was TRAF2 monitored after S100A9 cdc42 (Total) stimulation.

analyses showed that these cells were strongly positive When these cells were injected via tail vein, only the control for HMB-45 and MART-1 (Fig. 5E). Many melanoma cells B16 cells metastasized to lung in both wild and S100A9 Tg mice were found scattered around an artery that were veriﬁed with after 6 weeks observation (Supplementary Table S2 and Sup- anti-CD31 and anti-smooth muscle antibodies (Fig. 5E; Sup- plementary Fig. S5B). We could not ﬁnd any incidence for plementary Fig. S6). In some case, melanoma cells were dnEMMPRIN B16 cell metastasis in the lung. Interestingly, we embedded within the vessel wall. Relatively large numbers of observed 2 tumor nodules in the skin of S100A9 Tg mouse that melanoma cells were also found near hair follicle–like struc- were tail vein–injected with B16-BL6 cells (Supplementary tures (Fig. 5E). HMB-45–positive cells were not found in the Fig. S5C). skin of SK-MEL-5–injected S100A8 or S100A9 Tg mice (Sup- plementary Fig. S7). To elucidate further involvement of EMMPRIN in cancer cell Discussion metastasis, we carried out limited attempts using highly There have been many reports that both S100 proteins and invasive melanoma cell line, B16-BL6 and dnEMMPRIN B16. EMMPRIN are involved in malignancy, especially in cancer cell

178 Cancer Res; 73(1) January 1, 2013 Cancer Research

Identiﬁcation of a Novel Receptor of S100A9

metastasis (7, 31, 32). To date, the metastatic ability of these shown with PLA at the invasive edge of melanoma and the proteins is considered to be an independent phenomenon. In adjacent epidermis. the present study, we clarified for the first time the ligand– EMMPRIN, also known as CD147, is able to induce produc- receptor relationship between S100A9 and EMMPRIN. Binding tion of several MMPs (33). It is overexpressed in various of S100A9 to EMMPRIN was confirmed with (i) affinity isola- cancerous tissues (18, 34, 35). Although induction of MMPs tion-mass spectrometry analysis, (ii) immunoprecipitation has been regarded as evidence of the homophilic interaction study in cellular level, and (iii) dose-dependent binding to between EMMPRIN molecules, including a soluble form, our immobilized EMMPRIN. The activity of EMMPRIN as a func- results clearly show that the soluble EMMPRIN is able to bind tional receptor for S100A9 was further shown by the fact to S100A9 and prohibits S100A9-mediated MMP induction. that induction of both proinflammatory cytokines and MMPs Thus, MMP induction by EMMPRIN is, at least in part, caused was markedly suppressed in the EMMPRIN-knockdown cells. by the binding of the S100A9 or S100A8/A9 heterodimer In vivo association between S100A9 and EMMPRIN was also to EMMPRIN. In malignant melanoma, both MMP-9 and

S100A9 S100A9 + DAPI S100A9 S100A9+DAPI

EMMPRIN EMMPRIN + DAPI EMMPRIN EMMPRIN+DAPI C E Figure 3. Immunohistochemical Col VII analysis shows that EMMPRIN is expressed in the invasive edge of melanomas. A, normal skin showing no expression of S100A9 and EMMPRIN. B, pigmented nevus. C, HE immunohistochemistry for S100A9 EMMPRIN (red) and HMB-45 (green) in primary melanoma. Nuclear staining with DAPI (blue) is also shown. D, magniﬁed view of invasive region of melanoma from boxed area of C. E, immunostaining for collagen type VII S100A9+EMMPRIN+DAPI DAPI (Col VII) of normal human skin. F, loss of basement membrane in EMMPRIN-expressing melanoma lesion. An example of melanoma tissue stained with collagen type VII (Col VII, red) and EMMPRIN (green). F S100A9+HMB-45+DAPI Dotted line shows the edge of Col VII corniﬁed layer. Scale bars, 100 mm. D

EMMPRIN

EMMPRIN HMB45

DAPI

Merge Merge

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 179

Hibino et al.

A SK-MEL-2+ B SK-MEL-2 siEMMPRIN SK-MEL-2 SK-MEL-2 + siEMMPRIN

5 * 4 Medium 4 * 3 3 2 2 (fold increase)

1 intensity Relative 1 (fold increase) Relative intensity 0 0 Ctrl S100A8 S100A9 S100A8/A9 Ctrl S100A8 S100A9 S100A8/A9

+S100A8

C SK-MEL-3 SK-MEL-5 +S100A9 5 5 4 4 3 3 2 2 1 1 (fold increase) (fold increase) Relative intensity intensity Relative Relative intensity intensity Relative 0 0 +S100A8/A9 Ctrl S100A8 S100A9 S100A8/A9 Ctrl S100A8 S100A9 S100A8/A9

D E B16 control dnEMMPRIN-B16 B16 Control dnEMMPRIN-B16 3 3 2.5 * 2.5 2 2 Medium 1.5 1.5 1 1 (fold increase) (fold increase)

Relative intensity 0.5 0.5 Relative intensity 0 0 Ctrl S100A8 S100A9 S100A8/A9 Ctrl S100A8 S100A9 S100A8/A9

+S100A8 F G 80 1.5 70 mRAGE hRAGE 60 +S100A9 50 1 40 30 0.5 Fold increase 20 10 hRAGE/G3PDH 0 0 +S100A8/A9 B16 dnEMMPRIN B16

Figure 4. Chemotactic activity of S100A9 is dependent on EMMPRIN expression. A, SK-MEL-2 chemotaxis assay. Entire views of lower chemotaxis ﬁlter were shown for S100 proteins stimulated SK-MEL-2 and siRNA-treated SK-MEL-2. Scale bars, 1 mm. B, quantitative analyses for A. C, results for SK-MEL-3 and SK-MEL-5, low EMMPRIN expressers, were quantiﬁed (n ¼ 4). D, B16 chemotaxis assay. Similar experiments were carried out for B16 and dnEMMPRIN- B16. Scale bars, 1 mm. E, quantitative analysis of chemotaxis assays are shown (n ¼ 4). F, qPCR analysis of mouse RAGE expression in B16-BL6 and dnEMMPRIN B16. G, qPCR analysis of human RAGE expression in SK-MEL-2, SK-MEL-5, and keratinocytes.

EMMPRIN were variably expressed in the invasive radial MMP-9 and EMMPRIN (36). Our observation also supports growth phase but not in the vertical growth phase, and early these ﬁndings and suggests that peripheral keratinocyte– invasion of melanoma is associated with de novo expression of derived S100A9 promotes MMP production in neighboring

180 Cancer Res; 73(1) January 1, 2013 Cancer Research

Identiﬁcation of a Novel Receptor of S100A9

S100A8 S100A9 B Ctrl SK-MEL-2 SK-MEL-5 HMB45 Bright field HMB45 Bright field HMB45 Bright field Figure 5. Overexpression of 460 µm S100A9-induced melanoma cell migration in vivo. A, expression of S100A8 or S100A9 in Tg mouse skin. Involucrin promoter–driven S100A8 460 µm 460 µm (left) and S100A9 (right) Tg mouse 460 µm skin were immunostained with anti- human S100A8 antibody or anti- SK-MEL-2/S100A8 Tg human S100A9 antibody, Merge Merge Merge respectively. Scale bars, 100 mm. B, confocal analysis of the skin with C Ctrl SK-MEL-2 SK-MEL-5 epidermis-specific S100A8 Tg mice. HMB45 Bright field HMB45 Bright field HMB45 Bright field SK-MEL-2 or SK-MEL-5 cells were 460 µm injected into the tail vein, and 4 weeks later, skin was scanned for 460 460 mm2 at a time after HMB- 45 staining. Images of bright and merged fields from Control (Ctrl), SK- 460 µm 460 µm 460 µm MEL-2–injected, and SK-MEL-5– injected mouse tissue are shown. C, confocal analysis of the skin of Merge Merge Merge SK-MEL-2/S100A9 Tg epidermis-specific S100A9 Tg mice. D, Mason–Fontana staining of S100A9 Tg mouse skin injected with D E H&E H&E H&E SK-MEL-2 cells. Magnified view was also shown. E, SK-MEL-2–injected S100A9 skin stained for hematoxylin and eosin (H&E), HMB-45, and MART-1. Enlarged figures of the boxed areas were also shown. Scale HMB45 HMB45 HMB45 bars, 100 mm.

MART1 MART1 MART1

cancer cells via EMMPRIN signal transduction. Apparently, S100A9, thereby making it easier for tumor cells to home in on MMP production is required for efficient cancer cell metastasis the premetastatic sites. Our metastasis assays using S100A8 by degrading basement membrane and surrounding matrix and S100A9 Tg mice revealed that only the high EMMPRIN- proteins. We showed the disappearance of the basement expressing SK-MEL-2 cells migrated into the S100A9-expres- membrane just in the area where these proteins are sing skin. Low EMMPRIN expresser (SK-MEL-5), EMMPRIN- coexpressed. knocked down SK-MEL-2, and dnEMMPRIN B16 all failed to Some cancer cells, including melanoma cells, seem to influ- metastasize or to migrate in response to S100A9. Furthermore, ence distant tissues to establish a premetastatic niche before S100A9–EMMPRIN interaction induced cdc42 activation (Fig. metastasis occurs (8). Primary tumor cells secrete pro-inflam- 2E). cdc42 is known to promote filopodia formation, cell matory factors such as VEGF-A, TGF-b, and TNF-a, which polarity, and migration (37). This may be the reason why induce selective expression of chemoattractants S100A8 and EMMPRIN is expressed at the invasive edge of melanomas

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 181

Hibino et al.

and not in the denser mass of melanoma cells. Indeed, when we anti-inflammatory factor (45), although it is a Janus-faced used the established B16 clone that overexpressed dnEMM- molecule functioning as a potent amplifier of inflammation PRIN failed to migrate after S100A9 stimulation. Our limited in autoimmunity as well as in cancer development and tumor attempts to assess the metastatic ability in vivo also showed spread (46). EMMPRIN is also involved in fetal development, that only the EMMPRIN-expressing B16 cells, but not dnEMM- retinal function, nervous system development, and plaque PRIN B16, metastasized to lung in mice. These lines of evidence formation in Alzheimer disease (33). Thus, although knock- further suggest the critical role of EMMPRIN in melanoma cell down of these molecules might be an effective therapeutic metastasis. option, there might be limitations to directly targeting S100A9 It is thought that S100 proteins actively participate in the or EMMPRIN. Interfering with the S100A9–EMMPRIN inter- modulation of inflammation and immune responses by sig- action would be a novel strategy to block metastatic activity of naling via RAGE (32, 38, 39). On the other hand, EMMPRIN is malignant melanomas. also known as a signaling receptor for cyclophilines A and B – (40). Recent studies have shown a role for cyclophilin EMM- Disclosure of Potential Conflicts of Interest PRIN interactions in the regulation of inflammatory responses No potential conflicts of interest were disclosed. (41). Although RAGE and EMMPRIN seem to have multiple ligands, S100A9–EMMPRIN interaction is of particular rele- Authors' Contributions vance to cancer cell biology. Conception and design: T. Hibino, N.-H. Huh In the present study, we clearly showed that EMMPRIN and Development of methodology: T. Ito RAGE have distinct properties in S100A9-induced signal trans- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Hibino, M. Sakaguchi, S. Miyamoto, M. Yamamoto, duction. EMMPRIN recruited TRAF2 as an adaptor molecule A. Motoyama, J. Hosoi, R. Tsuboi in its signaling pathway, whereas RAGE required MyD88, Analysis and interpretation of data (e.g., statistical analysis, biostatistics, TIRAP, and TRAF6 (Fig. 2). The fact that EMMPRIN failed to computational analysis): M. Sakaguchi, S. Miyamoto, A. Motoyama, R. Tsuboi, N.-H. Huh form a heterodimer with RAGE further supports the difference Writing, review, and/or revision of the manuscript: T. Hibino, M. Sakaguchi, in signal transduction and physiologic functions in these R. Tsuboi, N.-H. Huh Administrative, technical, or material support (i.e., reporting or orga- molecules. nizing data, constructing databases): T. Hibino, M. Sakaguchi, T. Shimokata, Because of the involvement of EMMPRIN in the invasion and T. Ito, N.-H. Huh metastasis processes of many types of cancers, its downregula- Study supervision: T. Hibino, R. Tsuboi, N.-H. Huh tion would seem to be a logical method of preventing cancer cell invasion (42, 43). Indeed this idea is supported by reports Acknowledgments that transfection of EMMPRIN cDNA into breast cancer cells The authors thank Mr. Tatsuo Maeda (Department of Dermatology, Tokyo resulted in increased incidence of metastasis (44) and that Medical University) for his excellent support in metastasis assays and preparation of tissue samples. EMMPRIN-targeting siRNA inhibited the metastatic activity The costs of publication of this article were defrayed in part by the payment of of malignant melanoma in a nude mouse model of pulmo- page charges. This article must therefore be hereby marked advertisement in nary metastasis (35). However, S100A9 and EMMPRIN are both accordance with 18 U.S.C. Section 1734 solely to indicate this fact. pleiotropic molecules that play a critical role in various phys- Received November 29, 2011; revised August 30, 2012; accepted September 20, iologic reactions. S100A9 also works as an anti-infective and 2012; published OnlineFirst November 7, 2012.

References 1. Sgambato A, Cittadini A. Inflammation and cancer: a multifaceted link. cade establishes a pre-metastatic phase. Nat Cell Biol 2008;10: Eur Rev Med Pharmacol Sci 2010;14:263–8. 1349–55. 2. Wang D, Dubois RN, Richmond A. The role of chemokines in intestinal 9. Leclerc E, Fritz G, Vetter SW, Heizmann CW. Binding of S100 proteins inflammation and cancer. Curr Opin Pharmacol 2009;9:688–96. to RAGE: an update. Biochim Biophys Acta 2009;1793:993–1007. 3. Porta C, Larghi P, Rimoldi M, Totaro MG, Allavena P, Mantovani A, et al. 10. Bierhaus A, Stern DM, Nawroth PP. RAGE in inflammation: a new Cellular and molecular pathways linking inflammation and cancer. therapeutic target? Curr Opin Investig Drugs 2006;7:985–91. Immunobiology 2009;214:761–77. 11. Clynes R, Moser B, Yan SF, Ramasamy R, Herold K, Schmidt AM. 4. Nukui T, Ehama R, Sakaguchi M, Sonegawa H, Katagiri C, Hibino T, Receptor for AGE (RAGE): weaving tangled webs within the inflam- et al. S100A8/A9, a key mediator for positive feedback growth stim- matory response. Curr Mol Med 2007;7:743–51. ulation of normal human keratinocytes. J Cell Biochem 2008;104: 12. Donato R. RAGE: a single receptor for several ligands and different 453–64. cellular responses: the case of certain S100 proteins. Curr Mol Med 5. Yao R, Lopez-Beltran A, Maclennan GT, Montironi R, Eble JN, Cheng L. 2007;7:711–24. Expression of S100 protein family members in the pathogenesis of 13. Sakaguchi M, Murata H, Yamamoto K, Ono T, Sakaguchi Y, Motoyama bladder tumors. Anticancer Res 2007;27:3051–8. A, et al. TIRAP, an adaptor protein for TLR2/4, transduces a signal from 6. Saleem M, Kweon MH, Johnson JJ, Adhami VM, Elcheva I, Khan N, RAGE phosphorylated upon ligand binding. PLoS One 2011;6:e23132. et al. S100A4 accelerates tumorigenesis and invasion of human 14. Cecil DL, Appleton CT, Polewski MD, Mort JS, Schmidt AM, Bendele A, prostate cancer through the transcriptional regulation of matrix metal- et al. The pattern recognition receptor CD36 is a chondrocyte hyper- loproteinase 9. Proc Natl Acad Sci U S A 2006;103:14825–30. trophy marker associated with suppression of catabolic responses 7. Harpio R, Einarsson R. S100 proteins as cancer biomarkers with focus and promotion of repair responses to inflammatory stimuli. J Immunol on S100B in malignant melanoma. Clin Biochem 2004;37:512–8. 2009;182:5024–31. 8. Hiratsuka S, Watanabe A, Sakurai Y, Akashi-Takamura S, Ishibashi S, 15. Robinson MJ, Tessier P, Poulsom R, Hogg N. The S100 family Miyake K, et al. The S100A8-serum amyloid A3-TLR4 paracrine cas- heterodimer, MRP-8/14, binds with high affinity to heparin and heparan

182 Cancer Res; 73(1) January 1, 2013 Cancer Research

Identiﬁcation of a Novel Receptor of S100A9

sulfate glycosaminoglycans on endothelial cells. J Biol Chem 2002; 31. Gebhardt C, Nemeth J, Angel P, Hess J. S100A8 and S100A9 in 277:3658–65. inflammation and cancer. Biochem Pharmacol 2006;72:1622–31. 16. Srikrishna G, Panneerselvam K, Westphal V, Abraham V, Varki A, 32. Ghavami S, Rashedi I, Dattilo BM, Eshraghi M, Chazin WJ, Hashemi M, Freeze HH. Two proteins modulating transendothelial migration of et al. S100A8/A9 at low concentration promotes tumor cell growth via leukocytes recognize novel carboxylated glycans on endothelial cells. RAGE ligation and MAP kinase-dependent pathway. J Leukoc Biol J Immunol 2001;166:4678–88. 2008;83:1484–92. 17. Gabison EE, Hoang-Xuan T, Mauviel A, Menashi S. EMMPRIN/CD147, 33. Iacono KT, Brown AL, Greene MI, Saouaf SJ. CD147 immunoglobulin an MMP modulator in cancer, development and tissue repair. Biochi- superfamily receptor function and role in pathology. Exp Mol Pathol mie 2005;87:361–8. 2007;83:283–95. 18. Nabeshima K, Iwasaki H, Koga K, Hojo H, Suzumiya J, Kikuchi M. 34. Kanekura T, Chen X. CD147/basigin promotes progression of malig- Emmprin (basigin/CD147): matrix metalloproteinase modulator and nant melanoma and other cancers. J Dermatol Sci 2010;57:149–54. multifunctional cell recognition molecule that plays a critical role in 35. Chen X, Lin J, Kanekura T, Su J, Lin W, Xie H, et al. A small interfering cancer progression. Pathol Int 2006;56:359–67. CD147-targeting RNA inhibited the proliferation, invasiveness, and 19. Motoyama A, Venable JD, Ruse CI, Yates JR 3rd. Automated ultra- metastatic activity of malignant melanoma. Cancer Res 2006;66: high-pressure multidimensional protein identification technology 11323–30. (UHP-MudPIT) for improved peptide identification of proteomic 36. van den Oord JJ, Paemen L, Opdenakker G, de Wolf-Peeters C. samples. Anal Chem 2006;78:5109–18. Expression of gelatinase B and the extracellular matrix metalloprotei- 20. Motoyama A, Xu T, Ruse CI, Wohlschlegel JA, Yates JR 3rd. Anion and nase inducer EMMPRIN in benign and malignant pigment cell lesions cation mixed-bed ion exchange for enhanced multidimensional separa- of the skin. Am J Pathol 1997;151:665–70. tions of peptides and phosphopeptides. Anal Chem 2007;79:3623–34. 37. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature 21. Carroll JM, Albers KM, Garlick JA, Harrington R, Taichman LB. Tissue- 2002;420:629–35. and stratum-specific expression of the human involucrin promoter in 38. Leclerc E, Fritz G, Weibel M, Heizmann CW, Galichet A. S100B and transgenic mice. Proc Natl Acad Sci U S A 1993;90:10270–4. S100A6 differentially modulate cell survival by interacting with distinct 22. Broome AM, Ryan D, Eckert RL. S100 protein subcellular localization RAGE (receptor for advanced glycation end products) immunoglobulin during epidermal differentiation and psoriasis. J Histochem Cytochem domains. J Biol Chem 2007;282:31317–31. 2003;51:675–85. 39. Wolf R, Howard OM, Dong HF, Voscopoulos C, Boeshans K, Winston 23. Piao L,LiY,KimSJ, ByunHS,Huang SM,HwangSK, etal.Associationof J, et al. Chemotactic activity of S100A7 (Psoriasin) is mediated by the LETM1 and MRPL36 contributes to the regulation of mitochondrial ATP receptor for advanced glycation end products and potentiates inflam- production and necrotic cell death. Cancer Res 2009;69:3397–404. mation with highly homologous but functionally distinct S100A15. 24. Toole BP. Emmprin (CD147), a cell surface regulator of matrix metal- J Immunol 2008;181:1499–506. loproteinase production and function. Curr Top Dev Biol 2003;54: 40. Yurchenko V, Constant S, Bukrinsky M. Dealing with the family: CD147 371–89. interactions with cyclophilins. Immunology 2006;117:301–9. 25. Eue I, Konig S, Pior J, Sorg C. S100A8, S100A9 and the S100A8/A9 41. Yurchenko V, Constant S, Eisenmesser E, Bukrinsky M. Cyclophilin- heterodimer complex specifically bind to human endothelial cells: CD147 interactions: a new target for anti-inflammatory therapeutics. identification and characterization of ligands for the myeloid-related Clin Exp Immunol 2010;160:305–17. proteins S100A9 and S100A8/A9 on human dermal microvascular 42. Liang Q, Xiong H, Gao G, Xiong K, Wang X, Zhao Z, et al. Inhibition of endothelial cell line-1 cells. Int Immunol 2002;14:287–97. basigin expression in glioblastoma cell line via antisense RNA reduces 26. Leukert N, Sorg C, Roth J. Molecular basis of the complex formation tumor cell invasion and angiogenesis. Cancer Biol Ther 2005;4: between the two calcium-binding proteins S100A8 (MRP8) and 759–62. S100A9 (MRP14). Biol Chem 2005;386:429–34. 43. Wang L, Wu G, Yu L, Yuan J, Fang F, Zhai Z, et al. Inhibition of CD147 27. Amano S, Akutsu N, Matsunaga Y, Nishiyama T, Champliaud MF, expression reduces tumor cell invasion in human prostate cancer cell Burgeson RE, et al. Importance of balance between extracellular matrix line via RNA interference. Cancer Biol Ther 2006;5:608–14. synthesis and degradation in basement membrane formation. Exp Cell 44. Zucker S, Hymowitz M, Rollo EE, Mann R, Conner CE, Cao J, et al. Res 2001;271:249–62. Tumorigenic potential of extracellular matrix metalloproteinase 28. Seltzer JL, Eisen AZ, Bauer EA, Morris NP, Glanville RW, Burgeson RE. inducer. Am J Pathol 2001;158:1921–8. Cleavage of type VII collagen by interstitial collagenase and type IV 45. Hsu K, Champaiboon C, Guenther BD, Sorenson BS, Khammani- collagenase (gelatinase) derived from human skin. J Biol Chem 1989; vong A, Ross KF, et al. Anti-InfectiveProtectivePropertiesofS100 264:3822–6. Calgranulins. Antiinflamm Antiallergy Agents Med Chem 2009;8: 29. Roth J, Vogl T, Sunderkotter C, Sorg C. Chemotactic activity of 290–305. S100A8 and S100A9. J Immunol 2003;171:5651. 46. Ehrchen JM, Sunderkotter C, Foell D, Vogl T, Roth J. The endogenous 30. Li ZH, Bresnick AR. The S100A4 metastasis factor regulates cellular Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate motility via a direct interaction with myosin-IIA. Cancer Res 2006;66: amplifier of infection, autoimmunity, and cancer. J Leukoc Biol 2009; 5173–80. 86:557–66.

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 183

S100A9 Is a Novel Ligand of EMMPRIN That Promotes Melanoma Metastasis

Toshihiko Hibino, Masakiyo Sakaguchi, Shoko Miyamoto, et al.

Cancer Res 2013;73:172-183. Published OnlineFirst November 7, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-11-3843

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2012/11/05/0008-5472.CAN-11-3843.DC1

Cited articles This article cites 46 articles, 13 of which you can access for free at: http://cancerres.aacrjournals.org/content/73/1/172.full#ref-list-1

Citing articles This article has been cited by 8 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/73/1/172.full#related-urls

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

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/73/1/172. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.