Bone Sialoprotein Peptides Are Potent Inhibitors of Breast Cancer Cell Adhesion to Bone'

[CANCER RESEARCH56, 1948-1955. April 15. 19961 Bone Sialoprotein Peptides Are Potent Inhibitors of Breast Cancer Cell Adhesion to Bone'

Gabri van der Pluijm,2 Hans J. M. Vloedgraven, Boris Ivanov, Frank A. Robey, Wojciech J. Grzesik, Pamela Gehron Robey, Socrates E. Papapoulos, and Clemens W. G. M. LÃ¶Wik

Department of Endocrinology and Metabolic Diseases C4-89, University Hospital, P. 0. Box 9600. 23tX@RC Leiden, the Netherlands 1G. v. d. P.. H. I. M. V., S. E. P., C. W. G. M. LI, and Bone Research Branch 1W. J. G., P. G. Ri, and Peptide and Immunochemistry Unit, Laboratory of Cellular Development and Oncology (B. I., F.A.R.J, National institute of Dental Research, NIH, Bethesda. Maryland 20892

ABSTRACT bronectin, type I collagen, vitronectin, thrombospondin, OPN,3 and BSP, only the last is almost exclusively restricted to bone tissue (18). Bone and bone marrow are important sites of metastasis formation in This sialic acid-rich glycoprotein and its mRNA are found predomi breast cancer. Extracellular matrix proteins with attachment properties nantly in the mature, matrix-producing osteoblastsand osteocytes are generally believed to play a key role in tumorigenesis and metastasis (18â€”24).The BSP gene shows a distinct expression pattern in bone formation. We have investigated whether mammary carcinoma cells (MDA-MB-231) can recognize constructs of the fairly bone-specific hu (23â€”25).Inaddition,epiphysealcartilagecellslikehypertrophicchon man bone sialoprotein, which encompass the RGD sequence (EPRGD drocytes (18, 26) osteoclasts (18, 26, 27), and marrow stromal cells NYR). Exogenouslyaddedboneslaloprotein peptideswith this amino acid (28)alsoproduceBSP.Thehumanintegrmn-bindingregionofBSPhas sequenceIntheir backbonestructure, but not the more commonfibronec the more rare GRGDN rather than the more common GRGDSIT tin-derivedGRGDSpeptide,stronglyinhibitedbreastcancercelladhesion sequence. to extracellular bone matrix at micromolar concentrations. Most cyclic Several studies have shown that extracellular matrix proteins, like derivatives with the EPRGDNYR sequence were more effective inhibiters BSP and OPN, may be involved in the recognition and adhesion of oftumor cell adhesion to bone than their linear equivalents. Furthermore, osteoclasts and their precursors to bone matrix, a process mediated changesIn the RGD-tripeptlde of the backbone structure of the con through the vitronectin (aV@33)receptor(29â€”35).The0Lj33receptor is stnacts, removal of the NYR flanking sequence,or a different tertiary believed to play a key role in osteoclast formation (36) and osteoclas cyclic structure significantly decreasedtheir inhibitory potencies.In ad dition, the RGE-analogue EPRGENYR was capable of inhibiting breast tic resorption (29â€”36). cancer cell adhesionto bone,albeit to a lesserextent. Besides a variety of functions in normal physiology, adhesion We conclude,therefore, that the Inhibitory potency of the bone sialo receptors in tumor cells appear to play important roles along the protein-derived peptides on breast cancer cell adhesion to bone is not metastatic cascade of events leading to the establishment of distant solely due to a properly positioned RGD-motif alone but is also deter metastases (9, 10, 37â€”41).Recently, we have shown that adhesion @ mined by Its flanking regions, together with the tertiary structure of the receptors of the (3@and integrmn family may be functionally EPRGDNYR peptide. Synthetic cyclic constructs with the EPRGDNYR involved in adhesion of mammary carcinoma cells to extracellular sequencemay,therefore, be potentially useful as antiadhesiveagentsfor bone matrix (constituents; Refs. 42 and 43). Individual components of cancer cells to bone in vivo. extracellularbone matrix may, therefore,be involved not only in the preferredbonehomingof normalcells (like osteoclastprecursorsand INTRODUCTION osteoprogemtors)tobonebut may also provide a fertile environment for breast cancer cells. Breast cancer metastasizes frequently to the skeleton, leading to In this study,we examinedthe attachmentcharacteristicsofbreast serious clinical complications such as bone pain, fractures, spinal cord cancer cells to conformationally constrained peptide analogues de compression, and hypercalcemia (1â€”4).The mechanisms underlying signed to mimic the cell attachment site of human BSP. Several the apparent preference of mammary carcinoma cells for bone remain, constructs that encompassthe BSP-derived RGD-cell attachment mo however, poorly understood. The interactions of tumor cells with the tif EPRGDNYR in linear and cyclical conformations were tested (44). bone microenvironment,togetherwith the anatomicalpropertiesof In addition,the effectsof theseexogenouslyaddedBSP peptideson the skeleton, are generally believed to be decisive during the final breast cancer cell adhesion to bone were studied in three in vitro steps in the metastatic cascade, leading to the establishment and models of extracellular bone matrix. Results presented in this paper invasive growth of skeletal metastases (5â€”10). provide evidence that several peptide analogues that are based on the The organic matrix of bone is composed of collagenous proteins (RGD-contaiing) cell attachment site of BSP are recognized by (type I collagen fibers) representing 85â€”90%of total bone protein, breast cancer cells and may be potentially useful as antiadhesive whereas noncollagenousproteins comprise the remaining 10â€”15% agents for cancer cells to bone. (1 1â€”13).The list of proteins that can be isolated from mineralized bone matrix with cell attachment activity (adhesion proteins) is best MATERIALS AND METHODS headedby the proteinsthatcontainROD (Arg-Gly-Asp) motifs, a cell attachment recognition consensus sequence for several adhesion re Breast Cancer Cell Lines. Thebreastcancercell line MDA-MB-231 was ceptors of the integrin family (14, 15). Of the six identified RGD purchasedfromtheAmericanTypeCultureCollection(Rockville,MD). This containing extracellular bone matrix proteins (11, 13, 16, 17), fi estrogen receptor-negative cell line was established from a single pleural effusionobtainedfrom a 51-year-oldwhite womanwith poorly differentiated adenocarcinoma (45). MDA-MB-23l cells injected into the left heart ventricle Received12/18/95;accepted2/16/96. The costsof publicationof this article weredefrayedin partby the paymentof page of nudemicehavebeenshownto form osteolyticmetastasesinvivo (8, 46).@ charges. This article must therefore be hereby marked advertisement in accordance with Cells were cultured in RPM! 1640 + 10% fetal bovine serum + penicillin/ 18 U.S.C. Section 1734 solely to indicate this fact. streptomycin (Life Technologies, Breda, the Netherlands) in a humidified @ This work wassupported,in part, by a Grant 1KW93-580from the Dutch Cancer Society. Part of this work was presented at the 17th Annual Meeting of the American Society for Bone and Minced Research, Baltimore, Mb, September 9-13. 1995. 3 The abbreviations used are: OPN, osteopontin; BSP, bone sialoprotein; SFCM, 2To whomrequestsforreprintsshouldbe addressed.Phone:(31)71-5263076;Fax: serum-free conditioned medium. (3 1) 7 1-5248 136; E-mail: [email protected]. 4 Unpublished observations. 1948

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1996 American Association for Cancer Research. BSP@PEPT1DE5ANDBREAST CANCER CELL ADHESION TO BONE incubator at 37Â°Cat 5% CO2 until confluency. For attachment assays, tumor the figures of this report. To study the importance of the RGD adhesion cells werecultureduntil approximately90%confluencyandweredissociated recognition sequenceand the relative contribution of ROD flanking regions in into single-cell suspensions from the tissue culture flasks using 0.125% tryp CNB, the tripeptide sequence was replaced by ROE, resulting in the peptide sin-O.05% EDTA in PBS for 3 mm (see attachment assays). CCP. Synthetic Peptides,Proteins, and Reagents.BSA was purchasedfrom Cell AttachmentAssays:ProteinsCoatedontoPlastic.Cell attachment Miles Scientific, Inc. (Naperville, IL). SFCM was obtained from confluent assays were performed in bacteriological 96-well plates dotted with glycopro normal,primaryhumantrabecularbonecellsincubatedfor24 h in serum-free teins, as described previously (16). In brief, 10 pi of SFCM or peptide medium supplemented with 0.5% ITS@[insulin, transferrin, and selenium (2.5 constructin PBScontaining1 mMCa2@wasapplied,resultingin a protein gxg/ml), 0.5 mg/mI BSA, and 0.5 gxg/ml linoleic acid; Collaborative Research, coated area (â€œdotâ€•)of0. 12 cm2. After 16 h at 4Â°C,the fluid was removed, and Inc., Bedford, MAI. SFCM contains a mixture of various extracellular bone 100 @xlof60% methanol were added to each well for 2 h at 4Â°C.The methanol matrix components, including BSP, and reflects the diversity and composition fixation was found to improve the stability and durability of the protein-coated of extracellular bone matrix in vivo as described earlier (1 1, 16, 47). area without altering the biological activity of the substrate (16). The methanol The linear and cyclic peptides used in this study were synthesized using an was removed, and wells were washed for 30 mm at 4Â°Cwith washing buffer automatedsolidphasepeptidesynthesizerasdescribedearlier(44). We have [50 mM Tris-HC1 (pH 7.8), 1 10 mM NaCI, 5 nmi CaCl2, 0.1 mi@iphenylmeth used three linear synthetic BSP peptides (BSA-BA6, BSA-LBP, and CP-3), ylsulfonyl fluoride, 1% BSA, and 0.1 p.Msodium azide) to block unbound sites five cyclic BSP peptides (BSA-CNB, CNB, CCP, Ac-CNB, and C-BC3; Fig. on the plastic.After removalof the washingbuffer, the wells were washed 1), and two unrelated peptides C-CB1 and GRGDS (Calbiochem, La Jolla, three times with serum-free medium (RPMI 1640) supplemented with gluts CA). C-CB1 was reportedearlierto have an antithromboticactionby inter mineand0.5%ITS@. feting with the heterodimericglycoproteinintegrin receptorGPIIbIIIa (48). Cells were precultured in RPMI 1640 (+ HEPES) + 10% fetal bovine The GPllbllIa receptor, like several members of the mtegrin family, contains serum + penicillin/streptomycin in a humidified incubator at 37Â°Cat5% CO2 a binding site for the tripeptide Arg-Gly-Asp (RGD). until 90% confluency.For attachmentassays,tumorcells were dissociated BSA, GRADS, and GRGES were used as negative controls and did not from the flasks using 0.125% trypsin-O.O5% EDTA solution in PBS (pH 7.2) affect adhesion of mammary carcinoma cells to the various substrates tested. for 3 mm. Cells were washed and resuspended in serum-free medium (RPMI Forreasonsofclarity, BSA, GRGES,andGRADScontrolsarenotdepictedin 1640 containing 0.5% ITS@) and seeded at a density of 10,000/cm2 (2800 cells/l50 p1)on thematrixcoated96 wells. The96-well dishes wereincubated at 37Â°Cat5% CO2for a maximumof 3 h. Eachwell waswashedthreetimes with serum-free medium, and attached cells were fixed for 20 mm with 80% Peptide designation Peptide structure methanol at 4Â°Cand stained with AmidoBlack. Two nonoverlapping micro scopic fields within each protein-coatedareawere counted. Each group was performed in 4-fold, and experiments were repeated three times. BSA-BA6 EPRGD-DPA-cM-BsA The adhesion of mammary carcinoma cells to constructs coated onto plastic was determined at various concentrations. Coating efficiencies to bacteriolog ical plastic differed between the tested constructs. Significantly detectable BSA-LBP EPRGDNYR-DPA-cM-BSA coating was obtained only with BSP-derived peptides conjugated to BSA (maximal coating efficiencies about 65% at 1 p@MROD content), whereas coating efficiencies of other peptides were low (< 10%) or nondetectable at CP3 EPRGENYR various concentrations tested (0.01â€”10 @xM).At concentrations higher than 1.0 ELM,the absolute amount ofplastic bound peptides (BSA-CNB, BSA-BA6, and BSA-LBP) remained constant (results not shown). AdhesionofBreastCancerCellstoBovineCorticalBoneSlices.Bovine BSA-CNB BSA-CM-A-DPA-EPRGDNYRCySNH2 cortical bone slices were a kind gift from Dr. Tuna Laitala (University of Oulu, I @Ã¸CM1 Oulu, Finland). Tissue culture plates (24 wells) were coated with 500 @xlof1% CNB agarose in PBS (pH 7.2) to prevent non specific adhesion to the tissue culture DPA-EPRGDNYRCysNH2 plastic.Aftertheagarosecooleddownandbecamesolid,a dropof melted agarosewasaddedtoeachwell on theagaroselayer,andthebonesliceswere I @lI-CM-i placed on top of this drop, resulting in a bone slice fixed to the agarose underlay. Subsequently, the wells were washed twice with 1 ml of serum-free ccP c-EPRGENYR-BPA culturemediumandpreincubatedfor30 mm.Tumorcellswereseededontop of theseslicesata densityof 100,000cells/500pAandincubatedfor3 h. After I @iulCMJ incubation, the bone slices were removed from the wells and washed three times in PBS and fixed for 30 mm with 500 pi 3% paraformaldehyde in PBS Ac-CNB Ac-DPA-EPRGDNYRCYSNH2 containing 2% sucrose. Bone slices were stained for 8 mm with 1% toluidine I @I-CM@@J blue in 1% sodium borate and, after fixation, were washed three times with PBS and mounted on glass coverslips with Histomount (National Diagnostics Brunschwig Chemie, Amsterdam, the Netherlands). Stained bone slices were C-BC3 EPRGDNYRCysNH2 kept dark beforehistologicalexamination.Cellslocatedwithin two nonover lapping microscopic fields were counted at the center of each bone slice. Each I @tCMJ experimentalgroupwasperformedin4-fold, andexperimentswererepeatedat leastthreetimes. C-CB1 dYRGDCYSNH2 Adhesionof Breast Cancer Cells to Frozen Sectionsof Developing L@ICMJ Trabecular Bone. Two-day-oldneonatalmice (SwissAlbino) were killed, and their tails were dissected, stretched in Tissue Tek (Miles Scientific, Naperville, II..), frozen in liquid N2 for at least 10 mm, and stored at â€”80Â°C. Fig. I. Designated abbreviations and structures of linear and cyclic peptides used in Five-@xmsections were cut at â€”20Â°Cusinga cryostat (Microm HM 500 M) this study. Six cyclical (BSA-CNB, CNB, CCP, Ac-CNB, C-BC3, and C-CB1) and three andsubsequentlytransferredto3-aminopropyltriethoxy-silane(SigmaChem linear (BSA-BA6, BSA-LBP, and CP3) were studied.Single letter codesare usedfor ical Co, St. Louis, MO)-coated glass coverslips and stored at â€”80Â°Cuntil use. amino acids, except for cysteineamide (CysNH2).GRGDS was used as an RGD control. Prior to adhesion experiments, the frozen sections were air dried at room GRADS, GRGES. and BSA were used as negative control peptides/proteins. Ac, acetyl; DPA, aj3-i-diaminopropionic acid; BPA, N@-bromoacetyldiaminopropionic acid; CM. temperature for 30 mm. Around each tissue section, a (hydrophobic) line was S-carboxymethylcysteine. drawn with a DAKO pen (DAKO A/S, Olostrup, Denmark) to avoid spreading 1949

BSP.PEFflDES AND BREASTCANCERCELL ADHESIONTO BONE

@@@ of the cell suspension over the entire glass coverslip area. Subsequently, Tissue . .â€¢,, .@ @ Tek wasremovedbyincubatingthesectionsfor5 nunin a largevolumeof A @, SFC@v1 @ PBS The sections were incubatedat 4 C with 100 @tlofPBS + 2% w/v j' I @4 BSA fraction V for 2 h to avoid nonspecific bmding Eighty thousand breast cancer cells in 80 ,.d serum free medium (RPMI 1640 + flS@) were added per section in the presence or absence of 2 @Msyntheticpeptides (Fig 1) andâ€¢ for 1 h at 37 C in a humidified incubator(5% C02) After mcuba tion, the sectionswerewashedthreetimeswith PBS(to removenonadherent @@@@@ cells), and attached cells were fixed in 4% paraformaldehyde for 30 mm. .. .: @: ) @@@ ,â€˜ â€¢ â€˜@ â€˜1@ Subsequently, the sections were washed with PBS and stained with hematox @ ylin for 1 mm. The hematoxylin was removed, and the stained sections were . P@ ; @. . . . @ washed in running tap water for 5 mm. The sections were mounted in @a , @ .coverslips.ICellHistomount (National Diagnostics-Brunschwig Chemie) and covered with@. â€˜ .

numbers were determined by counting nonoverlapping microscopic

4determinedfields for each central section of vertebra at an original X200 magnification. . â€¢.@ 4 The mean value of two microscopic fields within one vertebral section was.

@ within eachsection(n = 5). The centralsectionsof the tail were @@@ made from the same animal and used for control and expenmental groups â€˜,@i.., Eachexpenmentwasrepeatedatleastthreetimes(n = 3) 1) @A-CN B @@@ ;@ ..@.:.â€˜@ â€˜ , . s Inhibition ofBreast CancerCell Adhesionto Extracellular BoneMatrix by SyntheticPeptides.Breastcancercell attachmenttobonematrixwas @@@@@ determined in the continuous presence of synthetic peptides at various con \ ../@ .. - . @@@ .Statistics. . â€¢ / .. centrations, and IC5@swere calculated.. Significances between experimental groups within each experi â€˜ . . . . @ mentweredeterminedusingafactorialone-wayanalysisof variance,followed . .â€œ . . \,@: @ by a Fisher's PLSD test. \@ I \ ,

@RESULTS â€˜@@..@! . I. .@ . @: ,,@â€˜CoatedAdhesion of Breast Cancer Cells to Varying Concentrations ofâ€˜ â€˜@ BSP Peptide Conjugates. MDA-MB-23l cells were mom @ â€¢plastictored for their ability to adhere to different concentrations of synthetic . BSP peptides conjugated to BSA that were coated onto bacteriological,,,..@ todependent(96-well plates). Peptide structures are based on the ROD Fig. 3. The morphological appearance of MDA-MB-231 cells following adhesion cell attachment site of human bone sialoprotein. Mammary SFCM (A) and a cyclic EPRGDNYR constructconjugatedto BSA, BSA-CNB (B). The carcinoma cells bound to immobilized BSP peptides and subsequently weilsof a edcmtiter@late were coated with 2 iz@iPeptide or 1:1 diluted SFCM in PBS x200.sion containing2 msi Ca (see MatenalsandMethods). spread in. a dose-dependent manner (Fig.. 2). Maximal. cellular adhe of mammary carcinoma cells was obtained at peptide concentra

tions of 1 p.Mand higher (Fig. 2). However, differences were found in . .. . . ofnormalhumantrabecularbonecells(Fig.3A,positivecontrol)and the ability of MDA-MB-23l cells to adhere to the various peptides. . . BSA-CNB (Fig. 3B) when coated onto plastic. BSA-CNB promoted cellular adhesion most strongly, followed by . . . . Effects of Exogenously Added Peptides on Attachment of BSA-LBP and BSA-BA6 respectively. Fig. 3 depicts the morpholog . . . . . Breast Cancer Cells to Extracellular Bone Matrix. Three in vitro ical appearance of MDA-MB-23l cells following adhesion to SFCM. . attachment assays were used to investigate whether exogenously toextracellularbonematrix.MDA-MB-231cellsadheredstronglytoadded BSP peptides can modulate the adhesion of breast cancer cells immobilized SFCM of normal human trabecular bone cells. Exog cells/area enously added GRGDS peptide, at concentrations up to 300 @aM (IC50 400,.aM;Table1),didnotsignificantlyaffecttheattachment of MDA-MB-231 cells to SFCM. In contrast to GRGDS, seven of eight tested synthetic BSP-peptides significantly inhibited adhesion to various degrees (Table 1). This is further illustrated in Fig. 4 with equimolar concentrations of the tested peptides (2 p.M).At this concentration, the cyclic peptides BSA-CNB and CNB were more potent inhibitors of adhesion (Â±80% inhibition) than the linear peptides BSA-LBP or BSA-BA6 with similar or shortened amino acid sequences (50 and 30%, respectively). Further more, blocking of the N'@2terminus by the addition of an acetyl (Ac) group decreased the inhibitory potency of CNB. A change in tertiary structure by modification of the ring size, leading to the smaller cyclic C-BC3 peptide with identical EPRGD @aMproteinduringcoating NYR amino acid sequence, resulted in total loss of the inhibitory

Fig. 2. Dose-dependent attachment patterns of breast cancer cells (MDA-MB-23l) to effectontheadhesionofbreastcancercellstobonematrix(Fig.4; the synthetic BSP-peptides BSA-CNB, BSA-BA6, and BSA-LBP that are coated onto Table 1). Furthermore, replacement of the ROD tripeptide by RGE plastic.A representativeexperimentis shown.The experimentwasrepeatedthreetimes. (CCP) significantly decreasedthe inhibitory potency of CNB (Fig. 4; 1950

BSP-PEPT1DESANDBREAST CANCER CELL ADHESIONTO BONE

TableI Haif-maxinwi inhibiwry concentrations(lC@s)ofexogenouslyaddedpeptides, susceptibility of tumor cells to various BSP-peptides increased during constructs. and proteins during adhesion oIMDA-MB-231 cells to SFCM of normal human trabecular bone cells maturation of trabecular bone (Fig. 6), resulting in a significant decreasein attachment of the breast cancer cells to the mature remod Peptide ICan(pi@i)forcellular adhesion eled bone (the proximal tail vertebrae 1â€”11;Fig. 6). ROD control peptide Figs. 7â€”9depict the the inhibitory effects of exogenously added GRODS 400 CNB during attachment of MDA-MB-23l cells to various SFCM Negativecontrols BSA-V >500 (Fig. 7), cortical bone slices (Fig. 8), and vertebrae (Fig. 9). The GRADS >500 micrographs show that both adhesion of MDA-MB-23l cells to GRGES >500 extracellular bone matrix and subsequent spreading are strongly Linear peptides/constructs decreased. BSA-BA6 2â€”20 BSA-LBP 2â€”20 CP3 400 DISCUSSION Cyclic peptides/constructs BSA-CNB 0.002-0.02 Several extracellular matrix proteins with cell attachment prop CNB 0.02-0.2 Ac-CNB 2 erties have been isolated from skeletal tissue (1 1, 13, 16â€”22, CCP 2â€”20 49â€”51). These include fibronectin, type I collagen, vitronectin, C-BC3 >500 thrombospondin, OPN, and BSP. Bone adhesion proteins are either C-CB1 >500 produced by bone cells and/or derived from extraskeletal sources and are involved in a variety of functions, such as cellular differ

attachedcells entiationandproliferationofbonecells,theirmigration,and (%of control) attachment (11â€”13,16â€”19,23â€”25,30, 31, 51). The bone glyco proteins BSP and OPN are differentially expressed during matu ration of bone, especially at the onset of calcification (1 1, 16,

1' 18â€”25,31,49, 52). Furthermore, cells in bone (osteoclasts, osteo blasts, osteocytes, and other stromal cells) can recognize these extracellular molecules or synthetic peptide sequences that encom pass the ROD sequence (16, 29, 31â€”36,53â€”55).Evidence is mounting that osteoclasts that highly express the vitronectin re ceptor a@f33usethis integrin to adhere to these extracellular bone matrix proteins like BSP, and it has been suggested that that this adhesion receptor is a prerequisite for osteoclastic bone resorption @oPi and osteoclast development (29â€”36). ,Â°@ @@ ci << LI u@

â€”cyclic â€” linear â€” adherent Fig.4. InhibitoryeffectsofexogenouslyaddedBSP-peptides(2@asi)onadhesionof cells breast cancer cells (MDA-MB-23l) to extracellular bone matrix (SFCM coated onto plastic).Two @LMGRODSwereusedto studythe ROD-dependentnatureof tumor cell attachment.BSA,GRADS,andORGESwereuseasnegativecontrolsanddid notchange adhesionofmammarycarcinomacellsto bonematrix(resultsnotshown).Eachgroupwas @ performed in 4-fold, and experiments were repeated three times; bars, SEM. Â°,P 0.05; @@ **. 0.005; ***, 0.0005.

Table 1). However, this ROE analogue of CNB was still capable of inhibiting breast cancer cell adhesion to bone, albeit to a lesser extent. Removal of the NYR flanking sequenceof the peptide BSA-LBP, onbreastcancercellattachment.Inaddition,adifferent(smaller)resulting in BSA-BA6, significantly decreasedits inhibitory potency EPRGDNYR ring structure, resulting in C-BC3, also decreased the inhibitory potency of the cyclic CNB peptide (Fig. 4; Table 1). Cycizing the ROD motif with non-BSP-specific flanking regions was .@ C,, â€œC @ ineffective (C-CB1; Fig. 4; Table 1). @z: @z; < @ Similarly, as shown in Fig. 5, breast cancer cell adhesion to bovine C.) C.) â€˜-ii c3@ â€˜@ corticalbonesliceswassignificantly inhibitedby thetestedcyclic and C@ Cl) Cl, linear BSP-peptides (BSA-CNB, CNB, BSA-LBP, and BSA-BA6) but not with GRODS or negative control peptides (GRADS, ORGES, and BSA; data not shown). In this assay as well, the cyclic BSP 2@M peptides (BSA-CNB and CNB) were more effective inhibitors than Fig. 5. Inhibitory effects of exogenously added BSP-peptides (2 @M)onadhesion of the linear ones (BSA-LBP and BSA-BA6). breast cancer cells (MDA-MB-23l) to extracellular bone matrix (bovine cortical bone In line with the results obtained in the above described in vitro slices). Two p.@GRGDS were used to study the ROD-dependent nature of tumor cell adhesion assays, exogenously added BSP-peptides were capable of attachment. BSA, GRADS, and GRGES were use as negative controls and did not change adhesion of mammary carcinoma cells to bone matrix (results not shown). Each group was @ inhibiting breastcancercell attachmentto cryostatsectionsof trabe performedin 4-fold, andexperimentswererepeatedthreetimes;bars,SEM. Â°,P 0.05; @ cular bone (2-day-old neonatal mouse tail vertebrae; Fig. 6). The 5*, p 0.005; P 0.0005. 1951

BSP-PEPT!DES AND BREAST CANCER CELL ADHESION TO BONE

0 control Fig. 6. Inhibitory effects of exogenously added BSP-peptides (2 @LM)onadhesion of breast cancer cells (MDA-MB-23l) to extracellularbonematrix . (neonatalmousetail vertebrae).The number of a attached cells per area (2.54 mm2) on vertebrae 24, 18and 11, representing hypertrophic cartilage, cal r2 cified cartilage. and mature remodeled trabecular bone, respectively, were counted. Two psi GRGDS was used to study the ROD-dependent nature of tumor cell attachment. A representative experiment is shown. BSA. GRADS. and ORGES were use as negative controls and did not change vertebrae adhesion of mammary carcinoma cells to bone 24 18 matrix (results not shown). Values are expressedas I vertebranumber means (n = 5). Each experiment was repeated at @ least three times; bars, SEM. Â°,P 0.05; Â°Â°, distal @11 proximal P 0.005. I .1.

@hypertrop

In analogyto osteoclasts,ithasbeenshownthatinvasivemammary carcinomas express the aj33 receptor (37, 39, 40, 42, 43), and it was @ .. , , I ;. .â€˜._-@ ..@ reported recently that breast cancer cells show a selective increase in â€¢, @1 , j i.@.'-. â€¢.-â€˜ ( - f33integnns in skeletal metastases(56). Furthermore, recent evidence 2pM BSA suggests that highly invasive primary breast carcinomas can express ,â€˜â€˜byâ€” :@S@;@' . :@ @@@ high levels of BSP when compared to noninvasive breast cancers and - â€”.. @1 .â€˜@:;.::i.â€¢:@@@..â€”.'(.. - normal breast tissue (57). We have also shown recently that various @ ; @ mammary carcinoma cell lines have high affinities for the BSP i'@ ,, â€˜.;....,./â€”@: : @: â€˜.. p molecule in vitro (42).@We hypothesized, therefore, that mammary i,Â°.@ @qâ€¢, . ,@ -:@. . carcinomacellsusemechanism(s)ofbonerecognitionthatarecomI -â€¢@: -@ ,_. - If parable to those used by normal cells in bone. In this study, we describeinteractionsof breastcancercells with @ synthetic BSP peptides. Our data show that mammary carcinoma cells ; (MDA-MB-231) recognize various immobilized (and solubilized) synthetic BSP sequences, which include the ROD motif and its flanking regions. Furthermore, adhesion of breast cancer cells to #t@ @. .@ -, extracellular matrix of bone was significantly inhibited by nearly all peptides containing (parts of) the EPRODNYR sequence(BSA-CNB, CNB > Ac-CNB, BSA-LBP > BSA-BA6 > C-BC3). The cyclic B . +2pMCNB peptides BSA-CNB, CNB, and Ac-CNB were generally more potent inhibitors than linear peptides with similar or shortened amino acid @ sequences (BSA-BA6 and BSA-LBP). This strongly suggests that . :@, @ tertiary structures of the peptides are important for their ability to : @:1., :@: @ interfere with tumor cell adhesion to bone matrix. Our observations . ,-,44I'@..- are in line with earlier publications using different ROD-containing . _1&@@ â€˜.. cyclic and linear peptides and other cell types. Several ROD-contain ing cyclic (and linear) peptides have proven to be strong and selective inhibitors of cellular adhesion in vitro (15, 58) and metastasisforma tion in vivo (59) under different experimental conditions. Further . â€˜ , @ - more, it was shown recently that human trabecular bone cells adhere and spreadon immobilized constructscontainingthe EPRGDNYR motif of BSP (44), which is in agreement with our data on breast cancer cells. The inhibitoryeffectsof the testedBSP-peptideanaloguesontumor Fig. 7. Micrographs of MDA-MB-231cells in the absence (A) or presence (B) of 2 psi CNB peptide during 3 h incubation onto SFCM of human trabecular bone cells coated onto plastic. X200. 5 o. van der Pluijm, H. Vloedgraven, S. Papapoulos. C. Ldwik, L. van der Wee-Pals, W. Grzesik, 3. Kerr, and P. Gehron Robey, manuscript in preparation. 1952

B5P-PEPTIDES AND BREAST CANCER CELL ADHESION TO BONE

Fig. 8. Micrographs of MDA-MB-231 :@t::& cellsin theabsenceorpresenceof2 psi CNB peptide during 3 h incubation on bovine cor tied bone slices(B).A, the negative control (2 ,LM BSA). X200. B â€˜. +2pMCNB

@ I. â€˜-- .

@- .@

S â€¢,

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cell adhesion to bone, together with the lack of an effect of the on breast cancer cell adhesion to bone matrix. Therefore, proper synthetic non-BSP-derived ROD-peptides ORODS and C-CB 1 (up to positioning of the ROD motif within the framework of its 100-fold higher concentrations) indicate that the ROD sequence by BSP-flanking regions strongly enhancesthe inhibitory potency of itself is not the sole prerequisite for blocking breast cancer cell the synthetic peptides on breast cancer cell adhesion to bone adhesion to bone matrix under these experimental conditions. Moth matrix. fication of the @2andCOOH-terminal ends of the EPRODNYR In conclusion, we have demonstrated that breast cancer cells sequence(see Ac-CNB versus CNB and BSA-BA6 versus BSA-LBP) recognize synthetic peptides encompassing the ROD motif of negatively affected the inhibitory potency of the peptides. This sug human BSP in various conformations. Attachment of cancer cells gests that the presence of the NH2-terminal cationic and COOH to three in vitro models for extracellular matrix of bone (serum terminal anionic groups can modify the functional responsemediated conditioned medium of bone cells, cortical bone slices, and frozen by its receptor(s), as has been reported earlier for osteoclasts (60). sectionsoftrabecularbone)waseffectivelyinhibitedbyexog Furthermore, our data support the notion that ROD flanking enously added BSP peptides. This inhibition is most likely medi regions of BSP (EP-ROD-NYR), which differ from the more ated through adhesion receptors of the integrin family that recog common ROD recognition sequence of fibronectin (G-ROD-SPC), nize ROD motifs and/or its flanking regions. Clearly, further may additionally determine the inhibitory potency of the peptides studies are warranted to establish the relevance of these observa 1953

BSP-PEPT!DES AND BREAST CANCER CELL ADHESION TO BONE

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Gabri van der Pluijm, Hans J. M. Vloedgraven, Boris Ivanov, et al.

Cancer Res 1996;56:1948-1955.

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