Mucin 16 is a functional selectin ligand on pancreatic cancer cells

The FASEB Journal • Research Communication

Mucin 16 is a functional selectin ligand on pancreaticcancer cells

Shih-Hsun Chen,* Matthew R. Dallas,*,†,‡ Eric M. Balzer,*,†,§

and Konstantinos Konstantopoulos*,†,‡,§,�,1

*Department of Chemical and Biomolecular Engineering, †Institute for NanoBioTechnology,‡Cancer Nanotechnology Training Center, §Physical Sciences-Oncology Center, and �Center forCancer Nanotechnology Excellence, Johns Hopkins University, Baltimore, Maryland, USA

ABSTRACT Selectins promote metastasis by mediat-ing specific interactions between selectin ligands ontumor cells and selectin-expressing host cells in themicrovasculature. Using affinity chromatography inconjunction with tandem mass spectrometry and bioin-formatics tools, we identified mucin 16 (MUC16) as anovel selectin ligand expressed by metastatic pancreaticcancer cells. While up-regulated in many pancreaticcancers, the biological function of sialofucosylatedMUC16 has yet to be fully elucidated. To address this,we employed blot rolling and cell-free flow-based adhe-sion assays using MUC16 immunopurified from pan-creatic cancer cells and found that it efficiently bindsE- and L- but not P-selectin. The selectin-binding deter-minants are sialofucosylated structures displayed on O-and N-linked glycans. Silencing MUC16 expression byRNAi markedly reduces pancreatic cancer cell bindingto E- and L-selectin under flow. These findings providea novel integrated perspective on the enhanced meta-static potential associated with MUC16 overexpressionand the role of selectins in metastasis.—Chen, S.-H.,Dallas, M. R., Balzer, E. M., Konstantopoulos, K. Mucin16 is a functional selectin ligand on pancreatic cancercells. FASEB J. 26, 1349–1359 (2012). www.fasebj.org

Key Words: MUC16 � metastasis � shear stress

Metastasis is a multistep process in which cancer-ous cells separate from a primary tumor and enter thevascular system, where they interact extensively withhost cells and tissues. Tumor cells that successfully

arrest in distant sites may then exit the vasculature andestablish micrometastatic colonies. Mounting evidencesuggests that vascular selectins (E-, L-, and P-selectin), afamily of C-type lectins, play a prominent role in thisprocess by facilitating the interaction of circulatingtumor cells (CTCs), which express selectin ligands, andselectin-expressing host cells. Mice deficient in P-and/or L-selectin display reduced metastasis (1, 2),which may result from diminished aggregation of CTCswith P-selectin-expressing platelets (3–5) and L-selec-tin-bearing neutrophils (6, 7). E-selectin has also beenshown to support metastatic spread in vivo, as E-selectinexpressed on the surface of activated endothelial cellsfacilitates adhesion of free-flowing CTCs and thus theireventual exit from the vasculature and lodging in targetorgans (8–11).

Selectins recognize sialofucosylated oligosaccharides,such as sialyl Lewis x (sLex) and its isomer, sialyl Lewisa (sLea) (12). Overexpression of these epitopes isassociated with pancreatic tumor growth and metastasis(13–15). In fact, sLex is absent from healthy pancreatictissue, but its expression progressively increases withhigher-grade pancreatic intraepithelial neoplasia (Pa-nIN) lesions and pancreatic adenocarcinoma (14, 16).Both sLex- and sLea-decorated glycoconjugates canmediate selectin-dependent CTC adhesion to host cells(12, 17). Despite the importance of selectin-mediatedbinding to sialofucosylated target molecules as a poten-tial metastatic determinant for pancreatic cancer, selec-tin ligands on pancreatic cancer cells have not beenwell characterized other than by general classifications(i.e., sialofucosylated glycoproteins). The binding affin-ity of selectins for isolated sLex and sLea is markedlylow. As a consequence, neither sLex nor sLea itselfcorrelates with the properties of endogenous selectinligands on cellular targets. As pointed out in theliterature (18), the “functional” selectin ligand shouldfulfill certain requirements: the ligand should bind

1 Correspondence: Department of Chemical and Biomolec-ular Engineering, Johns Hopkins University, 3400 N. CharlesSt., Baltimore, MD 21218, USA. E-mail: [email protected]

doi: 10.1096/fj.11-195669This article includes supplemental data. Please visit http://

www.fasebj.org to obtain this information.

Abbreviations: AP, alkaline phosphatase; benzyl-GalNAc, ben-zyl-2-acetamido-2-deoxy-�-d-galactopyranoside; CHO-E cell, E-selectin-expressing Chinese hamster ovary cell; CHO-P cell,P-selectin-expressing Chinese hamster ovary cell; CTC, circulat-ing tumor cell; DMJ, deoxymannojirimycin; D-PBS, Dulbecco’smodified medium-PBS; EDTA, ethylenediaminetetraacetic acid;HRP, horseradish peroxidase; IP, immunoprecipitation; mAb,monoclonal antibody; MUC16, mucin 16; MUC16-KD, MUC16-knockdown; MS, mass spectrometry; OSGE, O-sialoglycoproteinendopeptidase; PE, phycoerythrin; SDS-PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis; shRNA, short hair-pin RNA; siRNA, short interfering RNA; sLea, sialyl Lewis a; sLex,sialyl Lewis x.

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with some selectivity and relatively high affinity; re-moval of it should prevent cell adhesive interaction.

In this study, we sought to identify novel selectinglycoprotein ligands on pancreatic carcinoma cells byutilizing a combination of immunoaffinity chromatog-raphy and tandem mass spectrometry (MS/MS). Ouranalysis identified a member of the mucin family ofglycoproteins, mucin 16 (MUC16), as an E- and L-selectin ligand overexpressed on metastatic pancreaticcancer cells.

MUC16 is a large (2500–5000 kDa), heavily glycosy-lated protein that is expressed in mucous membranesof various tissues, including the upper respiratory tractand reproductive organs (19, 20). MUC16 contains anextracellular N terminus adjacent to glycosylated tan-dem repeats, a transmembrane region, and a shortcytoplasmic tail (21, 22). In a clinical context, MUC16is significantly up-regulated in �80% of patients withovarian tumors, and the MUC16 epitope CA125 is anaccepted tumor marker for ovarian cancer (23, 24).Recent work also suggests that MUC16 is not ex-pressed in the normal pancreatic ducts but is stronglyup-regulated in pancreatic cancer and may play apotential role in the progression of this disease (25).Despite this strong clinical correlation, a biologicalmechanism for how MUC16 enhances the tumorprogression has yet to be delineated beyond its roleas a mesothelin ligand (26).

To define its functional role as a selectin ligand, weconducted the following set of experiments: blot roll-ing and cell-free flow-based adhesion assays revealedthat MUC16 possesses high E- and L- but low P-selectin-binding activity. Treatment of pancreatic cancer cellswith specific glycosidases and metabolic inhibitors re-vealed that the selectin-binding determinants forMUC16 are sialofucosylated structures displayed onboth O- and N-linked glycans. Furthermore, silencingMUC16 with short hairpin RNA (shRNA) significantlysuppresses binding to immobilized E- and L-selectinunder flow. Collectively, our results suggest that up-regulation of sialofucosylated MUC16 may enhance theability of CTCs to tether and adhere to host tissues thatexpress E- and L-selectin ligands. Our findings providea unified perspective for the enhanced metastatic po-tential associated with overexpression of sialofucosy-lated structures and MUC16 in pancreatic cancers andthe role of selectins in metastasis.

MATERIALS AND METHODS

Cell culture

The human nonmalignant pancreatic epithelial hTERT-HPNE cells and the pancreatic cancer cell lines CFPAC-1 andSW1990 were obtained from the American Type CultureCollection (Manassas, VA, USA), and Pa03C (27) was agenerous gift from Dr. Anirban Maitra (Johns HopkinsSchool of Medicine, Baltimore, MD, USA). hTERT-HPNE wascultured in medium containing 3 vol of glucose-free DMEM,1 vol of Medium M3 base (InCell, San Antonio, TX, USA), 5%

FBS, 5.5 mM glucose, 10 ng/ml human recombinant EGF,and 50 �g/ml gentamicin at 37°C under air with 5% CO2.SW1990 and Pa03C were cultured in DMEM with 10% FBS.CFPAC-1 was cultured in IMEM with 10% FBS. Before celllysis, cells were detached from culture flasks using enzyme-free cell dissociation medium (15 min at 37°C; Chemicon,Phillipsburg, NJ, USA). For flow cytometry and flow-basedadhesion assays, cells were harvested by mild trypsinization[0.05% trypsin/ethylenediaminetetraacetic acid (EDTA) for5 min at 37°C] and subsequently incubated (107 cells/ml) at37°C for 2 h to allow for regeneration of surface glycoproteins(3, 28). E- and P-selectin-expressing Chinese hamster ovary(CHO-E and CHO-P) cells, stably transfected with cDNAencoding full-length E- or P-selectin, respectively, were gen-erously provided by Affymax (Palo Alto, CA, USA) andprocessed as described previously (29, 30).

Antibodies, reagents, and adhesion molecules

Anti-sLea monoclonal antibody (mAb) KM231 was purchasedfrom Millipore (Temecula, CA, USA). Anti-MUC16 mAbX306 was from Santa Cruz Biotechonology (Santa Cruz, CA,USA), X75 was from Abcam (Cambridge, MA, USA), and M11was from Dako (Burlington, ON, Canada). Anti-MUC1 (VU-4-H5) and MUC4 (1G8) mAbs were from Invitrogen (Carls-bad, CA, USA). Phycoerythrin (PE)-conjugated secondaryantibody was from Vector Laboratories (Burlingame, CA,USA). Alkaline phosphatase (AP)- and horseradish peroxi-dase (HRP)-conjugated anti-mouse IgG and AP-conjugatedanti-rat IgM were from Southern Biotech (Birmingham, AL,USA). All other unlabeled antibodies were from BD Biosci-ences Pharmingen (San Jose, CA, USA), unless otherwisestated. The chimeric form of E-, L-, and P-selectin-IgG Fc (E-,L-, and P-selectin) consisting of the lectin, EGF, and consen-sus repeat domains for human E-, L-, and P-selectin, respec-tively, linked to each arm of human IgG1, were purchasedfrom R&D Systems (Minneapolis, MN, USA).

Glycoprotein purification and MS analysis

As described previously (31, 32), the putative selectin ligandcorresponding to the glycoproteins with a molecular mass �460 kDa, which carries HECA-452-reactive sialofucosylatedoligosaccharides such as sLex and sLea, were isolated fromSW1990 cell lysate by affinity chromatography using recom-binant protein G agarose supports (Invitrogen) cross-linkedwith bis(sulfosuccinimidyl) suberate (Pierce Biotechnology,Rockford, IL, USA) to the anti-sLea mAb KM231. Elutedproteins were then separated by 3–8% sodium dodecyl sul-fate–polyacrylamide gel electrophoresis (SDS-PAGE) gels (In-vitrogen) and stained in gel with ProQ Emerald 300 glyco-protein stain (Invitrogen), which binds only to carbohydrategroups at glycosylation sites, thereby leaving the polypeptidecore intact (31, 32). The stained band with a molecularmass � 460 kDa was then excised, and trypsin-digested gelfragments were submitted for analysis by nanoflow HPLCinterfaced to electrospray ionization MS/MS (HPLC-MS/MS)using a ThermoFinnigan LTQ mass spectrometer (refs. 31,32; ThermoFinnigan, San Jose, CA, USA). The MS data weresearched against all taxonomies in the National Center forBiotechnology Information (NCBI; Bethesda, MD, USA)nonredundant protein database with a 95% significancethreshold (P�0.05) using Mascot (Matrix Science, Boston,MA, USA) and with a P � 0.01 confidence using theBioWorks 3.3 software featuring the SEQUEST algorithm(ThermoFinnigan).

1350 Vol. 26 March 2012 CHEN ET AL.The FASEB Journal � www.fasebj.org

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Western blot analysis

Whole-cell lysate or immunopurified MUC16 was diluted withreducing sample buffer and separated using 3–8% SDS-PAGEgels. Resolved proteins were transferred to Sequi-blot orImmun-blot PVDF membrane (Bio-Rad Laboratories, Hercu-les, CA, USA) and blocked with StartingBlock (Pierce Bio-technology) for 30 min. Blots were stained with HECA-452 oranti-MUC16 (X306) mAbs and rinsed with TBS-0.1% Tween20. Blots were incubated with appropriate AP- or HRP-conjugated secondary antibodies. Western blue AP substrate(Promega) and SuperSignal West Pico chemiluminescentsubstrate (Pierce Biotechnology) were used to develop theAP- and HRP-conjugated antibody-stained immunoblots, re-spectively.

Cell lysis and immunoprecipitation (IP) of MUC16

Whole-cell lysate was prepared by membrane disruption using2% Nonidet P-40 followed by differential centrifugation (30–33). MUC16 was purified via IP from SW1990 and Pa03C celllysate with an anti-MUC16 mAb (X306, X75, or M11), usingrecombinant protein G agarose spheres (30–33).

Blot rolling assay

Western blots of SW1990 and Pa03C cell lysate or immuno-purified MUC16 from the lysate were stained with anti-MUC16 or HECA-452 mAbs and rendered translucent byimmersion in 90% Dulbecco’s modified medium-PBS (D-PBS)-10% glycerol as described previously (34). The blotswere placed under a parallel-plate flow chamber, and humanperipheral blood lymphocytes or CHO transfectants, resus-pended at 5 � 106 cells/ml in 90% D-PBS-10% glycerol, wereperfused at a shear stress of 0.5 dyn/cm2 (30–33). Molecularmass markers were used as guides to aid placement of the flowchamber over stained bands of interest. The number ofinteracting cells per lane was averaged over 5 �10 fields ofview (0.55 mm2 each) within each stained region. Nonspecificadhesion was assessed by perfusing 5 mM EDTA in the flowmedium.

Preparation of MUC16-coated microspheres

MUC16 purified from SW1990 and Pa03C cell lysate wasdiluted to desired concentrations with binding buffer (0.2 Mcarbonate/bicarbonate buffer, pH 9.2) and incubated with 10�m polystyrene microspheres (2.5�107 microspheres/ml;Polysciences, Warrington, PA, USA) overnight at 4°C withconstant rotation, as described previously (30–33). Micro-spheres were washed 2 times with D-PBS and subsequentlyblocked with D-PBS/1% BSA for 30 min at room tempera-ture. Microspheres were resuspended (106 microspheres/ml)in D-PBS/0.1% BSA for use in flow cytometric and flowchamber assays. Site densities of MUC16-coated microsphereswere determined by flow cytometry (30–33).

Enzymatic and inhibitor treatments

MUC16-coated microspheres (1�107 microspheres/ml) weretreated with 0.1 U/ml Vibrio cholerae sialidase (Roche Molec-ular Biochemicals, Indianapolis, IN, USA) for 90 min at37°C to remove terminal sialic acid residues (30, 32, 33). Inselect experiments, MUC16-coated microspheres suspensions(5�106 microspheres/ml) were incubated for 2 h at 37°Cwith 120 �g/ml of O-sialoglycoprotein endopeptidase(OSGE; Accurate Chemical & Scientific, Westbury, NY, USA)

to specifically cleave glycoproteins with O-glycosylation onserine and threonine residues (35). To cleave N-linked gly-cans from MUC16, MUC16-coated microspheres were treatedwith PNGase F (New England Biolabs, Beverly, MA, USA)according to the manufacturer’s protocol. For metabolicinhibitor studies, cell suspensions (107 cells/ml) were pre-treated with 0.1 U/ml sialidase for 60 min at 37°C to removeterminal sialic acid residues and ensure de novo synthesis ofnewly generated HECA-452-reactive carbohydrate structures(30, 32, 33). Complete removal of sialylated structures wasconfirmed via flow cytometry using the mAb HECA-452 thatrecognizes sialic acid-bearing epitopes. Subsequently, cellswere cultured for 48 h at 37°C in medium containing either2 mM benzyl-2-acetamido-2-deoxy-�-d-galactopyranoside (benzyl-GalNAc) to inhibit O-linked glycosylation or 1 mM deoxyman-nojirimycin (DMJ) to disrupt N-linked processing (30, 32,33); DMSO dilution was used for control untreated cells. Sitedensities of MUC16 adsorbed on microspheres followingenzymatic or inhibitor treatments were determined by flowcytometry before used in flow-based adhesion assays.

Flow cytometry

MUC16 and HECA-452 site densities on microspheres wereassessed by single-color immunofluorescence and flow cytom-etry (FACSCalibur; BD Biosciences) using primary anti-MUC16 mAb (X306) with appropriate PE-conjugated second-ary antibodies or FITC-conjugated HECA-452 mAb. Similarly,surface expression of MUC1, MUC4, and sLea was monitoredusing anti-MUC1 (VU-4-H5), MUC4 (1G8), or sLea (KM231)mAbs with appropriate PE-conjugated secondary antibodies.CD29 expression on cells was monitored by using the PE-conjugated anti-CD29 antibody (MAR4). Background levelswere determined by incubating cell or microsphere suspen-sions with properly matched FITC- or PE-conjugated isotypecontrol antibodies (30, 32, 33).

Flow-based adhesion assays

Wild-type, mammalian scramble control, and MUC16-knock-down (MUC16-KD) cells or MUC16-coated microspheressuspended in D-PBS-0.1% BSA (1�106/ml) were perfusedover immobilized E-, L-, or P-selectin-coated dishes at pre-scribed wall shear stresses using a parallel-plate flow chamber(250 �m channel depth, 5.0 mm channel width) as describedpreviously (3, 5). The number of interacting cells or micro-spheres was then quantified by averaging over multiple �10fields of view during a 2-min period. In select experiments, E-,L-, or P-selectin-coated dishes were preincubated with the 20�g/ml anti-E-, -L-, or -P-selectin function-blocking mAb for1 h before being used in flow assays. Nonspecific adhesion wasassessed by adding 5 mM EDTA to the perfusion medium.

Preparation of MUC16 short interfering RNA (siRNA)oligonucleotides

siRNA oligonucleotides targeting MUC16 were generated usingthe siRNA design program from Whitehead Institute (Massachu-setts Institute of Technology, Cambridge, MA, USA) as de-scribed previously (30, 31). The siRNA sequences were used toconstruct 60-mer shRNA oligonucleotides, which were thensynthesized (Invitrogen) and ligated into the pSUPER.puroexpression vector (Oligoengine, Seattle, WA, USA) under thecontrol of the H1 promoter. The following oligonucleotide wasused: 5�-GATCCCCCAGCAGCATCAAGAGTTATTTCAAGAGAATAACTCTTGATGCTGCTGTTTTTA-3� (underscored, senseand antisense sequences; boldface, restriction enzyme sites;italicized, polymerase III termination signal; boldface italicized,

1351MUC16 AS A FUNCTIONAL E- AND L-SELECTIN LIGAND

loop with linker). The ligated product was transformed intocompetent DH5� Escherichia coli cells and amplified in thepresence of ampicillin, and the plasmid was purified using theEndoFree Maxi kit (Qiagen, Valencia, CA, USA). Sequenceinsertion was verified by restriction digestion and confirmed bydirect sequencing. A mammalian scramble sequence (Oligoen-gine) was used as a negative control in all siRNA experiments.

Generation of stable MUC16-KD pancreatic carcinoma celllines

As described previously (30, 31), SW1990 and Pa03C cellswere plated in 100-mm dishes and grown to reach 90%confluence. Cells were then transfected with pSUPER.puro.MUC16 using Lipofectamine 2000 (Invitrogen) according tomanufacturer’s instructions. On reaching confluence, trans-fected cells were passed and 106 cells/Petri dish were seededin growth medium in triplicate. After 24 h, the medium wasreplaced by a fresh aliquot containing 0.5 �g/ml puromycin(Invitrogen). Cells were then grown continually withoutpassaging for 2 wk, replenishing the puromycin-containingmedium every 3 d. Single-cell colonies were isolated andcultured using standard techniques.

Statistical analysis

Data are expressed as means � se for �3 independentexperiments. Statistical significance of differences betweenmeans was determined by ANOVA. The statistical significancewas set at probability values of P � 0.05.

RESULTS

Sialofucosylated MUC16 is expressed on metastaticpancreatic cancer cells

Sialofucosylated glycoproteins presented on the surfaceof metastatic pancreatic cancer cells are importantmediators of selectin-dependent adhesion (36, 37).However, the selectin ligands on pancreatic cancer cellshave yet to be identified. Using a flow-based adhesionassay, we observed that metastatic pancreatic cancercells, such as SW1990, Pa03C, and CFPAC-1, express-ing sialofucosylated glycoproteins (Supplemental Fig.S1A), adhered to E-selectin and transiently tethered toL-selectin in shear flow (Table 1). In contrast, thenonmalignant hTERT-HPNE pancreatic epithelial cellsare devoid of sialofucosylated structures (Supplemental

Fig. S1A) and failed to interact with selectins (Table 1).In an attempt to characterize the sialofucosylated gly-coproteins that function as selectin ligands on meta-static pancreatic cancer cells, SW1990 cells were initiallychosen for further studies because of their potentselectin binding activity. Western blot analysis of wholeSW1990 cell lysate was carried out to examine theexpression of all sialofucosylated (i.e., HECA-452-reac-tive) glycoproteins. Two HECA-452-reactive proteinbands were identified in a 3–8% gradient gel: anintense band broadly distributed at �460 kDa and aweak signal at �200 kDa (Fig. 1A). With the use of ablot rolling assay, the selectin binding activity of theseHECA-452-reactive bands was evaluated. This tech-nique identifies selectin-ligand interactions under phys-iological shear conditions (34). To this end, CHO-Eand CHO-P cells and L-selectin-expressing lymphocyteswere perfused at 0.5 dyn/cm2 over HECA-452 immu-nostained blots, which were rendered translucent byimmersion in 90% D-PBS/10% glycerol (34). CHO-E(Fig. 1B) and lymphocytes (not shown) bound predom-

TABLE 1. Extent of adhesion of pancreatic nonmalignant or carcinoma cells to E-, L-, andP-selectin under physiological fluid flow

Cell line Pancreatic cell type

Interaction with selectins

E L P

hTERT-HPNE Nonmalignant CFPAC-1 Metastatic carcinoma * SW1990 Metastatic carcinoma *** Pa03C Metastatic carcinoma **

Pancreatic nonmalignant cells or pancreatic cancer cells were perfused at a wall shear stress levelof 0.5 dyn/cm2 over the surface coated with 20 �g/ml E-, L-, and P-selectin. , Firm cell adhesion; �,transiently cell tethering/rolling; , no interaction.

Figure 1. Sialofucosylated glycoproteins expressed bySW1990 pancreatic carcinoma cells support selectin-mediatedadhesion. A) Western blot of SW1990 cell lysate was stainedwith a HECA-452 mAb. Two major bands were observed at�200 and �460 kDa. B) Selectin-dependent adhesion toSDS-PAGE resolved SW1990 lysate under physiological flowconditions. CHO-E cells were perfused at the wall shear stresslevel of 0.5 dyn/cm2 over the HECA-452 stained Westernblots. Number of interacting cells per square millimeter wastabulated as a function of molecular weight to compile anadhesion histogram. Data represent means � se of n � 5experiments.


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inantly to the �460- and �200-kDa regions, whereasCHO-P cells failed to interact with either band.

We recently reported that sialofucosylated CD44variant isoforms, carcinoembryonic antigen, and podo-calyxin are functional selectin ligands on colon carci-noma cells (29–33). Because of their lower molecularmasses (�150–180 kDa), it is unlikely either of thesemolecules constitutes the HECA-452-reactive band ob-served in the �460-kDa region. To identify this novelselectin ligand, we purified the putative sialofucosylatedglycoprotein from SW1990 cell lysates by affinity chro-matography using protein G agarose beads coated withan anti-sLea mAb (Fig. 2). Eluted proteins were sepa-rated by SDS-PAGE and stained in gel with the ProQEmerald 300 stain, which fluorescently labels periodate-oxidized glycans while leaving the polypeptide back-bone intact (31, 32). The gel band corresponding tothe �460-kDa HECA-452-reactive protein was excisedand digested in gel with trypsin. Extracted peptideswere analyzed by HPLC-MS/MS (Fig. 2). Bioinformat-ics analysis identified peptide fragment matches forMUC16 (NP_078966.2).

A series of experiments was then carried out toconfirm the MS data. Western blot analysis using ananti-MUC16 mAb, X306, revealed the presence ofMUC16 in SW1990 cell lysates, and MUC16 was re-tained in the affinity chromatography eluate (Fig. 3A).Flow cytometry confirmed the expression of MUC16 onthe surface of SW1990 cells (Supplemental Fig. S1B). Inaddition, sLea and sLex epitopes on MUC16 weredisclosed by staining immunopurified MUC16 withHECA-452 mAb (Fig. 3A).

We next sought to confirm the ability of MUC16 tomediate selectin binding under physiological levels ofshear stress. To this end, CHO-E and CHO-P cells andL-selectin-expressing human peripheral blood lympho-cytes were perfused over the immunopurified MUC16band at a wall shear stress of 0.5 dyn/cm2. CHO-E cellsfirmly adhered to the MUC16 protein band (Fig. 3B);lymphocytes tethered transiently to immunopurifiedMUC16, which is in line with the nature of selectin-mediated binding, whereas negligible binding ofCHO-P cells was detected (Fig. 3B). The specificity ofthese adhesive interactions was confirmed by incubat-

ing CHO-E, CHO-P, or lymphocyte suspensions with ananti-E-, -P-, or -L-selectin function-blocking mAb, re-spectively (Fig. 3B). Moreover, CHO-E cells or lympho-cytes suspended in flow medium containing EDTA,which sequesters Ca2 (indispensable in the selectin-ligand interaction), failed to adhere to any region ofthe blot (data not shown). Taken together, the datasuggest that MUC16 possesses E- and L- but not P-selectin ligand activity. These observations are consis-tent with flow-based adhesion assays using intactSW1990 cells, which revealed appreciable binding to E-and L- but not P-selectin (Table 1).

To eliminate the presence of potential non-MUC16selectin ligands in the �460-kDa region (Fig. 1A),whole SW1990 cell lysate was depleted of MUC16 byrepeated IP and the immunodepleted fraction wasmeasured for selectin ligand activity in blot rollingassays (29). After 4 rounds of IP with anti-MUC16 mAb,

Figure 2. Identification of MUC16as the sialofucosylated glycopro-tein � 460 kDa expressed bySW1990 cells. 1) HECA-452-reac-tive molecules were purified fromSW1990 cell lysate by immunoaffin-ity chromatography using anti-sLea

mAb immobilized on recombinantprotein G agarose supports. HECA-452-reactive molecules were elutedwith low-pH elution buffer. 2) Sam-ples were separated by SDS-PAGEand stained in gel with ProQ Emer-ald 300 glycoprotein stain. Fluores-cently labeled band �460 kDa was

subsequently excised from the gel. 3) Proteins were extracted, trypsin-digested, and subjected to tandem MS. 4, 5) bioinformatics analysisof the MS data revealed the presence of MUC16 in the HECA-452-reactive band �460 kDa.

Figure 3. MUC16 carries sialofucosylated glycans and sup-ports selectin-mediated adhesion. A) Western blots of whole-cell lysate or IP MUC16 or sLea from SW1990 pancreaticcarcinoma cells. HECA-452 (lanes 1, 3, 5) or anti-MUC16(lanes 2, 4, 6) mAbs were used to stain Western blots of celllysate (lanes 1, 2), IP MUC16 (lanes 3, 4), and IP sLea (lanes5, 6) from the cells. B) Selectin-dependent adhesion toSDS-PAGE resolved immunopurified MUC16 from SW1990lysate. CHO-P cells, lymphocytes, or CHO-E cells were per-fused at the wall shear stress level of 0.5 dyn/cm2 overWestern blots of MUC16 immunopurified from SW1990lysate. For control experiments, CHO-P cells, lymphocytes, orCHO-E cells were pretreated with anti-P-, L-, or E-selectinfunction-blocking mAbs (20 �g/ml), respectively, before blotrolling assays. This concentration was maintained throughoutthe experiment. Data represent means � se of n � 3experiments. *P � 0.05 vs. untreated cells.


no MUC16 glycoprotein and HECA-452 reactivity weredetectable by Western blot analysis (Fig. 4A). In addi-tion, blot rolling assays revealed the number of inter-acting CHO-E cells over the �460-kDa region of theMUC16-depleted blot was essentially abolished (Fig.4B). As a control, no significant change was detected inthe extent of selectin-mediated adhesion to the �200-kDa region of the MUC16-depleted blot (Fig. 4B).Collectively, these observations suggest that MUC16 isthe main selectin ligand in the �460-kDa HECA-452-reactive species from SW1990 cell lysates. Of note,

other membrane-bound mucins often implicated incancer progression are either modestly expressed (e.g.,MUC1) by or absent (e.g., MUC4) from SW1990 cells(Supplemental Fig. S1C).

Selectin-binding determinants of MUC16 onpancreatic carcinoma cells are displayed onsialofucosylated O- and N-linked glycans

We next investigated the mechanism of interactionbetween MUC16 and selectins using a cell-free flow-based adhesion assay (30–33). Microspheres werecoated with MUC16 immunopurified from SW1990 andperfused over E-, L-, and P-selectin-coated substrates ata wall shear stress of 1 dyn/cm2. In agreement with blotrolling assays (Fig. 3B), a substantial number ofMUC16-coated microspheres bound to E- and L-selec-tin (Fig. 5A). The specificity of MUC16-selectin bindingin these assays was confirmed by the use of nonspecificIgG-bearing microspheres and by preincubating theselectin-coated dishes with the respective anti-selectinfunction-blocking mAb prior to perfusion of MUC16-coated microspheres (Fig. 5A). As an additional con-trol, MUC16-coated microspheres failed to adhere toany of 3 different selectin substrates in the presence ofEDTA (Fig. 5A).

To determine the location of the sialofucosylatedglycans responsible for selectin binding of MUC16,MUC16-coated spheres were treated with enzymes thatselectively cleave carbohydrate moieties and examinedfor their ability to interact with E- and L-selectin inshear flow. After treatment of MUC16-coated sphereswith sialidase, which cleaves the sialic acid residue onsLex and sLea, adhesion of MUC16-bearing micro-

Figure 4. Immunodepletion of MUC16 eliminates selectin-mediated binding �460 kDa. A) Western blots of SW1990lysate depleted of MUC16 by sequential IP. Anti-MUC16mAbs (X306 or X75) were used to immunodeplete MUC16from the cell lysate by 4 rounds of IP. Anti-MUC16 mAb andHECA-452 were used to stain the untreated lysate (ctrl) andsequentially depleted lysates (depletions 1–4). B) CHO-Ecells were perfused at the wall shear stress level of 0.5dyn/cm2 over 2 major HECA-452-reactive bands on SDS-PAGE Western blots of SW1990 cell lysate depleted of MUC16by sequential rounds of IP. Number of interacting cells persquare millimeter was tabulated as described in Fig. 1. Datarepresent means � se of n � 3 experiments. *P � 0.05;ANOVA.

Figure 5. MUC16-coated microspheres interact with immobilized selectins, and the binding determinants of MUC16 aresialofucosylated structures displayed on both O- and N-linked glycans. A) Extent of interaction of microspheres (106/ml) coatedwith nonspecific IgG or MUC16 purified from SW1990 cells with E-, L-, or P-selectin (20 �g/ml) at a wall shear stress level of1.0 dyn/cm2 for 2 min. In control experiments, MUC16-coated microspheres were incubated with anti-E-, L-, or P-selectinfunction-blocking mAbs (20 �g/ml) before use in flow assays. Nonspecific adhesion was assessed by adding EDTA (5 mM) tothe perfusion medium. B) Extent of interaction of MUC16-coated microspheres pretreated with V. cholera sialidase, PNGase F,or OSGE with E- or L-selectin at a wall shear stress level of 1.0 dyn/cm2. C) Extent of interaction of microspheres coated withMUC16 immunopurified from SW1990 cells cultured in DMJ or benzyl-GalNAc-containing medium with E- or L-selectin at a wallshear stress level of 1.0 dyn/cm2. Data represent means � se of n � 3–4 experiments. *P � 0.05 vs. control MUC16-coatedmicrospheres.


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spheres to E- and L-selectin was reduced by �90% (Fig.5B). The removal of sialic acid residues in sialidase-treated MUC16-coated microspheres was confirmed byflow cytometry whereas control MUC16-bearingspheres retained their HECA-452 reactivity (Supple-mental Fig. S2A). To assess the relative contributions ofN- vs. O-linked glycans on MUC16-selectin binding,MUC16-bearing spheres were treated with either PN-Gase F, which cleaves N-linked glycans from the proteinbackbone, or OSGE, which cleaves the polypeptidebackbone of heavily O-glycosylated glycoproteins, fol-lowed by flow-based adhesion assays, as described pre-viously (32, 35). PNGase F treatment reduced theHECA-452 reactivity of MUC16-coated spheres (Supple-mental Fig. S2A) and diminished the interaction be-tween MUC16-coated spheres and E- and L-selectin(Fig. 5B). OSGE treatment of MUC16 completely elim-inated sLex and sLea detection on the bead surface inflow cytometric analysis (Supplemental Fig. S2A), re-sulting in dramatic reduction in adhesion of MUC16-coated spheres to E- and L-selectin (Fig. 5B). Tovalidate these data, microspheres were coated withMUC16 immunopurified from SW1990 cells pretreatedwith metabolic inhibitors DMJ, an N-linked processinginhibitor, or benzyl-GalNAc, which interferes with O-glycosylation. The site density of MUC16 on micro-spheres from DMJ- or benzyl-GalNAc-treated SW1990cells was assessed to be similar to those of untreatedcontrols (data not shown). However, the expression ofsLex and sLea epitopes on MUC16 was partially reducedafter DMJ incubation or treatment with benzyl-GalNAc(Supplemental Fig. S2B). Microspheres coated withMUC16 from DMJ- or benzyl-GalNAc-derived lysatesdisplayed reduced interaction with E- or L-selectincompared with spheres treated with vehicle controllysates (Fig. 5C). To ensure the generality of ourbiochemical observations, MUC16 was also immunopu-rified from metastatic Pa03C pancreatic cancer cellsand used to coat microspheres. Pa03C MUC16-coatedmicrospheres displayed significant E- and L-selectin-binding activity in shear flow (Supplemental Fig. S3).These adhesive interactions were markedly reduced bysialidase treatment (Supplemental Fig. S3). Further-more, N-linked glycosidase PNGase F and O-linkedprocessing inhibitor benzyl-GalNAc attenuated thebinding of Pa03C MUC16-bearing microspheres to E-and L-selectin (Supplemental Fig. S3). Taken together,these data suggest that the selectin-binding determi-nants on MUC16 immunopurified from metastaticSW1990 and Pa03C pancreatic cancer cells are sialofu-cosylated epitopes displayed on both O- and N-linkedglycans.

MUC16 is a functional E- and L-selectin ligand onpancreatic carcinoma cells

To assess the functional role of MUC16 in the adhesionof pancreatic carcinoma cells to selectins under flowconditions, stable MUC16-KD cell lines were generatedby transfecting wild-type SW1990 cells with MUC16

shRNA plasmids. As shown in Fig. 6A, this procedureresulted in the generation of MUC16-KD cells withmarkedly reduced MUC16 surface expression (�92%decrease in mean fluorescence intensity) relative toSW1990 cells transfected with scramble control ormock-transfected cells. The specificity of this interac-tion was confirmed by flow cytometric analysis of CD29,for which expression levels remained unaltered (Fig.6A). MUC16 knockdown efficiency was confirmed byWestern blot (Fig. 6B). Probing MUC16-KD with aHECA-452 mAb showed a dramatic reduction in stain-ing intensity at the �460-kDa region (Fig. 6C, lane 2),whereas the HECA-452-reactive band at the �200-kDaregion remained unchanged between wild-type andMUC16-KD cells. When CHO-E cells were perfusedover the �460-kDa region of Western blots ofMUC16-KD cell lysate, a marked reduction in CHO-Ebinding was observed in comparison with binding toblots of wild-type cell lysate. In contrast, the interactingnumber of cells over the �200-kDa region remainedunaltered (Fig. 6D). Similar observations were madeusing L-selectin-expressing human peripheral lympho-cytes (data not shown).

To investigate whether MUC16 is a functional selec-tin ligand on pancreatic cancer cells, MUC16-KDSW1990 cells were perfused in flow-based adhesionassays. The results showed that SW1990 cells transfectedwith mammalian scramble control plasmids interactedwith E- and L-selectin substrates under flow at levelscomparable with those of wild-type controls (Fig. 6E, F).However, MUC16-KD cells displayed a markedly re-duced capacity to bind to E-selectin under a shear stressof 0.5 and 1.0 dyn/cm2 (�50% of control; Fig. 6E).Similarly, two distinct MUC16-KD clones exhibited�40% reduction in cell tethering to L-selectin rela-tive to appropriate controls under shear (Fig. 6F). Togeneralize these observations, MUC16 was alsoknocked down in Pa03C cells by shRNA. The knock-down efficiency was confirmed by flow cytometric andWestern blotting analysis (Fig. 7A, B). Immunoblotsstained with HECA-452 mAb revealed the absence of animmunoreactive band at the �460-kDa region inMUC16-KD Pa03C cell lysates (Fig. 7C). Blot rollingassays revealed a �90% decrease in selectin-dependentadhesion to the �460-kDa region of Western blots ofMUC16-KD cell lysate relative to that observed in thecorresponding band of the wild-type cell lysate (Fig.7D). Furthermore, a marked reduction in the numberof interactions between MUC16-KD Pa03C cells and E-or L-selectin was observed under flow (Fig. 7E, F).Altogether, these data provide clear evidence thatMUC16 is a functional E- and L-selectin ligand inpancreatic carcinoma cells.

DISCUSSION

CA125, an epitope expressed on the extracellular do-main of MUC16, is the focus of active research as aclinical marker for the diagnosis of epithelial cancers,


due to its selective up-regulation in tumor tissue (23,24). It is presently the only serum biomarker usedroutinely for the diagnosis and prognosis of ovarian

cancer (38). Recent work reveals that MUC16 is nearlyabsent from normal pancreatic ducts but is stronglyup-regulated in pancreatic cancer (25), suggesting a

Figure 6. Knockdown of MUC16 decreases E- and L-selectin-mediated interaction of SW1990 cells. A) Representative flowcytometric histograms of MUC16 and CD29 surface expression by wild-type, mammalian scramble control, and 2 clones ofMUC16-KD SW1990 cells. Cells were stained by indirect single-color immunofluorescence using the anti-MUC16 or CD29 mAbsand their isotype control antibodies. B) Western blot of whole-cell lysate from wild-type and MUC16-KD SW1990 cells usinganti-MUC16 or anti-�-actin antibody. C) Western blot of whole-cell lysate from wild-type and MUC16-KD SW1990 cells usingHECA-452 mAb. D) Selectin-dependent adhesion under physiological flow conditions to SDS-PAGE resolved wild-type andMUC16-KD SW1990 cell lysates. CHO-E cells were perfused at the wall shear stress level of 0.5 dyn/cm2 over the HECA-452stained Western blots. Number of interacting cells per square millimeter was tabulated as described in Fig. 1. E) Extent ofadhesion of wild-type, mammalian scramble control, and MUC16-KD SW1990 cell lines (106/ml) to E-selectin (20 �g/ml) underphysiological flow conditions at the wall shear stress levels of 0.5 and 1.0 dyn/cm2. F) Extent of tethering of wild-type,mammalian scramble control, and MUC16-KD SW1990 cell lines (106/ml) to L-selectin (20 �g/ml) under flow conditions atshear stress levels of 0.5 and 1.0 dyn/cm2. Data represent means � se of n � 3–5 experiments.*P � 0.05 vs. wild type.

Figure 7. MUC16 is a functional E- and L-selectin ligand on metastatic Pa03C pancreatic cancer cells. A) Representative flowcytometric histograms of MUC16 and CD29 surface expression by wild-type, mammalian scramble control, and 2 clones of MUC16-KDPa03C cells. Cells were stained by indirect single-color immunofluorescence using the anti-MUC16 or CD29 mAbs and their isotypecontrol antibodies. B) Western blot of whole-cell lysate from wild-type and MUC16-KD Pa03C cells using anti-MUC16 or anti-�-actinantibody. C) Western blot of whole-cell lysate from wild-type and MUC16-KD Pa03C cells using HECA-452 or anti-�-actin antibody.D) Selectin-dependent adhesion under flow conditions to SDS-PAGE resolved wild-type or MUC16-KD Pa03C cell lysates. CHO-E cellswere perfused at the wall shear stress level of 0.5 dyn/cm2 over the anti-MUC16 mAb-stained Western blots. E) Extent of adhesion ofwild-type and MUC16-KD Pa03C cell lines (106/ml) to E-selectin (20 �g/ml) at the wall shear stress levels of 0.5 dyn/cm2. F) Extentof tethering of wild-type and MUC16-KD Pa03C cell lines (106/ml) to L-selectin (20 �g/ml) at the wall shear stress level of 0.5dyn/cm2. Data represent means � se of n � 3 experiments. *P � 0.05 vs. wild type.


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role for MUC16 in the progression of this disease. Asfor a biological mechanism underlying this clinicalpresence, MUC16 has been implicated in differentadhesive processes. It has been shown that MUC16binds mesothelin, which is expressed on the surface ofperitoneal mesothelial cells, providing an adhesive ca-pacity for metastasizing carcinoma cells (26). In addi-tion, recent studies have reported that MUC16 may actin an immune-suppressing capacity by binding to galec-tin-1 and Siglec-9, both of which are expressed byhuman immune cells. This binding may mask MUC16-expressing tumor cells from immunosurveillance(39, 40).

While the adhesive properties of MUC16 have beenbroadly appreciated as regulators of tumor progression,the potential interaction between MUC16 and selectinsin the vascular system has been largely overlooked.Several lines of evidence reveal the role of selectins andtheir cognate ligands during metastasis (2, 8, 41, 42),both of which are important for a thorough under-standing of the metastatic process. Although differenttypes of tumor cells may express selectin-binding sialo-fucosylated glycans on distinct protein backbones, iden-tifying these specific ligands, especially those overex-pressed by tumor cells, is important for the design ofchemotherapeutics that target circulating and dissemi-nating tumor cells.

sLea antigen, also known as CA19–9, is the onlydiagnostic marker approved by the U.S. Food and DrugAdministration for pancreatic cancer (43), and itsexpression can mediate selectin-dependent carcinomacell adhesion to vascular endothelium during hematog-enous metastasis (12, 17). In addition, sialylated oligo-saccharides have been reported to be overexpressed onmucins from pancreatic tumors (44). The importanceof the mucin family in selectin adhesion has beenindirectly demonstrated, as treatment of cultured pan-creatic cancer cells with the mucin-rich sera of pancre-atic cancer patients reduced tumor cell binding toE-selectin (37). sLea was shown to be vital in thisinteraction through treatment of SW1990 cells withanti-sLea antibodies. This intervention resulted in thedramatic reduction of SW1990 binding to E-selectin-expressing endothelial cells and inhibition of livermetastasis (36). Nevertheless, the protein carriers ofthese sLea epitopes remained unidentified before ourstudy. Without this vital information, therapeuticsaimed at disrupting tumor cell-selectin binding may notbe possible, as targeting all sLex and sLea epitopeswould also disrupt leukocyte-selectin interactions (45),thereby compromising an otherwise healthy immunesystem. In support of this targeting strategy, severaltherapeutic antibodies or vaccines are designed totarget carcinoma mucins, such as MUC1, and tumormucin-associated oligosaccharides, such as O-linked gly-can sialyl Tn antigen, as a strategy for cancer treatment(46). With the use of immunoaffinity chromatographyand MS analysis, MUC16 was identified as the sialofu-cosylated glycoprotein with a molecular mass � 460

kDa and confirmed as a functional ligand of selectins in2 distinct metastatic pancreatic cancer cell lines.

MUC16 is heavily glycosylated with both O- andN-linked oligosaccharides (47); however, the biologicalsignificance of the glycans expressed on MUC16 is notfully understood. It has been shown that the binding ofMUC16 to galectin-1 depends on its �-galactose-termi-nated, O-linked glycans (39), whereas the MUC16-mesothelin interaction is dependent on N-linked oligo-saccharides (48). Treatment of pancreatic cancer cellswith glycosidases or carbohydrate metabolic inhibitorsdemonstrated that the sialofucosylated epitopes fromMUC16 are displayed on both O- and N-linked glycans.This finding is supported by prior MS data, whichsuggested the presence of sialyl Lewis epitopes on O- aswell as N-linked glycans on MUC16 (47). Taken to-gether, these data suggest that the ability of MUC16 tosupport selectin-mediated adhesion may help to confermetastatic potential to MUC16-expressing tumor cells.

It has been argued that distinctions must be madebetween ligands that can bind to selectins under staticcondition in vitro and functional ligands that interactwith selectins under dynamic condition in vivo (18).Consequently, the functional role of MUC16 in selec-tin-dependent adhesion warrants further exploration.We achieved this by stably silencing MUC16 expressionin pancreatic cancer cells, which bound to E-selectinwith markedly reduced capacity. These cells also dis-played a significantly decreased ability of transientlytethering to L-selectin. To our knowledge, this is thefirst characterization of the capacity for MUC16 tointeract with E- and L-selectin in a dynamic flowenvironment. It is noteworthy that depletion of MUC16from SW1990 or Pa03C pancreatic cancer cells resultedin a 40–60% inhibition of cell binding to E- andL-selectin in shear flow. Thus, other molecules may alsocontribute to the selectin-dependent adhesion of met-astatic pancreatic cancer cells. Immunostaining ofSW1990 (Fig. 1) or Pa03C (data not shown) cell lysateswith HECA-452 mAb reveals two distinct bands: onecorresponding to MUC16 at �460 kDa and another at�200 kDa. The latter band corresponds to podocalyxin(32, 42, 49) and is capable of supporting selectin-dependent adhesion (Fig. 4B). In addition, SW1990cells express MUC1 but at lower levels relative toMUC16 (Supplemental Fig. S1C). Thus, MUC1 mayalso contribute, albeit modestly, to selectin-dependentadhesion under dynamic flow conditions. Finally, wecannot exclude the possibility that glycolipids may playa role in E-selectin-dependent binding (50).

In summary, by employing biochemical/bioengi-neering approaches involving SDS-PAGE analysis, blotrolling, and flow-based adhesion assays, we demonstratethat MUC16 is a functional E- and L-selectin ligand onpancreatic carcinoma cells. Our findings offer a unify-ing perspective on the apparent enhanced metastaticpotential associated with the overexpression of MUC16and sialofucosylated structures on many types of tumorcells, including pancreatic carcinoma, and the criticalrole of selectins in metastatic spread (Table 1). Our


data enhance our understanding of this ubiquitousmolecule, and warrant further investigation into thedevelopment of novel therapies which target MUC16 tocombat metastasis.

The authors thank Drs. Robert Cole and Tatiana Boronina,who performed the peptide analysis and protein identifica-tion in the Mass Spectrometry and Proteomics Facility atJohns Hopkins School of Medicine with support from theInstitute for Cell Engineering. The authors also thank Dr.Anirban Maitra (Department of Pathology, Johns HopkinsSchool of Medicine) for the generous gift of the Pa03Cpancreatic carcinoma cell line. This work was supported bygrants from the U.S. National Cancer Institute (awards U54-CA143868 and RO1-CA101135).

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Received for publication September 7, 2011.Accepted for publication November 21, 2011.


Mucin 16 is a functional selectin ligand on pancreatic cancer cells

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