RESEARCH ARTICLE

O-glycosylation of MUC1 mucin in prostate cancerand the effects of its expression on tumor growthin a prostate cancer xenograft model

Pushpa Premaratne & Karin Welén & Jan-Erik Damber &

Gunnar C. Hansson & Malin Bäckström

Received: 9 July 2010 /Accepted: 15 September 2010# International Society of Oncology and BioMarkers (ISOBM) 2010

Abstract MUC1 mucin is up-regulated and aberrantlyglycosylated in many human epithelial carcinomas. Over-expression of MUC1 has also been implicated in prostatecancer, but neither the role of MUC1 in the cancerprogression nor the mucin O-glycosylation has been fullyelucidated. In this study, we characterized the O-glycans onMUC1 when over-expressed in the human prostate cancercell line C4-2B4. We found that the main O-glycanconsisted of the neutral core 2 oligosaccharide Galβ3(Galβ3/4GlcNAcβ6)GalNAc-ol, with minor componentsbeing fucosylated and sialylated variants of the same core 2oligosaccharide. Small amounts of the shorter core 1O-glycans were also detected.We then used the MUC1over-expressing cell lines to study the effects of MUC1 onprostate cancer cell behavior. The results demonstrate thatover-expression of MUC1 did not affect the cells’ prolif-eration, but led to a decreased adhesion to the extracellularmatrix components fibronectin and collagen I in vitro.When inoculated in BALB/c nude mice, C4-2B4 cellsexpressing MUC1 showed a tendency to form fewer tumorsthan wt cells and the tumors also grew more slowly, butthere was a large variation between different tumors. Thesefindings suggest that MUC1 may not have the same cancer-promoting effect in prostate cancer cells that is commonly

seen in other epithelial cancers such as breast, colon, andpancreatic cancer.

Keywords Mucin .MUC1 .O-glycosylation . Prostatecancer . Xenograft

Introduction

Prostate cancer is one of themost common cancers amongmenand also a common cause of cancer deaths in Western society[1]. It is a heterogeneous disease that displays a broad rangeof clinical behavior from tumors with very slow growth totumors that progress to more aggressive and metastatic statesthat no longer respond to hormone therapy. In these cases, thepatient survival is generally less than a year.

Mucins are large, highly O-glycosylated proteins thathave been implicated in the biologic behavior and progres-sion of several cancers, including those of the breast, colon,and pancreas [2, 3]. They are present at the surface of mostepithelial cells and are involved in the protection andlubrication of the epithelium [3]. The expression of mucingenes is organ- and cell-type-specific. The increasedexpression and altered glycosylation of mucins are associ-ated with the development of cancer and influence cellulargrowth, differentiation, transformation, adhesion, and im-mune surveillance [4]. This is especially true for MUC1, amembrane-bound mucin which is expressed on the apicalside of the epithelial cells of the pancreas, breast, lung,gastrointestinal, and reproductive tracts [5]. In carcinomacells, the polarized expression of MUC1 is often lost andassociated with high expression levels over the entire cellsurface [5, 6], and this is associated with a poor prognosisof breast cancer [7].

P. Premaratne :G. C. Hansson :M. Bäckström (*)Department of Medical Biochemistry and Cell Biology,University of Gothenburg,Box 440, 405 30 Gothenburg, Swedene-mail: malin.backstrom@medkem.gu.se

K. Welén : J.-E. DamberLundberg Laboratory for Cancer Research, Department ofUrology, Institute of Clinical Sciences, University of Gothenburg,Gothenburg, Sweden

Tumor Biol.DOI 10.1007/s13277-010-0114-9

The N-terminal ectodomain of MUC1 consists of avariable number of tandem repeats, with many serinesand threonines that serve as attachment sites forO-glycans [8, 9] and an SEA domain that is auto-catalytically cleaved, but holds the two MUC1 subunitsin a non-covalent association [10–12]. MUC1 also con-tains a trans-membrane domain and a cytoplasmic tail withseveral tyrosines that can be phosphorylated and that areinvolved in cell signaling [13].

In the prostate, MUC1 is expressed apically in theepithelial cells lining the lumen of the normal glands [14].Some studies indicate that MUC1 also may play a role inprogression and metastasis of prostate cancer, with alteredcell-surface expression patterns and aberrant glycosylation.However, the results are variable and often depend onwhich antibody was used to detect MUC1 [15–20]. Geneexpression profiling using DNA microarrays has shownthat MUC1 correlates with metastasis and disease recur-rence in prostate cancer [21]. One study, using a glycan-specific antibody, has suggested that increased sialylationof MUC1 O-glycans has a role in prostate cancerprogression [22]. To our knowledge, no structural informa-tion on MUC1 O-glycans in prostate cancer is howeveravailable. This study aimed to determine the structures ofthe O-glycans on MUC1 in a model prostate cancer cellline. We also investigated the effects of over-expression ofMUC1 in this cell line, both on the in vitro growth andadhesion and in vivo tumor formation.

Materials and methods

Cell culture

C4-2B4, an androgen-independent sub-line of the humanprostate cancer cell line LNCaP [23], was maintained inRPMI 1640 (Lonza, Verviers, Belgium) supplemented with10% fetal bovine serum (Lonza, Verviers, Belgium) (RPMI+FBS) and incubated at 37°C in 5% CO2. The hybridoma forthe monoclonal antibody (mAb) 214D4 against MUC1 waskindly provided by Dr. John Hilkens, The NetherlandsCancer Institute, Amsterdam and was cultured in a BDCL1000 flask with BD cell MAb medium (BD Biosciences,San José, CA). All cells were free from mycoplasma asdetected with MycoAlert (Lonza, Verviers, Belgium).

Recombinant plasmid

pcDNA3.1(+)MUC1FL, which expresses full-length MUC1with 32 tandem repeats from the CMV promoter, was a giftfrom Dr. J. Taylor-Papadimitriou, Cancer Research UK,London. The sequence of the non-tandem repeat regions ofMUC1 was identical to the MUC1 consensus sequence, as

confirmed by DNA sequencing (Eurofins MWG Operon,Ebersberg, Germany).

Antibodies

214D4 mAb against MUC1 was used as hybridomasupernatant or purified on a HiTrap protein G HP 5 mlcolumn (GE Healthcare, Uppsala, Sweden). Armenianhamster mAb CT2 against MUC1 cytoplasmic tail waskindly provided by Dr. Sandra Gendler, Mayo Clinic,Scottsdale, AZ and anti-actin mAb was from SigmaAldrich, St Louis, MO.

Transfection and selection of stable cell lines

C4-2B4 cells were seeded in six-well culture plates (Nunc,Roskilde, Denmark) and grown to 80% confluency, whentransfections were performed with Lipofectamine 2000(Invitrogen, Carlsbad, CA) according to manufacturer’sinstructions. After 72h, each well was passaged intoRPMI+FBS containing 400 μg/ml G418 (Invitrogen,Carlsbad, CA) for selection of stable transfectants. After7 to 10 days, cells were serially diluted, and single coloniesthat started to grow were picked and cultured individually.The cloning procedure was repeated once. The MUC1expression was determined by flow cytometry and verifiedby Western blot analysis. Four selected clones weremaintained in RPMI+FBS containing 400 μg/ml G418and used for the experiments.

Flow cytometry

Cells (1×105) were incubated with mAb 214D4 for 30 min.After washing with phosphate-buffered saline (PBS), thecells were incubated with APC-conjugated goat anti-mouseIg (BD Biosciences, San José, CA) for 30 min. The cellswere washed again and resuspended in PBS+2% bovineserum albumin (BSA) and 7AAD (BD Biosciences, SanJosé, CA) to exclude dead cells. All steps were carried outon ice. Cells were analyzed on a FACSCalibur (BDBiosciences, San José, CA) using CELLQUEST software(BD Biosciences, San José, CA). Cells were gated byforward and side light scatter and 7AAD staining toeliminate dead cells and debris.

Immunoblotting

Cells grown to 70% confluency were rinsed twice with ice-cold PBS and then lysed with lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl and 1% Triton X-100)supplemented with complete protease inhibitor cocktail(Roche Diagnostics, Mannheim, Germany). Lysed cellswere harvested and clarified by centrifugation at 16,000×g

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for 15 min at 4°C. Protein concentrations were measuredusing BCA protein assay (Thermo Scientific, Rockford, IL).

Protein (50 μg) were loaded in wells of 3–5% and 14%sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE). After separation, proteins were transferred toImmobilon-P membranes (Millipore, Bedford, MA) usingsemidry blotting in a Transblot SD (Bio-Rad) in 48 mMTris–HCl, 39 mM glycine, 1.3 mM SDS, and 10%methanol at 110 mA for 1 h. The membranes were blockedwith 5% dried non-fat milk in PBS+0.1% Tween 20 (PBS-T) and probed with 214D4 or CT2 in blocking buffer,washed with PBS-T, and then incubated with goat-anti-mouse Ig-alkaline phosphatase (AP) (Southern Biotech,Birmingham, AL) or goat-anti-Armenian hamster IgG-AP(Jackson ImmunoResearch, West Grove, PA). After washing,blots were developed with 5-bromo-4-chloro-3-indol-phosphate/nitro blue tetrazolium (Promega, Madison, WI).The bands in the developed blot were quantified using theImage J densitometry software (NIH, Bethesda, MD) andpresented in arbitrary density units.

O-glycan analysis by liquid chromatography-electrospraymass spectrometry

MUC1 was purified from cell lysates using an immunoaf-finity column with mAb 214D4. Purified mAb (1 mg) wascoupled to AminoLink Plus Resin as described in manu-facturer’s protocol (Thermo Scientific, Rockford, IL). Aftercoupling, ≤2 ml cell lysate of MUC1-expressing C4-2B4

clones were added to the column and incubated at roomtemperature for 2 h. After washing with PBS, boundproteins were eluted with 0.1 M glycine-HCl at pH 2.5.Fractions of 2 ml were collected and MUC1-containingfractions were pooled and concentrated using 30 kDVivaspin concentrators, (Sartorius, Gottingen, Germany).Before O-glycan release, MUC1 was separated in 3–5%SDS-PAGE and blotted onto Immobilon-P membranes(Millipore, Bedford, MA). MUC1-containing lanes weredivided by cutting, using one part of the lane to be stainedwith antibodies to localize MUC1. The remaining part ofthe lane was kept unstained for release of carbohydrates,from the membrane piece containing MUC1, by β-elimination using KOH/NaBH3, as previously described[24]. Purification of glycans were made as described bySchulz et al. [25]. O-glycan alditols were then analyzed bycapillary LC-MS and MS/MS using a LTQ linear quadro-pole ion trap mass spectrometer (Thermo Electron, SanJosé, CA), as previously described [24]. Three analyseswere performed for each MUC1-expressing clone.

The structure of the O-glycan alditols were elucidatedusing a combination of molecular mass, MS/MS fragmen-tation spectra, and LC elution times. Hexoses were alwaysinterpreted as galactose (Gal) and N-acetylhexosamines as

N-acetylglucosamine (GlcNAc), unless at the inner coreposition, where they were interpreted as N-acetylgalactos-amine (GalNAc). Deoxyhexoses were interpreted as fucose.

Cell proliferation assay

Cell proliferation was evaluated using Cell Titer 96 AQueous

One Solution Cell Proliferation Assay kit (Promega,Madison, WI). Briefly, cells were plated in four 96-welltissue culture plates (Nunc, Roskilde, Denmark) in tripli-cates. At 24 h intervals, assays were performed by adding20 μl per well of the Cell titer 96 AQueous One SolutionReagent, and the plate was further incubated for 1 h at 37°Cin a humidified, 5% CO2 atmosphere, and the absorbancewas read at 490 nm using a 96-well plate reader (Victor2,Perkin Elmer, Upplands Väsby, Sweden). When plotted, theseeded cell numbers were normalized to 5×103 cells/well,with the actual numbers ranging from 3.3×103 to 6.3×103

cells/well.

Adhesion assay

Cell culture 96 well-plates were coated with fibronectin(10 μg/ml) (Cohesion, Palo Alto, CA) or collagen I (10 μg/ml)(PureCol, Advanced Biomatrix, San Diego, CA) at 4°Covernight. Plates were blocked by incubating at 37°C for 2 hwith 1% BSA (Sigma Aldrich, St Louis, MO). Cells weresuspended in serum-free RPMImedium containing 0.1% BSAand 5 mM MgCl2 and plated in triplicate in fibronectin- orcollagen-coated plates (2×104 cells/well). After incubation at37°C, 5% CO2 for 2 h, wells were washed with PBS, whilethe input control wells were not washed. The cells were thenquantified by absorbance readings at 570 nm, after 15%crystal violet staining in 70% ethanol for 30 min. Thepercentage of attached cells was calculated by dividing theoptical density obtained with collagen and fibronectin withthat of the respective input control. The experiment wasrepeated three times.

Mouse experiments

Male athymic BALB/c nude mice were purchased fromCharles River Laboratories (Wilmington, MA). The micewere housed and maintained under specific pathogen-freeconditions and used for experiments when they were 10–12 weeks of age. The use of animals was approved by theanimal ethical committee in Gothenburg.

Wt and transfected C4-2B4 cells were harvested with0.25% (w/v) trypsin containing 0.04% (w/v) EDTA. Twomillion cells suspended in 150 μl of RPMI+FBS and150 μl Matrigel (BD Bioscience, Bedford, MA) wereinoculated subcutaneously in the flank of the mice. Tenmice were used for each clone. Tumor growth was

Tumor Biol.

evaluated every week, and the tumor diameter wasmeasured using a caliper. Mice were euthanized when thetumor volume reached 1,300 mm3. Tumors were excisedand one part was fixed in formalin for paraffin embedding,and the other part was snap-frozen in liquid nitrogen andstored at −80°C prior to preparation of lysates.

Immunohistochemistry

Tissue sections (4 μm) were deparaffinized in xylene andrehydrated with a graded series of ethanol. For antigenretrieval, the sections were heated in antigen unmaskingsolution (Vector Laboratories, Burlingame, CA). Endoge-nous peroxidases were blocked with 0.3% hydrogenperoxide (H2O2) in methanol for 30 min. Immunohisto-chemistry was performed using the Vectastain ABC kit(Vector Laboratories, Burlingame, CA) according to themanufacturer’s protocol. Briefly, unspecific binding wasblocked with normal horse serum and sections wereincubated with mAb 214D4-anti MUC1 overnight at 4°C.Detection was performed with biotinylated secondaryantibody followed by ABC reagent. The chromogenicreaction was visualized by incubating the slides with 3, 3-diaminobenzidine solution. The sections were counter-stained with hematoxylin and mounted. The primaryantibody was omitted as a negative control.

Preparation of tumor lysates

Tumor lysates were prepared by homogenizing the tumorsusing an Ultra-Turrax T8 (Ika-Werke, Staufen, Germany) inlysis buffer with protease inhibitors. Lysed tumor tissue wasthen centrifuged at 16,000×g for 10 min, and supernatantswere stored at −20°C until analysis.

Statistical analyses

Data are presented as mean±SEM. Kruskal–Wallis testfollowed by Mann–Whitney U test was used to comparedifferences between groups. Differences in tumor incidencebetween groups were analyzed with the Chi-square test.P values ≤0.05 were considered statistically significant.All statistical analyses were performed using SPSS forWindows, version 15.

Results

Exogenous expression of MUC1 in C4-2B4 cells

To investigate the effects of MUC1 on C4-2B4 prostatecancer cells, full-length MUC1 was exogenously expressedin C4-2B4 cells. This cell line is naturally deficient in

MUC1 and is therefore suitable for studying the effects ofover-expression of MUC1 in prostate cancer cells. In total,40 stable MUC1-expressing clones were generated byrepeated cloning. FACS analysis was used to determinethe MUC1 cell-surface expression in these clones, usingmAb 214D4 that recognizes an epitope in the tandem repeatin the extracellular domain of MUC1. From these results,four clones with good amounts of surface-expressed MUC1(C4-2B4/MUC1.1–4) were selected (Fig. 1a). To determinethe total amounts of MUC1 in these clones, immunoblot-ting was performed with both mAb 214D4 and mAb CT2,specific for an epitope in the cytoplasmic tail of MUC1.MUC1 were detected in lysates of C4-2B4/MUC1 clones,with the predicted size of >250 kDa band for theextracellular domain and multiple bands around 25 kDafor the processed cytoplasmic tail of MUC1. No MUC1was detected in wt or mock-transfected cells (Fig. 1b).Although FACS analyses detected similar surface expres-sion levels in all clones, different amounts of total MUC1were detected by Western blot, indicating a difference in theintracellular processing of MUC1 during biosynthesis in thedifferent clones (Fig. 1b, c). The higher levels of totalMUC1 in clones 1–2 indicate that there is more intracellularMUC1 in these clones, in contrast to clone 4 where almostall MUC1 is expressed on the surface (Fig. 1).

O-glycosylation of MUC1 from C4-2B4 prostate cancercells

The structure of the O-glycans on MUC1 expressed in C4-2B4 prostate cancer cells were determined by LC-ESI MS.The O-glycans were released from MUC1 that had beenimmunoaffinity-purified from cell lysates, using mAb214D4. A large proportion of the oligosaccharides releasedfrom MUC1 from C4-2B4 cells were core 2 structures, withthe main O-glycan being the non-sialylated and non-fucosylated oligosaccharide Galβ3(Galβ3/4GlcNAcβ6)GalNAc-ol (m/z 749). In addition, smaller peaks of thesame oligosaccharide but with the addition of fucose (m/z895), sialic acid (m/z 1040), or both (m/z 1186) were alsoidentified. A minor part of the oligosaccharides were core1 structures, with some NeuAcα3Galβ3GalNAc-ol (m/z675) and NeuAcα3Galβ3(NeuAcα6)GalNAc-ol (m/z966) being detected. Figure 2 shows a typical LC-ESIchromatogram for O-glycans released from C4-2B4/MUC1.3, but analyses from the other clones gave similarresults (data not shown). A similar O-glycosylation patternwas also found on MUC1 over-expressed in LNCaP cells(data not shown).

The similar pattern of oligosaccharides in the fourMUC1-expressing clones indicated that the expression ofthe glycosylating enzymes in the Golgi had not beengrossly affected by the transfections.

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Effects of MUC1 on in vitro cell proliferation

To determine the effect of MUC1 over-expression on thegrowth of C4-2B4 cells, we first assessed the in vitroproliferation of MUC1-expressing clones. All C4-2B4/MUC1 clones, with the exception of clone MUC1.1 whichgrew slightly slower, behaved as wt and mock-transfectedcells, and all grew at a similar rate (Fig. 3a).

Effect of MUC1 on in vitro cell adhesion

Next, we examined the effect of MUC1 over-expression oncell adhesion to fibronectin and collagen I, two definedcomponents of extracellular matrix (ECM). It was foundthat over-expression of MUC1 decreased the adhesion ofC4-2B4 prostate tumor cells to both collagen and fibronectinin vitro, compared to cells that did not express MUC1(Fig. 3b) (P≤0.05).

In tissue culture, we also noted that MUC1 over-expressing cells were much more readily detached relativeto wild-type and mock-transfected cells. While C4-2B4 wtcells assumed an elongated morphology, MUC1-expressingcells exhibited a rounded shape, especially in clone 2 and 4(data not shown).

�Fig. 1 Expression of MUC1 in transfected C4-2B4 clones. a Flowcytometric analysis of MUC1-expressing cells stained with mAb214D4 (solid lines) or C4-2B4 wt cells stained with mAb 214D4(dashed lines) or wt cells stained with only goat anti-mouse Ig-APC(dotted line, upper left panel). b Western blot of cell lysates from C4-2B4 wt, C4-2B4 mock-transfected cells, C4-2B4/MUC1.1, C4-2B4/MUC1.2, C4-2B4/MUC1.3, C4-2B4/MUC1.4 were subjected toimmunoblotting with two anti-MUC1 mAbs: mAb 214D4 (upperpanel) or CT2 (middle panel). Anti-actin (lower panel) antibody wasused as a control to confirm equal loading of protein for all samples. cDensitometric scanning of MUC1 staining in CT2 immunoblots oflysates of the different C4-2B4/MUC1 clones. The band intensities forthree individual determinations were quantified using the Image Jsoftware and plotted as mean±SEM

Fig. 2 A representative base peak LC-ESI MS chromatogram of O-glycans released from MUC1 from C4-2B4/MUC1.3. Each peakrepresents one O-glycan alditol, the structure of which was elucidatedby a combination of molecular mass, MS/MS spectra (data notshown), and elution time. Gal galactose, GalNAc N-acetylgalactos-amine, GlcNAc N-acetylglucosamine, Fuc fucose, NeuAc N-acetyl-neuraminic acid

Tumor Biol.

Effect of MUC1 on xenograft tumor growth in vivo

To determine how the over-expression of MUC1 affectedthe tumorigenesis of the cells in vivo, two C4-2B4/MUC1clones, mock-transfected, and wt cells were injectedsubcutaneously into nude mice. All (10/10) of the micethat received C4-2B4 wt cells and 9 of the 10 mice injectedwith C4-2B4 mock-transfected cells developed xenograft

tumors (Fig. 4a), with no difference in tumor volumebetween these two groups (Fig. 4b). However, miceinjected with the two MUC1-expressing clones respondedvery differently both in terms of tumor incidence andgrowth. The tumor incidence was 9/10 for C4-2B4/MUC1.1cells, and the tumors showed a similar growth rate as in thecontrol groups (Fig. 4a, b). In contrast, C4-2B4/MUC1.2gave a tumor incidence of only 3/10 (where one tumor wasvery small) (Fig. 4a), and the tumors grew more slowlyrelative to control tumors (Fig. 4b). Since the results of thisfirst round of animal experiments were inconclusive, thestudy was extended with the other two MUC1-expressingclones. This time, the number of tumors was slightly lowerin both groups receiving MUC1-expressing cells comparedto the control group, with the tumor incidence being 7/10for both MUC1-expressing clones at the end of theexperiment (Fig. 4c). Moreover, mice implanted with C4-2B4/MUC1.4 formed significantly smaller tumors relativeto the mice in the control group, while tumors developingfrom C4-2B4/MUC1.3 cells behaved quite similar to thecontrol cells, but with a slightly slower growth in thebeginning of the experiment (Fig. 4d). There was noobvious difference in the histological appearance betweenthe tumors formed in the different groups.

Taken together, these data suggest that MUC1 over-expression has a negative influence on the xenograft tumorincidence in three out of the four clones tested (clonesMUC1.2–4), but only one of the clones (C4-2B4/MUC1.2)showed a statistically significant decrease in the number oftumors formed (p=0.02; Chi-square test). There was,however, a delay in the time until the tumors wereestablished in these three clones (Fig. 4a, c), and theaverage growth rates also appeared lower in these tumors(Fig. 4b, d).

Expression and localization of MUC1 in xenograft tumors

To confirm that the cells expressed MUC1 also at the end ofthe in vivo experiment, and to compare the intensity andlocalization of MUC1 in the different tumors, immunohisto-chemistry staining was performed. MUC1 was detected intumors from all C4-2B4/MUC1-expressing clones (Fig. 5a–d). All stained tumor sections were positive for MUC1 witha predominant localization in the apical membrane in themajority of tumors. In two out of seven tumors from cloneC4-2B4/MUC1.3 and four out of seven tumors from cloneC4-2B4/MUC1.4, a more diffuse, cytoplasmic localizationof MUC1 was also found (Fig. 5c, d). MUC1-expressionwas undetectable in C4-2B4 wt and mock-transfected tumors(Fig. 5e, f).

To quantify MUC1 in the tumors, tumor lysates wereanalyzed by Western blot with mAb CT2 against the MUC1cytoplasmic tail. A representative blot with one control

Fig. 3 In vitro characteristics of C4-2B4 cells expressing MUC1. aCell proliferation. Triplicates containing 5×103 cells of C4-2B4/MUC1.1–4, C4-2B4/pcDNA3.1, and C4-2B4 wt were evaluated forproliferation at 24 h intervals. Values are expressed as means±SEM ofthree individual experiments with triplicates. b Cell adhesion to theECM components fibronectin (upper panel) and collagen I (lowerpanel). Values are means±SEM of three individual experiments withtriplicates (*P≤0.05)

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tumor and four tumors from C4-2B4/MUC1 is shown inFig. 6a, and the staining intensities of MUC1 from alltumors are shown in Fig. 6b. In clone C4-2B4/MUC1.1, theexpression levels of MUC1 in the tumors were considerablylower than those found in the cells before inoculation intothe mice (Fig. 6b compared to Fig. 1c). This reduction wasnot seen in the other clones. Negative control tumors didnot show any detectable MUC1 expression.

It is obvious that the expression level of MUC1 in thetumors showed a large variation among tumors bothbetween the groups but also within each group. The highestexpression levels were found in the few tumors thatdeveloped from C4-2B4/MUC1.2. These cells alsoexpressed high levels of MUC1 before implantation(Fig. 1) and gave rise to few and more slowly growingtumors (Fig. 4).

Discussion

In this study, we investigated the effects of MUC1expression in a prostate cancer cell line, both in vitro andin vivo. MUC1 is up-regulated and has a changedglycosylation in several cancers, but how MUC1 effectscancer development in the prostate is less studied. More-over, the structure of the O-glycans on MUC1 in prostatecancer is to a large extent unknown. To elucidate this, we

stably expressed MUC1 in the prostate cancer cell line C4-2B4 and characterized the cells in vitro for MUC1O-glycosylation, cell proliferation, and cell adhesion andalso in vivo for the ability of these cells to form tumors innude mice. C4-2B4 is a sub-line of the prostate cancer cellline LNCaP that lacks endogenous MUC1 [26]. Therefore,these cells represent a good model to study the contributionof MUC1 over-expression in prostate cancer progression.

We report one of the first mass spectrometric structuraldeterminations of O-glycans of MUC1 in prostate cancer.Our LC-ESI MS methodology is very sensitive, needingvery low amounts of stating materials [24]. It also givesconsistent, reproducible results and is a highly competitivemethod when compared to other glycoanalytical techniquesavailable [27]. We released and analyzed the O-glycansfrom MUC1 that had been purified from cell lysates usingmAb 214D4 that recognizes a peptide epitope in the MUC1tandem repeats. This antibody was used because it isexpected to pick up a majority of the glycoforms of MUC1,as it is less sensitive to glycosylation than other antibodies[28]. The major O-glycan on MUC1 expressed in C4-2B4

cells was the non-sialylated and non-fucosylated core 2oligosaccharide Galβ3(Galβ3/4GlcNAcβ6)GalNAc-ol (m/z749), with minor peaks of the same oligosaccharide with theaddition of either fucose (m/z 895), sialic acid (m/z 1040), orboth (m/z 1186). This glycosylation pattern is different tothat typically found in breast cancer, where cancer cells have

Fig. 4 Subcutaneuos xenografttumor incidence and growth.Cells (2×106) from the differentclones were injected into nudemice at day 0 (n=10 in eachgroup). The upper panels repre-sent the first experiment and thelower panels the second experi-ment. a, c Tumor establishmentof C4-2B4 wt, C4-2B4/pCDNA3.1, and MUC1-expressing C4-2B4 clones.Results are expressed as thenumber of animals with a pal-pable tumor at different timesafter cell implantation. b, dTumor volumes at each timepoint. Data points representmean±SEM including onlythe animals with palpabletumors (n)

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a higher content of shorter core 1 O-glycans, of which wefound only small amounts in this study (NeuAcα3Galβ3-GalNAc-ol (m/z 675) and NeuAcα3Galβ3(NeuAcα6)GalNAc-ol (m/z 966)). This raises the possibility that thesame switch, from a predominance of core 2 in the normalmammary epithelium to core 1 in breast cancer, is not foundin prostate cancer. This is supported by our earlier analysis ofanother prostate cancer cell line, DU-145, which also had amajority of core 2 O-glycans on its endogenous MUC1,although with a higher degree of sialylation [24]. It would bevaluable to do the same type of chemical glycan analysisalso on prostate tumor tissue from patients, an analysiswhich has so far not been reported. Neither has theO-glycosylation of MUC1 in the normal prostate beendetermined. The complexity of prostate cancer and the factthat the needle biopsies that are often used contain a mixtureof tumor and normal material will, however, complicate suchanalysis. Arai et al. [22] have found the presence of the core1 O-glycan NeuAcα3Galβ3GalNAc-Thr in prostate cancer,

by using an antibody that detects this glycan epitope onMUC1, and this epitope correlated to progression of diseaseas well as to progression-free survival. This O-glycan was,however, only present in small amounts in the two prostatecancer cell lines that we have analyzed here or in an earlierstudy [24].

By gene expression analysis, MUC1 was associated withmore aggressive cases of prostate cancers and elevated riskof recurrence [21]. As MUC1 is a complex glycoprotein, itis likely that its glycosylation will also affect the impactMUC1 will have on tumor development. Often, antibodystaining has been used to detect MUC1 and the many anti-MUC1 mAbs available will react differently with MUC1depending on the carbohydrates added to the polypeptidechain. Therefore, different studies may give seeminglyconflicting results because different anti-MUC1 antibodieswere used. In prostate cancer, some studies which usedantibodies to under-glycosylated MUC1 show increasedlevels of MUC1 in more advanced stages [22, 29], whereas

Fig. 5 Immunohistochemicalstaining of MUC1 expressionin xenograft tumors. Subcellulardistribution of MUC1 recog-nized by mAb 214D4 in tumorsections ranged from mostlyapical in a C4-2B4/MUC1.1 andb C4-2B4/MUC1.2, to bothapical and diffuse cytoplasmicin c C4-2B4/MUC1.3 and d C4-2B4/MUC1.4. Wild-type andmock-transfected C4-2B4 werenegative (e and f). Magnifica-tion, ×200 (a, b, e, and f)and ×400 (c and d)

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other studies that used antibodies detecting MUC1 withmore extensive glycosylation indeed report lower MUC1levels in more advanced stages [26, 30]. A high degree ofsialylation of glycans is often seen in cancer cells, and thiswill enhance tumor growth [31] and may also impairimmune responses against tumor cells. Here, we foundmainly non-sialylated core 2 O-glycans on MUC1 and it ispossible that the lack of sialylation has led to a reducedtumor formation and growth in this study, as compared toother more sialylated cancers.

We found that over-expression of MUC1 altered thebinding properties of tumor cells to known extracellularligands for tumor cells, as adhesion to collagen I andfibronectin were significantly decreased in MUC1 expressingcells compared to mock-transfected cells. MUC1 over-expression is known to decrease cell–cell interactions [32–34]and cell–substratum interactions [35]. As MUC1 has a longmucin domain that extends quite far from the cell membrane,it has been proposed that the anti-adhesive function ismediated by general steric hindrance [36]. Indeed, thetransfection of MUC1 cDNA into a human melanoma cellline reduced integrin-mediated adhesion to fibronectin,laminin, and collagen type I and IV and cadherin-mediatedadhesion to other cells [33, 36]. These features suggest thatincreased MUC1-expression can cause an inhibition of cell–

ECM adhesion, and this could contribute to the delay ofestablishment of tumors in vivo, which is a notable finding ofthis study. This is, on the other hand, in contrast to earlierstudies, where the presence of MUC1 has led to increasedtumor incidence and growth [4, 37]. However, these studieshave largely been made in breast cancer cells and tumors, andthe proposed mechanisms involve signaling activities of theMUC1 cytoplasmic tail [13, 38]. Our study is one of the firstto address the role of MUC1 in prostate cancer, a cell typethat could well have other intracellular signaling pathwaysand thus other outcomes of MUC1 expression. However, wealso observed that our MUC1-expressing clones had differentprocessing of MUC1, leading to different amounts of thecytoplasmic tail in the cells, something we could not relate tothe growth properties of the different clones.

We also saw a large variation in the development oftumors also within the groups receiving cells of the sameclone. Such a variation is probably due to clonal selectionof tumor cells in the animal, a well-known phenomenon incancer progression once the cells have lost control of theirgenomes [39]. We observed in some cases that thereseemed to be a delay before the tumor was established,with only a slow growth in the beginning before the tumorstarted to grow faster and became well established.Although it has not been possible to analyze the MUC1expression at these critical moments, the fact that thereseems to be less MUC1 in the fastest growing tumors(clone 1.1) might suggest that cells with a lower expressionof MUC1 had an advantage and grew faster. This may alsoexplain why there was a reduction of MUC1 expression inclone 1.1 in the tumors compared to what was found in thecells before inoculation into the mice. It is possible thatsome cells in this clone had reduced levels of MUC1, andthey were selected in the tumors because they grew faster,leading to a tumor with less MUC1 than what was found inthe original cell clone. A growth advantage of a sub-population of the tumor cells probably caused the change ingrowth behavior. As a majority of the clones in our studyformed tumors that grew more slowly than the cells notexpressing MUC1, and as the clone with the highest levelsof MUC1 was the only one leading to significantly lowernumbers of tumors, we can conclude that in our system andmaybe in prostate cells in general, the over-expression ofMUC1 does not seem to be of any growth advantage. Thelower tumorigenicity of MUC1 in prostate cancer comparedto other cell types can have several different reasons, asMUC1 is a protein with many functions. It may involve theextracellular domain of MUC1 with its extensive glycosyl-ation that can act as a shield for the immune system if it isnot being recognized as foreign, but may also involve thecytoplasmic tail which is known to interact with severalproteins involved in tumorigenesis, including β-catenin.The outcome of these interactions will then be dependent

Fig. 6 Quantification of MUC1 in xenograft tumors by Westernblotting using mAb CT2 against the cytoplasmic tail. a Western blotof tumor lysates from one wt and four C4-2B4/MUC1 tumors. bDensitometric scanning of MUC1 staining in CT2 immunoblots oflysates of all available tumors. The band intensities were quantifiedusing the Image J software and plotted as mean±SEM

Tumor Biol.

on the repertoire of these proteins in the host cell, whichmay be different in prostate cancer cells.

In conclusion, the over-expression of MUC1 in C4-2B4

prostate cancer cells gave a reduced adhesion to ECMcomponents and also showed a tendency of cells to giverise to fewer xenograft tumors that appeared later and grewmore slowly than tumors from wt cells. Indeed, none of theMUC1-expressing cells gave rise to tumors with enhancedgrowth compared to wt cells. Even though the experimentswere only performed in one prostate cancer cell line (with asingle glycosylation pattern) and even though the effectswere not seen in all clones, it indicates that MUC1 may nothave the same tumor-promoting effects in prostate cancer ashas previously been described in breast, colon, andpancreatic cancer.

Acknowledgements The skilful technical assistance by Anita Fae isgreatly acknowledged. We thank Dr D. Baeckström for advice aboutthe adhesion assay and the Mammalian Protein Expression CoreFacility at the University of Gothenburg for the hybridoma cellculture.

This work was supported by the Sahlgrenska University Hospital(LUA-ALF) and the foundations of Wilhelm and Martina Lundgren,Assar Gabrielsson, Magn Bergvall, Serena Ehrenström and Torstenand Sara Jansson to M.B. and also by the Swedish Research Council(no. 7461, 21027, and 342-2004-4434), The Swedish CancerFoundation, The Knut and Alice Wallenberg Foundation(KAW2007.0118), IngaBritt and Arne Lundberg Foundation, Torstenoch Ragnar Söderbergs Stiftelser, and The Swedish Foundation forStrategic Research—The Mucosal Immunobiology and VaccineCenter (MIVAC) and the Innate Immunity Program (2010–2014) toG.H.

Conflict of interest The authors declare that they have no conflict ofinterest.

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