Biochemical and Biophysical Research Communications 406 (2011) 423429

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Biochemical and Biophysical Research Communications

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Gangliosides expressed on breast cancer cells are E-selectin ligands

Venktesh S. Shirure a, Karissa A. Henson b, Ronald L. Schnaar c, Leonardo Nimrichter d, Monica M. Burdick a,b,
a

Department of Chemical and Biomolecular Engineering, Ohio University, Athens, OH 45701, United States Biomedical Engineering Program, Ohio University, Athens, OH 45701, United States c Department of Pharmacology and Molecular Sciences and Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States d Instituto de Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
b

a r t i c l e

i n f o

a b s t r a c t
Cancer cell adhesion to vascular endothelium is a critical process in hematogenous metastasis. We hypothesized that breast cancer cells express ligands that bind under blood ow conditions to E-selectin expressed by endothelial cells. At a hemodynamic wall shear rate, BT-20 and MDA-MB-468 breast cancer cells adhered to cytokine-activated human umbilical cord vein endothelial cells (HUVECs) but not to antiE-selectin monoclonal antibody treated HUVECs, demonstrating that adhesion was specically mediated by E-selectin. Characterization of glycans expressed on breast cancer cells by a panel of antibodies revealed that BT-20 cells expressed sialyl Lewis X (sLex) and sialyl Lewis A (sLea) but MDA-MB-468 cells did not, suggesting that the former possess classical glycans involved in E-selectin mediated adhesion while the latter have novel binding epitopes. Protease treatment of the breast cancer cells failed to signicantly alter the carbohydrate expression proles, binding to soluble E-selectinIg chimera, or the ability of the cells to tether and roll on E-selectin expressed by HUVECs, indicating that glycosphingolipids are functional E-selectin ligands on these cells. Furthermore, extracted breast cancer cell gangliosides supported binding of E-selectinIg chimera and adhesion of E-selectin transfected cells under physiological ow conditions. In summary, our results demonstrate that breast cancer cells express sialylated glycosphingolipids (gangliosides) as E-selectin ligands that may be targeted for prevention of metastasis. 2011 Elsevier Inc. All rights reserved.

Article history: Received 2 February 2011 Available online 15 February 2011 Keywords: Cell adhesion Sialyl Lewisx Sialyl Lewisa Metastasis Shear rate

1. Introduction Metastasis to distant organs involves complex yet systematic events, in which cancer cells disseminate from a primary tumor and journey through the vasculature to a secondary site, where they attach and extravasate into the tissue to form a new growth. Although a metastatic niche at the secondary site is undeniably important for successful colonization, a circulating tumor cell cannot invade without attaching to the vascular endothelium at the target site. Treatment strategies that prevent cancer cell adhesion to endothelium are thus attractive methods to inhibit metastasis, but rst an understanding of the molecular mediators involved is necessary to develop such interventions.

Abbreviations: CHO-E, E-selectin transfected Chinese hamster ovary cells; EIg chimera, recombinant mouse E-Selectin/human immunoglobulin Fc chimera; FBS, fetal bovine serum; FITC, uorescein isothiocyanate; HUVECs, human umbilical cord vein endothelial cells; mAbs, monoclonal antibodies; PE, phycoerythrin; sLea, sialyl Lewis A; sLex, sialyl Lewis X. Corresponding author. Address: Department of Chemical and Biomolecular Engineering, Russ College of Engineering and Technology, Ohio University, Stocker Center 168, Athens, OH 45701, United States. Fax: +1 740 593 0873. E-mail address: burdick@ohio.edu (M.M. Burdick). 0006-291X/$ - see front matter 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.02.061

It is hypothesized that cancer cells adhere to endothelium by a process similar to that of leukocyte homing. In this model, cells in ow are captured on the endothelial surface (also known as tethering), slowed by transient adhesive interactions with endothelial selectins (rolling), and rmly anchored on endothelium (rm adhesion) to enable entry into the underlying tissue. The selectins, particularly E-selectin, are recognized to mediate adhesion and thus potentiate metastasis of certain cancers [1,2]. E-selectin is expressed in most vascular beds in response to inammatory stimuli and is also constitutively expressed by endothelium of hematopoietic tissues such as bone marrow [3]. Notably, breast cancer frequently spreads to bone, and breast cancer cells have been reported to specically bind endothelial E-selectin under hematogenous shear conditions [4,5]. In complement, breast cancer lesions but not normal breast cells express sialofucosylated modications (e.g., sialyl Lewis X; sLex) in situ [6] that are recognized by E-selectin [1,2], with higher expression correlating with disease progression [6]. Despite persuasive evidence for the involvement of E-selectin and its ligands in breast cancer metastasis, breast cancer cell E-selectin ligands operative under physiological ow conditions have not been identied. In contrast, we have previously isolated and characterized numerous sialylated glycosphingolipid (ganglio-

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side) E-selectin ligands expressed by normal human leukocytes [7,8]. Also, in studies of E-selectin ligands on colon cancer, prostate cancer, and leukemia, gangliosides were identied as signicant mediators of cell adhesion [911]. The contribution of these molecules in breast cancer cell adhesion is currently unknown, which is surprising given their importance in various metastatic processes, including epithelial to mesenchymal transition (EMT) [12]. Therefore, the present study was designed to investigate the potential role of gangliosides as uid shear-resistant E-selectin ligands expressed by invasive breast cancer cell lines.

2.5. Thin layer chromatography (TLC) and immuno-overlay assay Ganglioside extracts from equivalent numbers of breast cancer cells were resolved on glass-backed silica gel TLC plates (Merck, Gibbstown, NJ) using chloroform/methanol/0.25% aqueous KCl (60:35:8 v/v/v) as the developing solvent. The ganglioside bands were visualized after spraying with resorcinol reagent (0.3% (w/v) resorcinol, 0.003% (w/v) cupric sulfate pentahydrate and 30% (v/v) concentrated HCl in water) [16], and subsequently heating the plates for 20 min at 125 C. To evaluate whether gangliosides from breast cancer cells are ligands for E-selectin, an immuno-overlay assay using EIg chimera was performed on gangliosides resolved by TLC. Dry TLC plates were immersed in a mixture of hexanes and then transferred to a solution of 1 mg/ml polyisobutylmethacrylate in hexanes [16]. The plates were dried, immersed in phosphate buffered saline (PBS) for 5 min and then transferred to blocking buffer (0.1% bovine serum albumin (BSA)/0.05% Tween 20 in PBS) for 30 min at room temperature (RT). The plates were overlaid with EIg chimera for 1 h at RT, followed by alkaline phosphatase conjugated anti-human IgG under the same conditions. After extensive washes with PBS and one wash with water, the TLC plates were immersed in nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution (SigmaAldrich, St. Louis, MO). A standard mixture of gangliosides (GM3, GM1, GD1a, GD1b, and GT1b), lacking glycans that support E-selectin binding, was used as a negative control. Brain gangliosides were purchased from Matreya (Pleasant Gap, PA) or puried from bovine brain extract [15], and GM3 was from SigmaAldrich. After development the TLC plates were washed with water and dried. Replicate plates were spotted and developed with sulfuric-resorcinol solution as described above. 2.6. Cell treatments Breast cancer cells were treated with 0.1 U/ml Vibrio cholerae sialidase (Roche Biochemicals, Indianapolis, IN) for 60 min at 37 C to remove terminal sialic acids. To cleave cell-surface proteins, breast cancer cells were treated with a broadly active protease, bromelain (SigmaAldrich), at 1% (w/v) for 60 min at 37 C. After enzyme treatments, cells were washed and resuspended in 0.1% BSA/Dulbeccos phosphate buffered saline (DPBS). 2.7. Shear-dependent cell adhesion assays Cancer cells, with or without enzyme treatment, were perfused over HUVECs at a bone marrow microvasculature wall shear rate of 80 s1 [17] using a parallel plate ow chamber (Glycotech, Rockville, MD). E-selectin expression was induced in HUVECs with 50 U/ml interleukin-1b (IL-1b; Calbiochem, San Diego, CA) at 37 C for 6 h [10,14], and E-selectin contribution was assessed by pretreating activated HUVECs with a function-blocking antiCD62E mAb [14]. To observe adhesion in real-time, the ow chamber was placed on a Nikon TE300 inverted microscope equipped with a video camera. Adhesion events were recorded for 2 min and analyzed ofine. Initial tethering was determined as the number of cancer cells attaching from the laminar ow stream, while cancer cells that remained stationary on HUVECs for more than 5 s were considered rmly adherent. Cell rolling velocity was calculated by determining the distance traveled by a cell in 5 s, using Image J software [10]. To determine whether gangliosides possess E-selectin ligand activity, CHO-E cells, untreated or treated with anti-CD62E mAb, were perfused over mock treated or enzyme treated immobilized ganglioside spots at 80 s1 using the parallel plate ow chamber. Gangliosides were mixed in 1:1 methanol/water containing 25 lM phosphatidylcholine and 25 lM cholesterol [8]. Approximately

2. Materials and methods 2.1. Cell culture The BT-20 breast invasive ductal carcinoma cell line and MDAMB-468 breast adenocarcinoma cell line [13] were obtained from the American Type Culture Collection (ATCC; Manassas, VA). BT-20 and MDA-MB-468 cells were cultured in MEM and DMEM (Invitrogen, Carlsbad, CA), respectively, supplemented with 10% fetal bovine serum (FBS) and 1 penicillinstreptomycin. E-selectin transfected Chinese Hamster Ovary (CHO-E) cells were a generous gift from Dr. Robert Sackstein (Harvard Medical School, Boston, MA). CHO-E cells were cultured in MEM supplemented with 10% FBS, 0.1 mM nonessential amino acids, and 1 penicillinstreptomycin. Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Allendale, NJ) and cultured as described previously [10,14].

2.2. Antibodies and recombinant proteins Anti-human CD43 (1G10), CD44 (515), PSGL-1 (KPL-1), CD62E (68-5H11), CD66 (COL-1), HECA-452 (recognizing several sialofucosylated epitopes correlating with E-selectin ligand activity), sialyl Lewis X (sLex; CSLEX-1) monoclonal antibodies (mAbs), and all isotype controls were obtained from BD Biosciences (San Jose, CA). Anti-sialyl Lewis A (sLea; KM-231) was from Calbiochem (San Diego, CA), and anti-human PCLP (3D3) and E-cadherin (67A4) mAbs were from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant mouse E-Selectin/human immunoglobulin Fc chimera (EIg chimera) and biotin-conjugated cholera toxin subunit B (CTB) were obtained from R&D systems (Minneapolis, MN) and Invitrogen (Carlsbad, CA), respectively. Fluorescein isothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated polyclonal secondary antibodies were purchased from Southern Biotech (Birmingham, AL).

2.3. Flow cytometry The procedure for cell labeling has been described previously [10,14]. A FACSort ow cytometer (BD Biosciences) was used for analysis.

2.4. Extraction of gangliosides The Svennerholm method using 4:8:3 chloroform/methanol/ water with phase partitioning was used to extract breast cancer cell gangliosides as described previously [8,15]. Following phase partitioning, samples were subjected to chromatography using Sep-Pak Plus C18 cartridges (Waters, Milford, MA) to obtain fractions enriched in gangliosides. Protein contamination was not detected when tested by BCA protein assay (Thermo Fisher Scientic, Rockford, IL).

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5 mm diameter (19.6 mm2) ganglioside spots were prepared on a Petri dish using material equivalent to a cellular surface area of 19.6 mm2 (BT-20 and MDA-MB-468 cell diameter are 18 2 and 20 2 lm, respectively). Dried ganglioside spots were then treated with sialidase (0.1 U/ml), bromelain (1% w/v) or buffer (mock treatment, no enzyme) for 30 min at 37 C. Dishes were blocked with 1% BSA at 37 C prior to use in adhesion assays. Observations of cell adhesion were made as described above. 2.8. Statistics Data are expressed as mean SEM of at least three independent experiments except where indicated. Statistical signicance was determined by the Students t-test with p 6 0.05 considered statistically signicant. 3. Results 3.1. BT-20 and MDA-MB-468 breast cancer cells express E-selectin ligands that mediate tethering on activated endothelium BT-20 and MDA-MB-468 cells were tested for E-selectin ligand activity under physiological ow conditions using the parallel plate ow chamber. Both breast cancer cell lines tethered (i.e., attached from the uid stream) on IL-1b-activated HUVECs, and attachment levels were maintained when the cancer cells were perfused over activated HUVECs pretreated with isotype control antibody. However, the cells failed to tether on anti-E-selectin monoclonal antibody (anti-CD62E mAb) treated (Fig. 1A) HUVECs or unactivated HUVECs (data not shown), thus establishing cancer cell binding was mediated specically by endothelial E-selectin. Additionally, a terminal sialylation requirement (characteristic of glycans supporting E-selectin adhesion) for attachment was veried by treating cancer cells with sialidase, which led to large and statistically signicant reductions in tethering of both cell lines (Fig. 1A). Treatment efcacy was conrmed by the complete loss of expression of sialic acid-dependent HECA-452 antigens by ow cytometry (data not shown). Therefore, sialylated glycans are required for optimal E-selectin-mediated tethering of breast cancer cells under ow conditions. 3.2. BT-20 but not MDA-MB-468 cells express sLex and sLea Breast cancer cell expression of glycans correlative with E-selectin ligand activity was characterized by immunostaining and ow cytometry. Labeling with mAbs specic to sialyl Lewis X (sLex; CSLEX-1) and sialyl Lewis A (sLea; KM-231) revealed that BT-20 cells strongly expressed these antigens, but MDA-MB-468 cells weakly expressed them (Fig. 1B). Similar trends were observed when cells were stained with HECA-452 mAb, which collectively recognizes sialofucosylated oligosaccharides including sLex and sLea (Fig. 2). These results imply that BT-20 cells express classical carbohydrate antigens correlating with E-selectin ligand activity, but MDA-MB-468 cells may express other glycans that bind E-selectin. 3.3. Protease-insensitive molecules possess E-selectin ligand activity Breast cancer cells were treated with a broadly active protease, bromelain, to assess the role of glycoproteins versus glycosphingolipids as E-selectin ligands. As shown in Fig. 2, protease treated BT-20 cells expressed similar levels of HECA-452 antigen compared to untreated cells. This signal persistence after enzymatic protein cleavage suggested that sialofucosylated antigens were present on glycosphingolipids. Protease treated MDA-MB-

Fig. 1. Breast cancer cell adhesion to IL-1b activated HUVECs is E-selectinmediated. (A) Untreated or sialidase treated (0.1 U/ml) BT-20 and MDA-MB-468 cells (1 106/ml) were perfused over activated, mIgG1 isotype treated, or antiCD62E mAb treated HUVECs for 2 min at wall shear rate of 80 s1. Data are mean number of breast cancer cell tethering on HUVECs SEM, n = 36. p < 0.05 with respect to untreated cell tethering. (B) Flow cytometric analysis of breast cancer cells labeled with anti-sLex or sLea mAbs. Open curves show isotype and lled curves show specic mAb reactivities.

468 cells expressed very low levels of HECA-452 antigen similar to untreated cells (Fig. 2). Consistent with earlier adhesion results (Fig. 1A), BT-20 as well as MDA-MB-468 cells exhibited reactivity with EIg chimera. Notably, EIg binding of both breast cancer cell lines did not decrease after protease treatment (Fig. 2), providing evidence of glycosphingolipids as E-selectin ligands. The efcacy of protein cleavage on both BT-20 and MDA-MB-468 cells was conrmed by ow cytometry through the complete loss of E-cadherin expression (data not shown). Additionally, the levels of GM1 on breast cancer cells (tested using cholera toxin B) were not changed, implying that protease treatment did not cleave glycosphingolipids. Collectively, these ndings strongly indicate that E-selectin ligands expressed by breast cancer cells are glycosphingolipids that are HECA-452 positive (BT-20) as well as negative (MDA-MB-468).

3.4. Gangliosides are E-selectin ligands In order to conrm sialylated glycosphingolipids as E-selectin ligands, gangliosides from breast cancer cells were extracted and used in adhesion and immuno-overlay experiments. When E-selectin-

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Fig. 2. Protease treatment does not alter the E-selectin activity of breast cancer cells. Untreated or protease (bromelain, 1% w/v) treated BT-20 and MDA-MB-468 cells were labeled with HECA-452 mAb, EIg chimera, or CTB and analyzed by ow cytometry. Open curves show isotype or negative controls, and lled curves show specic mAb reactivities.

transfected Chinese hamster ovary (CHO-E) cells were perfused over immobilized gangliosides, CHO-E cells tethered to mock treated BT20 and MDA-MB-468 ganglioside spots (Fig. 3A). The binding was E-selectin specic, as anti-CD62E mAb pretreated CHO-E cells failed to attach. When ganglioside spots were treated with protease, the number of tethering cells was the same as for mock treated spots, demonstrating that tethering was not due to contaminating proteins. Treatment with sialidase completely abolished CHO-E cell attachment, consistent with results in Fig. 1. These ndings denitively show that gangliosides expressed by breast cancer cells are ligands for E-selectin. Additional conrmation that gangliosides from breast cancer cells support E-selectin binding was achieved with immuno-overlay assays using EIg chimera (Fig. 3B and C). Although BT-20 and MDA-MB-468 cells expressed distinct ganglioside proles (Fig. 3B), reactive bands with the same TLC migration (rf) were detected by EIg chimera in both cells, suggesting that they may share the same E-selectin ligands (Fig. 3C). In addition, the higher staining intensity observed in BT-20 gangliosides corroborates ow cytometry analysis using HECA-452 mAb and EIg chimera (Fig. 2). 3.5. Gangliosides mediate tethering and rolling on endothelial E-selectin To assess the E-selectin ligand activity of glycosphingolipids in the native cell membrane, protease (bromelain) treated breast cancer cells were perfused over IL-1b-activated HUVECs. As shown in Fig. 4A, tethering of protease treated breast cancer cells were not

statistically different from that of untreated cells. Furthermore, protease treatment did not alter rolling velocities in a statistically signicant manner (Fig. 4B). These ndings demonstrate that breast cancer cell gangliosides are natural E-selectin ligands under physiological ow conditions. However, rm adhesion was significantly reduced after protease treatment as compared to rm adhesion of untreated cells (Fig. 4B). Altogether, these results indicate that breast cancer cells express gangliosides as E-selectin ligands required for cell tethering and rolling, but protein ligands facilitate rm adhesion. It is also notable that the percentage conversion of tethering into rm adhesion was signicantly higher for untreated BT-20 cells (70 11%) compared to that of untreated MDA-MB-468 cells (8 2%), and that the rolling velocities of BT-20 cells were signicantly lower than those of MDA-MB-468 cells (Fig. 4B). These data indicate that BT-20 cells express more and/or higher afnity E-selectin ligands than do MDA-MB-468 cells, which is consistent with earlier results (Figs. 2 and 3). 4. Discussion Several specic E-selectin ligands have been identied for a variety of cancer cell lines [14,1820]. However, knowledge of breast cancer cell E-selectin ligands is lacking. Due to the involvement of glycosphingolipids in a diverse array of breast cancer metastatic processes and their role as adhesion molecules in several other types of cancers, the E-selectin ligand activity of breast cancer cell glycosphingolipids is of particular interest. In the present

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Fig. 3. Breast cancer cell gangliosides are E-selectin ligands. (A) Gangliosides extracted from breast cancer cells were immobilized on Petri dishes. Untreated or anti-CD62E mAb treated CHO-E cells were perfused over ganglioside spots at a wall shear rate of 80 s1. Spots were treated with buffer (mock treatment), protease (bromelain, 1% w/v), or sialidase (0.1 U/ml). Data are mean number of CHO-E cells interacting SEM, n = 5. P < 0.05 with respect to untreated cell tethering. (B and C) A ganglioside standard mixture and breast cancer cell ganglioside extracts were resolved by TLC in replicate. Plates were separated in two, (B) stained with HCl-resorcinol or (C) overlaid with EIg chimera.

work, we have demonstrated that human BT-20 and MDA-MB-468 invasive breast cancer cell lines express gangliosides (sialylated glycosphingolipids) as E-selectin ligands that are operational under physiological ow conditions, irrespective of the presence of sialofucosylated epitopes recognized by classical carbohydrate antibodies. Our data clearly show that breast cancer cells specically and avidly attach to activated HUVECs via E-selectin. It is widely accepted that E-selectin binds to ligands bearing sialofucosylated oligosaccharides, including epitopes that can be detected by the HECA-452 mAb [21]. BT-20 cells expressed HECA-452 reactive antigens, particularly sLex and sLea oligosaccharides (Figs. 1B and 2). Hence, BT-20 cells likely express classical carbohydrate glycans as E-selectin binding domains. However, sLex, sLea and HECA-452 antigens are correlative markers but not necessarily the exact epitopes that bind E-selectin [8,9]; non-classical E-selectin ligands also exist [22]. To wit, human myeloid cells express non-sLex E-selectin ligands [9]. Such novel ligands may be expressed by the MDA-MB468 cell line, which possess E-selectin ligand activity while lacking signicant HECA-452 reactivity (Figs. 1 and 2). In turn, the types of glycans expressed on the breast cancer cells studied correlate with E-selectin ligand efciency. BT-20, the HECA-452-positive cell line, showed higher conversion of cell tethering into rm adhesion and slower rolling velocity than that of the HECA-452-negative MDAMB-468 cell line (Fig. 4A and B). Additionally, E-selectin binding afnity of BT-20 gangliosides, expressed on whole cells (Fig. 2), or

extracted (Fig. 3A and C), was higher than that of MDA-MB-468 cells. Thus the breast cancer cell lines studied represent two broader classes of E-selectin binding glycans, one consisting of high efciency sLex/a or sLex/a -like structures recognized by HECA-452 (BT-20), and the other expressing novel, low efciency glycans (MBA-MB-468). However, both require sialic acid to maintain Eselectin ligand function (Fig. 1). Our ndings do not discount the possibility of overlapping or redundant function of the various E-selectin ligands. Rather, adhesion of breast cancer cells is a complex interplay among different molecules, which is consistent with reports identifying distinct molecules as mediators of disparate adhesion events [10,17,23]. Whereas breast cancer gangliosides are clearly important for tethering and rolling on endothelium (similar to several other cancer cells and leukocytes [811]), proteins facilitate rm adhesion (Fig. 4). We tested BT-20 and MDA-MB-468 cells for known protein E-selectin ligands PSGL-1 [18], CD43 [1], CD66 [19] and PCLP [20], but none were detected (data not shown). E-selectin and its ligand CD44v4 have recently been shown to participate in transendothelial migration of breast cancer cells by static (no ow) assays [24]; investigation of CD44 as an E-selectin ligand (i.e., HCELL [14]) under physiologic ow conditions is currently ongoing in our laboratory. To the best of our knowledge, the present study is the rst to report that gangliosides expressed by breast cancer cells are Eselectin ligands under physiological ow conditions. This new role

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Benencia, Dr. Douglas Goetz, and Dr. China Kummitha (all of Ohio University) for helpful discussions and critical review of the manuscript. References
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Protease

Fig. 4. Effect of protease treatment on breast cancer cell adhesion to IL-1b activated HUVECs. Untreated or protease treated (1% w/v) BT-20 or MDA-MB-468 cells (1 106/ml) were perfused over activated HUVECs for 2 min at a wall shear rate of 80 s1. Adhesive interactions were identied as initial tethering or rm adhesion as described in Materials and Methods. (A) Data are mean number of breast cancer cells tethering or rmly adhering on HUVECs SEM, n = 36. P < 0.05 with respect to untreated cell adhesion. (B) Data represent mean rolling velocities SEM, n = 15 25. $P < 0.05 with respect to BT-20 cell rolling velocity.

for breast cancer glycosphingolipids is particularly signicant in light of a recent publication from the Hakomori group [12], in which it was clearly demonstrated that expression of gangliosides is altered during EMT [12], a process inducible by local factors at the metastatic site and may be essential for successful tumor spread [12,25]. Moreover, the gangliosides themselves may regulate EMT [12]. Altogether, breast cancer cell ganglioside expression appears to be a critically regulated process, in which certain gangliosides are present on circulating tumor cells for tissue-specic homing, while expression of others are altered post-homing. Further studies are warranted to investigate the regulation and synergy between different gangliosides in the metastatic cascade. In conclusion, we have shown that gangliosides on BT-20 and MDA-MB-468 breast cancer cell lines are E-selectin ligands that mediate adhesion to E-selectin expressing activated endothelium. These lipids are major participants in breast cancer cell tethering and rolling and cooperate with proteins to mediate rm adhesion. Moreover, there are breast cancer glycans not recognized by classic sialofucosylated epitope mAbs (e.g., HECA-452 and CSLEX-1), which are responsible for E-selectin ligand activity. Further efforts are required to reveal the identities of novel glycans and lipid backbones that form functional E-selectin ligands. Ultimately, the structural identication of E-selectin ligands and their correlation with disease progression may lead to new biomarkers and/or treatments against breast cancer metastasis. Acknowledgments This work was supported by a seed grant from the Ohio Cancer Research Associates (MMB), grants from CNPq and CAPES (LN), and a Fulbright Visiting Researcher Grant (LN). We thank Dr. Fabian

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