Ebfi

EMD

a

ARRA

K�GIPMM

I

pmNmIea

istot

taP

SP

0h

European Journal of Cell Biology 92 (2013) 355–362

Contents lists available at ScienceDirect

European Journal of Cell Biology

jo ur nal homepage: www.elsev ier .co m/locate /e jcb

xpression of integrins �3�1 and �5�1 and GlcNAc �1,6 glycanranching influences metastatic melanoma cell migration onbronectin

wa Pochec ∗, Marcelina Janik, Dorota Hoja-Łukowicz, Paweł Link-Lenczowski1,ałgorzata Przybyło, Anna Litynska

epartment of Glycoconjugate Biochemistry, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Krakow, Poland

r t i c l e i n f o

rticle history:eceived 11 July 2013eceived in revised form 23 August 2013ccepted 23 October 2013

eywords:

a b s t r a c t

Acquisition of metastatic potential is accompanied by changes in cell surface N-glycosylation. One of thebest-studied changes is increased expression of N-acetylglucosaminyltransferase V enzyme (GnT-V) andits products, �1,6-branched N-linked oligosaccharides, observed in the tumorigenesis of many cancers.In this study we demonstrate that during the transition from the vertical growth phase (VGP) (WM793cell line) to the metastatic stage (WM1205Lu line), �1,6 glycosylation of melanoma cell surface pro-

1-6 branchingnT-V

ntegrinsHA-Ligration

teins increases as a consequence of elevated expression of the GnT-V-encoding Mgat-5 gene. Treatmentwith swainsonine led to reduced cell motility on fibronectin in both cell lines; the effect was strongerin metastatic cells, probably due to the higher content of GlcNAc �1,6-branched glycans on the mainfibronectin receptors – integrins �5�1 and �3�1. Our results show that GlcNAc �1,6 N-glycosylation ofcell surface receptors, which increases with the aggressiveness of melanoma cells, is an important factor

ll mig

elanoma influencing melanoma ce

ntroduction

N-glycosylation regulates a wide range of physiological andathological processes. The major sugar components of the cellembrane are complex oligosaccharides, among them GlcNAc �1,6-glycans, which play a special role during malignant transfor-ation and carcinoma cell metastasis (Lau and Dennis, 2008).

nterestingly, they are also important in physiological processes,specially in T-cell maturation and activation (Dennis et al., 2009)s well as in the host immune response.

N-acetylglucosaminyltransferase V (GnT-V) is an enzymenvolved in complex-type N-glycan branching on common coretructure; it catalyzes a �1,6 linkage between core Man and GlcNAc,

he first monosaccharide in the antenna of tri- and tetraantennaryligosaccharides. Increased GnT-V expression on mRNA and pro-ein levels, and its increased activity, correlates positively with

Abbreviations: ECM, extracellular matrix; EMT, epithelial-to-mesenchymalransition; FN, fibronectin; GlcNAc, N-acetylglucosamine; GnT-V, � N-cetylglucosaminyltransferase V; LF, lectin fluorescence; Man, mannose; PHA-L,haseolus vulgaris agglutinin; SW, swainsonine; VGP, vertical growth phase.∗ Corresponding author. Tel.: +48 012 664 6467; fax: +480126645101.

E-mail address: ewa.pochec@uj.edu.pl (E. Pochec).1 Present address: Department of Medical Physiology, Faculty of Health Sciences,

chool of Medicine, Jagiellonian University, Michalowskiego 12, 31-126 Krakow,oland.

171-9335/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.ejcb.2013.10.007

ration.© 2013 Elsevier GmbH. All rights reserved.

the metastatic potential of various cancers (Dennis et al., 1999;Murata et al., 2000). The functional importance of this enzyme issupported by many studies of inhibition of complex-type N-glycansynthesis by swainsonine or by GnT-V-encoding gene (Mgat-5)knockouts or silencing, which all demonstrate that the lack ofGnT-V enzyme products alters cell adhesion and migration abili-ties. Mgat-5 knockout mice showed considerably slower mammarytumor growth induced by the Polyomavirus middle T oncogene anddecreased metastasis to the lungs (Granovsky et al., 2000). Swain-sonine (SW) is a plant alkaloid which inhibits Golgi �-mannosidaseII and thus redirects oligosaccharide synthesis towards hybrid andhigh-mannose-type N-glycans instead of complex structures. Theinhibitory effect of SW on the invasiveness of different cancer cellshas been demonstrated in a number of studies (Guo et al., 2002;Litynska et al., 2006; Pochec et al., 2006).

Altered N-glycosylation frequently affects protein functionvia changes in protein structure and the dynamics of ligandbinding. Emerging lines of evidence suggest that acquisition ofa metastatic phenotype is connected with enhanced expressionof GlcNAc �1,6 complex-type N-glycans on cell surface receptors(Ochwat et al., 2004; Pochec et al., 2003; Przybyło et al., 2007)Cancer cells invasion is dependent on loss of intercellular adhesion

and transmigration of cells through the basement membraneand the surrounding extracellular matrix (Rambaruth and Dwek,2011). Cell–ECM interactions are mediated by a large group ofheavily glycosylated adhesion receptors called integrins. Changes

3 al of C

ippnttuwtsorc

M

C

mE

PM(scaIpSfa(aL(alBc

C

w1avP

cs(fsfia

R

gRP2

56 E. Pochec et al. / European Journ

n integrin expression and in glycosylation patterns open newossibilities for cancer cells to interact with a variety of cellartners, enabling them to migrate efficiently and invade newiches. Heavy glycosylation of integrin subunits resulting fromhe presence of several potential N-glycosylation sites is believedo strongly affect integrin interactions with extracellular ligands,ltimately influencing cell behavior. The interaction of cancer cellsith fibronectin (FN) via integrins is especially important during

he transition from the vertical growth phase (VGP) to metastatictage, a key moment in melanoma progression. The main goalf the present study was to shed light on the role of fibronectineceptor glycosylation and its effect on migration of melanomaells during VGP-to-metastatic transition.

aterials and methods

ell lines and reagents

Early VGP WM793 and highly metastatic WM1205Lu humanelanoma cell lines were obtained through participation in the

STDAB project.Mouse monoclonal anti-GnT-V antibody was kindly supplied by

rof. Eiji Miyoshi and Prof. Naoyuki Taniguchi (Graduate School ofedicine, Osaka University, Japan). Primary antibodies against �3

AB1920), �5 (AB1928) and �1 (MAB2251, clone B3B11) integrinubunits were obtained from Millipore (Chemicon), rabbit poly-lonal anti-GAPDH (G9545) antibody from Sigma–Aldrich. Horsenti-mouse (7076S) and goat anti-rabbit (7074S) HRP-conjugatedgGs were purchased from Cell Signaling Technology, alkalinehosphatase-conjugated goat anti-mouse IgG (A1682) were fromigma–Aldrich and AP-conjugated sheep anti-rabbit IgG (AP322A)rom Millipore (Chemicon). Antibodies used in the functionalssays – mouse anti-�3 (CP11L, clone P1B5) and mouse anti-�5�1CBL497, clone NKI-SAM-1) – were purchased from Calbiochemnd Chemicon respectively. Alexa Fluor-conjugated lectin PHA-

(L-11270) was obtained from Invitrogen, biotinylated PHA-LB-1115) and agarose-bound PHA-L (AL-1113) from Vector Lab.,nd HRP-conjugated ExtrAvidin (E2886) from Sigma–Aldrich. Phal-oidin from Amanita phalloides labeled with tetramethylrhodamine

isothiocyanate (TRITC, P1951) and fibronectin (F2006) were pur-hased from Sigma–Aldrich.

ell culture conditions and cell lysate preparation

The cells were routinely maintained in RPMI1640 mediumith GlutaMAX (Invitrogen) supplemented with 10% FBS (Gibco),

00 U/ml penicillin and 100 �g/ml streptomycin (Sigma–Aldrich)t 37 ◦C in a CO2 incubator (Lab Line Instruments). Cells were har-ested upon 80% confluency using 0.25% trypsin and 0.02% EDTA inBS (Sigma–Aldrich).

The cells were lysed in RIPA buffer (Thermo Scientific, 89900)ontaining 25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1%odium deoxycholate, 0,1% SDS and protease inhibitor cocktailSigma–Aldrich, P2714). The mixture was centrifuged (18,000 rpm)or 30 min at 4 ◦C. Protein content was determined in the collectedupernatant using Total Protein Kit, Micro Lowry, Peterson’s Modi-cation (Sigma–Aldrich, TP0300). Aliquots of cell lysate were storedt −70 ◦C.

T-PCR for Mgat-5 expression

Total RNA was extracted using RNeasy Protect Mini Kit (Qia-

en) and reverse transcription was carried out using the OmniscriptT kit (Qiagen) according to the manufacturer’s instructions.CR was performed using Taq PCR Master Mix (Qiagen) in PTC-00 thermocycler (MJ Research). Each reaction consisted of 35

ell Biology 92 (2013) 355–362

cycles of denaturation (1 min at 94 ◦C), primer hybridization (1 minat 59.8 ◦C for GnT-V and 59.0 ◦C for GAPDH) and elongation(2 min at 72 ◦C). The reaction began from an initial denaturation(3 min at 94 ◦C) and the final step was the elongation (10 minat 72 ◦C). GAPDH was used as a housekeeping gene. Primersequences were as follows: 5′-GTGGATAGCTTCTGGAAGAA-3′

(GnT-V forward), 5′-CAGTCTTTGCAGAGAGCC-3′ (GnT-V reverse),5′-CCACCCATGGCAAATTCCATGGCA-3′ (GAPDH forward), and 5′-TCTAGACGGCAGGTCAGGTCCACC-3′ (GAPDH reverse).

The PCR amplification products were resolved on 2% agarose gelcontaining ethidium bromide and were visualized in a transillumi-nator (UVItec). Amplification product length was 857 bp for GnT-Vand 599 pz for GAPDH.

PHA-L precipitation

Cell lysate proteins (250 �g) in HEPES buffer (10 mM HEPES, pH7.5 containing 150 mM NaCl, 0.1 mM CaCl2 and 0.01 mM MnCl2)were added to 25 �l agarose-bound PHA-L lectin and incubatedovernight at 4 ◦C. After centrifugation (15,000 rpm, 2 min, 4 ◦C)the supernatants from lectin-treated lysates were collected andstored at −70 ◦C. The precipitates were washed three times inHEPES buffer and once in PBS, and glycoproteins were released fromthe glycoprotein–lectin–agarose complexes by boiling in Laemmlisample buffer (LSB, Bio-Rad, 161-0737) at 100 ◦C for 10 min. Aftercentrifugation the supernatants were collected and destined forintegrin subunit immunodetection.

Western blotting

GnT-V enzyme and integrin subunit expression was analyzed byWestern blotting. Equal amounts of proteins in LSB (for GnT-V, 5%�-mercaptoethanol was added) were separated on 10% SDS-PAGEgel, transferred to a PVDF membrane, and detected using the appro-priate antibodies. To detect GnT-V, after blocking in 1% BSA in TBST,membranes were incubated for 1 h with primary mouse antibodydiluted 1:2000 in 1%BSA in TBST at room temperature (RT). Rabbitanti-GAPDH IgG was diluted 1:4000 and incubated with a mem-brane in the same conditions. Then horse anti-mouse IgG and goatanti-rabbit IgG were applied in a 1:4000 dilution and incubationwas done at RT for 1 h. GnT-V was visualized by chemiluminescenceusing Western blotting luminol chemiluminescence reagent (SantaCruz Biotech.) in the ChemiDocTM XRS System (Bio-Rad). Visualiza-tion of integrin subunits was done by incubating the membraneswith BCIP and NBT substrates for AP (Roche).

Lectin blotting

The lectin reaction was done as described previously by Litynskaet al. (2001), with minor modifications. Briefly, the PVDF sheetswere incubated with 0.5% blocking reagent (Boehringer Mannheim)in TBST overnight at 4 ◦C. The membranes were probed withbiotinylated PHA-L lectin (1:4000) for 1 h at RT. The reaction withPHA-L blocked by 100 mM acetic acid was the control of lectinspecificity. Then the membranes were incubated with streptavidin-conjugated HRP (1:4000) for 1 h at RT. Glycoconjugate-bound lectinwas detected by chemiluminescence.

Lectin fluorescence confocal microscopy

The cells were seeded on coverglasses placed on the bottomof 4-well plates (Thermo Scientific Nunc) 24 h before staining. On

the day of staining, the cells were washed three times with PBS,fixed in 2% PFA in PBS for 20 min at RT, and incubated with AlexaFluor 488-conjugated PHA-L (1:100 in 2% BSA in PBS) overnight atRT. The slides were mounted upside down with Vectashield with

al of Cell Biology 92 (2013) 355–362 357

DpZ

F

aw4b

W

Bwiagama

fifihsZhm

S

mv

R

E

imtessltrm

tNnWWcm(

qt

Fig. 1. WM1205Lu metastatic melanoma cells show higher expression of GnT-Von the mRNA (A) and protein (B) levels, and higher expression of its products –complex-type N-glycans with �1,6 antennae (C) – than primary WM793 melanomacells do. (A) Fluorescence of 2% agarose gel-resolved, ethidium bromide-stained PCRproducts of the GnT-V target sequence and GAPDH (endogenous control). (B) Proteinsamples (20 �g) were electrophoresed on 10% SDS-polyacrylamide gel, electroblot-ted onto a PVDF membrane and incubated with primary mouse anti-GnT-V (1:2000)and rabbit anti-GAPDH (1:4000). After incubation with secondary HRP-conjugatedantibodies the reaction was visualized by chemiluminescence. (C) Protein extracts(10 �g) were SDS-PAGE separated on 10% gel, electroblotted onto a PVDF mem-brane and probed with biotin-conjugated PHA-L (1:4000). After incubation with

E. Pochec et al. / European Journ

API (Vector, H-1200) and the coverslips were sealed with nailolish and kept at 4 ◦C. Cell fluorescence was visualized using aeiss confocal microscope and analyzed in LSM Image Browser.

low cytometric analysis

The cells were harvested from cell culture by trypsynizationnd washed in cold PBS. All the steps were performed on ice. Cellsere incubated with Alexa Fluor 488-conjugated PHA-L (1:100) for

5 min, then centrifuged (1100 rpm, 5 min) and washed with PBSefore flow cytometric analysis (FACSCalibur, BD Biosciences).

ound-healing assay

The cells were seeded onto 6-well plate precoated with FN (BDiosciences). After reaching confluency, three straight scratchesere made in each well with a pipette tip. Migrating cells were

ncubated 24 h in the presence of swainsonine or the antibodiesgainst �3 and �5�1 integrins. The wound sites were photo-raphed in 10 random fields using Zeiss AxioVision Rel.4.8 imagenalysis software. The average extent of wound closure was deter-ined by measuring the width of the wound at the starting point

nd after 24 h.The procedure for the short wound-healing assays on

bronectin was similar to the 24 h assay. Slides were coated withbronectin by air-drying for ca. 2 h at room temperature. Threeours after wound-scratching the cells were fixed with 4% PFA,tained with TRITC-conjugated phalloidin and analyzed under aeiss LSM 510 META confocal microscope. The classical wound-ealing method was modified in this way in order to measure theigration of individual cells at the leading edge of the scratch.

tatistical analyses

The results are represented as means of three measure-ent ± SD. Duncan’s test was used to compare mean values. P

alues < 0.05 were considered significant.

esults

xpression of GnT-V and its product

The sugar-chain structures of surface glycoproteins are alteredn tumor cells. The changes in glycans may be related to abnormal

RNA expression of glycosyltransferases catalyzing synthesis ofhese oligosaccharides. To assess the expression of the Mgat-5 genencoding GnT-V enzyme, mRNA from both melanoma cell lines wasubjected to semiquantitative RT-PCR analysis. The amount of tran-cript significantly differed between the two melanoma cell lines,ower in WM793 than in WM1205Lu cells (Fig. 1A). Western blot-ing performed to detect GnT-V expression on the protein levelevealed that production of this enzyme was proportional to theRNA level (Fig. 1B).GnT-V enzyme products were determined by Western blot-

ing followed by reaction with a biotinylated lectin. Complex-type-glycans containing the GlcNAc �1,6 Man �1,6 group are recog-ized by PHA-L, an isolectin isolated from Phaseolus vulgaris. TheM1205Lu cells showed more PHA-L-positive glycoproteins inestern blot analysis (Fig. 1C), confirmed by PHA-L staining in flow

ytometry (Fig. 2A). Moreover, the WB glycoprotein bands frometastatic cell material showed a stronger reaction with the lectin

Fig. 1C).Swainsonine (SW) inhibits Golgi �-mannosidase II and conse-

uently obstructs the carbohydrate processing pathway prior tohe initiation of GlcNAc �1,6 branching. Treatment of cells with

streptavidin-HRP (1:4000) PHA-L-positive glycoproteins were visualized by chemi-luminescence. Arrow indicates nonspecific band as determined by the reaction withPHA-L blocked 2 h by 100 mM acetic acid.

SW results in the accumulation of high-mannose and hybrid-type structures (Moremen, 2002). We confirmed the action ofthis alkaloid in the analyzed melanoma cells by staining mem-brane glycoproteins with fluorescence-labeled PHA-L in confocalmicroscopy and flow cytometry. We observed a significant reduc-tion of fluorescence after SW treatment of both cell lines (Fig. 2A,B).

GlcNAc ˇ1,6-branched N-glycans promote melanoma cellmigration

Changes in N-glycosylation frequently affect cell behavior. It isknown from earlier studies that altered glycosylation may con-tribute to changes in cancer cells adhesion, migration and invasion.As shown in ESTDAB studies, WM793 and WM1205Lu cells differsignificantly in their ability to adhere to laminin, collagen IV andfibronectin (the European Searchable Tumour Cell-Line Database;IPD-ESTDAB: http://www.ebi.ac.uk/ipd/estdab/). To test migrationability we chose FN because the two melanoma lines differ com-pletely in their adhesion to this ECM protein, and a number ofstudies have made it clear that FN plays important roles in manyaspects of cancer progression (Allen and Jones, 2011; Kaplan et al.,2005; Labat-Robert, 2002; Yamada, 2000).

We examined cell migration in cell culture wound-healingassays. Both cell lines showed markedly impaired migration in thepresence of SW, but the delay in migration into the open spacewas greater for metastatic (46.8% of control cells rate) than for

non-metastatic cells (17.6% of control cells rate) (Fig. 3). Surpris-ingly, however, generally the primary cells migrated much moreefficiently than metastatic ones. We suggest that this is becauseindividual primary cells occupy a much larger area on the culture

358 E. Pochec et al. / European Journal of C

Fig. 2. The action of swainsonine leads to loss of PHA-L binding to the melanoma cellsurface. (A) SW-treated and control cells collected by trypsinization were stainedwith Alexa Fluor 488-labeled PHA-L (1:100) and fluorescence was analyzed witha FACSCalibur flow cytometer. (B) Some cells growing on FN-covered slides weretreated with SW (10 �g/ml RPMI medium supplemented with 10% FBS). After 24 h,SW-treated and control cells were fixed with 2% PFA and incubated with AlexaFluor 488-labeled PHA-L (1:100). The slides were mounted with Vectashield con-taining DAPI and fluorescence was analyzed with a Zeiss LSM 510 META confocalm

do

imacosBtmifl(tt

from the same patient. We chose the genetically related WM793

icroscope.

ish than metastatic cells do (Fig. 2B), especially after space ispened up by scratching.

To verify the results we used actin cytoskeleton staining, apply-ng phalloidin after a short period of wound repair to identify

orphological traits of migrating cells. It is well established thatctin polymerization and depolymerization play a crucial role inell movement. Changes in the actin cytoskeleton are hallmarksf migrating cells; one of them is the formation of protrusivetructures at the leading edge (Huttenlocher and Horwitz, 2011;ravo-Cordero et al., 2012). In our work the characteristic pro-rusions and extensions were visible at the leading edge of the

etastatic cells three hours after wounding, and were not observedn non-metastatic WM793 cells. In SW-treated primary cells onlyat cell edges were observed in the short wound-healing assay

Fig. 4). Actin staining of cells shortly after wounding confirmedhat the faster wound-closing by WM793 cells after 24 h was dueo the large area the primary cells occupied in the scratch, not to

ell Biology 92 (2013) 355–362

faster migration. That set of experiments also confirmed the effectof SW on cell migration.

Melanoma cell integrins ˛3ˇ1 and ˛5ˇ1 are substrates for GnT-V

Lectin precipitation showed the presence of integrin subunits(Fig. 5) among the PHA-L-positive proteins disclosed in lectin blot-ting (Fig. 1C). Densitometric analysis of WB revealed differencesbetween the two cell lines in the amount of GlcNAc �1,6 glycans onintegrin subunits �3 and �5. Subunit �3 possessed more structuresrecognized by PHA-L in WM1205Lu cells than in non-metastaticcells (Fig. 5), a result confirmed by immunodetection of this subunitin SW-treated and untreated cells (Fig. 6). Based on PHA-L precip-itation, the complex-type N-glycans on subunit �5 seemed more�1,6-branched in primary melanoma cells (Fig. 5), but immunode-tection in SW-treated and untreated cells indicated higher contentof these structures in WM1205Lu (Fig. 6). We estimated that theloss of �5 molecular weight after culture in the presence of SWwas greater in WM1205Lu cells (11.7% of glycoprotein MW) thanin WM793 (8.3%) (Fig. 6). Between the two melanoma cell linesthere were no statistically significant differences in PHA-L bindingto �1 integrin chains (Fig. 5), nor in glycoprotein MW loss after SWtreatment (Fig. 6).

Integrins ˛3ˇ1 and ˛5ˇ1 are necessary for metastatic melanomamigration

In order to study the participation of �5�1 and �3�1 integrinsin melanoma cell migration we performed 24-hour wound-healingassays in the presence of specific monoclonal antibodies blockingthese integrins. We used monoclonal antibodies against �3 sub-unit and �5�1 integrin complex. We referred the results obtainedwith anti-�3 IgG to the whole �3�1 integrin complex, because the�3 subunit forms a heterodimer only with the �1 integrin chain(Gu et al., 2009). We observed a quantifiable slowing of the migra-tion rate in metastatic melanoma cells treated with anti-�3 andanti-�5�1 antibodies; in contrast, the migration of melanoma pri-mary cells was not changed (Fig. 3). WM1205Lu melanoma cellmotility was inhibited by 23.6% and 51.1% versus the control inthe presence of antibodies against �3 and �5�1 integrins respec-tively. The higher content of GlcNAc �1,6 complex-type glycans onsubunits �3 and �5 of metastatic melanoma cells and their reducedmotility in the presence of SW offer strong support for the contri-bution these structures make to the interaction of both integrinswith fibronectin, which promotes WM1205Lu migration.

Discussion

Much research in recent years has been devoted to cancer-related N-glycan alterations and their possible consequences forcell behavior. Some structural changes of N-glycosylation areincreasingly well documented and seem to be present in the major-ity of cancers. Increased expression of GlcNAc �1,6 structures hasbeen reported in melanoma cells, especially those derived fromthe final stage of melanoma development (Ochwat et al., 2004;Pochec et al., 2003; Przybyło et al., 2007). Skin melanoma goesthrough a series of well-defined stages of neoplastic progression,facilitating study of the molecular and physiological changes dur-ing melanoma development (Li et al., 2002). In previous works onglycosylation in melanoma we used cell lines derived from tumorsof different patients (Litynska et al., 2001, 2006; Pochec et al.,2003; Przybyło et al., 2007). In the present study both lines came

and WM1205Lu melanoma cell lines, which together offer a goodmodel for tracking changes during melanoma progression. WM793originated from early VGP lesions, and its WM1205Lu metastatic

E. Pochec et al. / European Journal of Cell Biology 92 (2013) 355–362 359

Fig. 3. Differences in inhibition of melanoma cell migration on FN in the presence of swainsonine (SW) or antibodies against integrin �3 and �5. Cells were grown onF ) ando xpres

cs

taaomwt

mpstim2obtareWaetits

N-coated plates and scratched with pipette tips. Each wound was photographed (Af ten points along each scratch at the starting point and after 24 h (B). Results are e

ounterpart was obtained from lung metastases after several pas-ages in mice (Cornil et al., 1991).

Here we demonstrated that Mgat-5 expression and GnT-V pro-ein synthesis increase with melanoma cells aggressiveness (Fig. 1And B, respectively). The level of GnT-V enzyme in melanoma cellslso corresponds to the amount of its products – GlcNAc �1,6ligosaccharides. Using WB and FC methods we showed that bothelanoma cell lines profusely express complex-type structuresith �1,6 antennae (Fig. 2) and that their expression increases with

he cells’ acquisition of a metastatic phenotype (Fig. 1C).In research on cancer progression and especially on tumor cell

igration and invasion it is important to determine which surfaceroteins gain these structures (Meany and Chan, 2011). In previoustudies, several groups including our team have demonstratedhat among the substrates for GnT-V enzyme are transmembranentegrin receptors responsible for interaction with extracellular

atrix proteins (Guo et al., 2002; Ochwat et al., 2004; Pochec et al.,003; Przybyło et al., 2007, 2008; Zhao et al., 2006). Modificationf integrin conformation by sugar component influences cellinding to ECM ligands. Our current results provide evidence thathe carriers of these structures in WM793 and WM1205Lu cellsre integrins �5�1 and �3�1 (Figs. 5 and 6), which are adhesioneceptors for FN (Gu et al., 2009; Pochec et al., 2003, 2006; Zhengt al., 1994). Integrins are heavily glycosylated surface proteins.ithin the polypeptide sequences of �3, �5 and �1 subunits there

re 14, 14 and 12 potential N-glycosylation sites respectively (Gut al., 2012; Janik et al., 2010a,b). Lectin precipitation showed

hat each analyzed integrin subunit is GlcNAc �1,6 glycosylatedn both melanoma cell lines (Fig. 5). That is not surprising sincehe WM793 and WM1205Lu lines were derived from the last twotages of melanoma progression. There were differences in content

the rate of migration into the wound area was calculated based on measurementssed as means ± SD of three independent wounds, p < 0.05.

of PHA-L-positive structures, however; WM1205Lu expressedmore GlcNAc �1,6 N-glycans on �3 subunit than WM793 cells did(Figs. 5 and 6). This means that �1,6-branching of GlcNAc increasesas the melanoma cells acquire the ability to metastasize. Gil et al.(2011) reported an increase of �3�1 integrin expression duringthe epithelial-to-mesenchymal transition (EMT); to model thisprocess they used WM793 and WM1205Lu cells. That raises thequestion of whether the increase of GlcNAc �1,6 glycosylation weobserved was the result of the higher expression of the �3 subunitin the metastatic variant? The answer is found in the observationof lower glycoprotein molecular weight in the presence of SW,confirming higher GlcNAc �1,6-branched glycan content in the�3 subunit of metastatic cells (Fig. 6). We also observed increased�1,6 branching of subunit �3 glycans in earlier work; MALDI MSanalysis indicated that subunit �3 oligoaccharides from A375metastatic melanoma cells were more GlcNAc-branched thanthose from RGP-derived WM35 cells (Pochec et al., 2003).

In this study we also observed differences in �5 GlcNAc �1,6glycosylation between WM793 and WM1205Lu cells. PHA-L pre-cipitation gave a more intense protein band for primary melanomacells (Fig. 5), but a comparison of glycoprotein MW loss after SWtreatment indicated that the content of complex-type structures ishigher in metastatic cells (Fig. 6). In turn, flow cytometric analy-sis of �5 expression showed a higher amount of it in WM793 cells(data not shown), which may explain the results from PHA-L pre-cipitation (Fig. 5). In our model it appears that the reduction ofsubunit �5 expression is accompanied by increased �1,6 branch-

ing of the glycans of this integrin during the transitions from VGPto metastatic phase. Earlier studies described reduction or loss of�5�1 integrin expression in transformed cells and tumors (Felding-Habermann, 2003), and integrin suppression of the transformed

360 E. Pochec et al. / European Journal of Cell Biology 92 (2013) 355–362

Fig. 4. Inhibition of GlcNAc �1,6 branched N-glycan synthesis by swainsonine (SW) impairs protrusion formation in WM1205Lu but not in WM793 melanoma cells duringw ed wT cope.

p�wdfmuAuim

lmtSgacaLwoc(2ŁO�sw

ound closure on FN. Confluent cells growing on FN-coated coverslips were scratchRITC-labeled phalloidin and analyzed with a Zeiss LSM 510 META confocal micros

henotype of Chinese hamster ovary (CHO) cells under increased5�1 expression (Giancotti and Ruoslahti, 1990). In previous worke also found that expression of integrin �5 �1,6 oligosaccharidesepended on the stage of melanoma progression. The �5 subunitrom A375 (Ochwat et al., 2004), WM9 and WM239 metastatic

elanoma cell lines was GlcNAc �1,6 glycosylated, but the �5 sub-nit from WM35 primary melanoma was not (Przybyło et al., 2007).nother group reported that �1,6 branching is restricted to sub-nit �1 within �5�1 integrin complex, but that was shown only

n HT1080 human fibrosarcoma cells (Guo et al., 2002) and not inelanoma cells.Alteration of cell-surface glycoproteins has profound physio-

ogical effects (Fuster and Esko, 2005). The contribution of glycanodification to cell-cell and cell-ECM contacts is no less impor-

ant than that of protein-protein interactions (Litynska et al., 2006;aravanan et al., 2009). Many studies have shown that extensivelycosylation of extracellular integrin domains influences cell–cellnd cell–ECM interactions and triggers intracellular signaling cas-ades leading to changes in protein expression, cell proliferationnd migration (Gu and Taniguchi, 2008; Gu et al., 2012; Link-enczowski and Litynska, 2011). Effects of complex-type glycansith �1,6 branching on cell migration and invasion have been

bserved in many cancers, for example in HT1080 human fibrosar-oma cells (Guo et al., 2002), H7721 human hepatocarcinomaZhang et al., 2004), human hepatocellular carcinoma (Wei et al.,012), bladder cancer (Pochec et al., 2006) and melanoma (Hoja-ukowicz et al., 2013; Litynska et al., 2006; Przybyło et al., 2007).

ur present findings confirm the biological importance of GlcNAc1,6 glycans in human melanoma cell migration on FN. Swain-onine proved useful in determining the role of these glycans inound-healing assays: this alkaloid effectively inhibited GlcNAc

ith pipette tips. After 3 h the cells were fixed with 4% PFA; F-actin was stained withThe arrow indicates the formation of protrusion.

�1,6 glycan synthesis in both melanoma cell lines (Fig. 2). In thepresence of SW, both cell lines repaired wounds on FN-coveredplates more slowly than untreated cells did, but the inhibition per-centage was higher for metastatic ones (Fig. 3). Hoja-Łukowicz et al.(2013) observed an inhibitory effect of SW on the migration ofthese melanoma cells, but only on uncoated plate surfaces. Impor-tantly, inhibition of migration in both SW-treated melanoma cellswas significantly lower on uncoated surfaces than on FN (WM793:8.2% on plastic vs. 17,6% on FN; WM1205Lu: 14.6% on plastic vs.46,8% on FN). This suggests that GlcNAc �1,6 complex-type gly-cans are critical to melanoma cells’ interaction with FN and thattheir involvement increases with the stage of progression. In pre-vious work we also observed significant reduction of migration onFN in scratch wound assays and toward FN in the Boyden tran-swell migration assays in A375 metastatic cells treated with SW,and not in primary melanoma cells (Litynska et al., 2006). All theseresults confirm the importance of N-glycans with �1,6 antennae inmelanoma cell migration on fibronectin.

In an effort to determine whether integrins with �3 and �5 sub-units participate in melanoma cell migration, we used monoclonalantibodies in wound repair assay. Both antibodies significantlyimpaired metastatic cell motility but did not influence primary cellmigration on FN (Fig. 3). In WM793 primary cells, higher expres-sion of �5�1 integrin (data not shown) promotes stronger adhesionto FN (ESTBAD project data; http://www.ebi.ac.uk/ipd/estdab/)and, as we showed in scratch wound assay, this integrin is notengaged in primary melanoma migration (Fig. 3). On the other

hand, WM1205Lu cells with lower expression of �5�1 showed sig-nificantly weaker adhesion to FN, but as found in wound healingassays (Fig. 3) this integrin definitely is involved in the migrationof metastatic cells on FN. The �5�1 integrin had a greater effect on

E. Pochec et al. / European Journal of Cell Biology 92 (2013) 355–362 361

Fig. 5. Integrin subunits �3 and �5 differ in �1,6 glycan branching. Whole-celllysate proteins (250 �g) were precipitated with 25 �l agarose-bound PHA-L. Thecaptured glycoproteins were recovered by boiling in Laemmli sample buffer. PHA-Lprecipitates (P), cell homogenates (H) and supernatants (S) collected after precip-itation were resolved by 10% SDS-PAGE followed by Western blotting and probedwith specific anti-integrin chain antibodies: rabbit polyclonal anti-�3 (Chemicon,AB1920), rabbit polyclonal anti-�5 (Chemicon AB1928) and mouse monoclonalavm

W��pittebcsiMltd

tFtWuom

Fig. 6. Swainsonine (SW) treatment shows that complex-type N-glycan content inintegrin subunits �3 and �5 is higher in WM1205Lu metastatic cells. Cells treatedfor 24 h with SW and untreated cells were lysed in RIPA buffer. Protein extracts(20 �g) were SDS-PAGE separated on 10% gel, electroblotted onto a PVDF membraneand probed with specific primary antibodies: rabbit polyclonal anti-�3 (Chemicon,AB1920), rabbit polyclonal anti-�5 (Chemicon, AB1928) and mouse monoclonalanti-�1 (Chemicon, MAB2251, clone B3B11). Antibody-bound proteins were visu-

nti-�1 (Chemicon, MAB2251, clone B3B11). PHA-L-positive integrin subunits wereisualized by AP colorimetric reaction (A) and the glycoprotein band intensities wereeasured densitometrically (B). LP – lectin precipitation.

M1205Lu migration (ca. 50% inhibition of wound closure) than3 integrin (ca. 30% inhibition). This is understandable because5�1 integrin is the main fibronectin receptor (Gu et al., 2009);robably during acquisition of metastatic ability the role of this

ntegrin changes from participation in strong adhesion in early VGPo engagement in metastatic melanoma migration, perhaps dueo changes in �1,6 branching of this glycan (Figs. 5 and 6). Theffect of both integrins on cancer cell migration has been notedy other groups. CHO cells stably transfected with �5�1 integrinDNA showed impaired ability to migrate and form colonies inoft agar (Giancotti and Ruoslahti, 1990). �3�1 integrin partic-pates in human melanoma cell migration on laminin and also

atrigel invasion (Melchiori et al., 1995). Interactions of �3�1 withaminin-5 are important in melanoma (Tsuji et al., 2002) and gas-ric carcinoma (Saito et al., 2010) cell migration, invasion and ECMegradation.

Integrin function is modified by the glycans present in its struc-ure. The significant effect of �5�1 on WM1205Lu migration onN seems connected with the GlcNAc �1,6 glycosylation status ofhis integrin. The difference in GlcNAc �1,6 glycan content between

M1205Lu and WM793 cells was greater in the �5 integrin sub-nit (3%) than in �3 (1.5%) (Fig. 6), and despite the lower expressionf �5�1 in metastatic cells its contribution to WM1205Lu celligration was more significant (Fig. 3). Altered glycosylation does

alized by colorimetric AP reaction (A) and the loss of MW after SW treatment wascalculated based on the position of the integrin bands in relation to protein markers(B). M – protein markers.

change integrin function; in this case the more �1,6-branched�5�1 strongly influenced metastatic migration on FN. Akiyamaet al. (1989) and Zheng et al. (1994) demonstrated the importanceof glycans present on �5�1 integrin in interaction with fibronectin.The role of N-glycans in �5�1-mediated cell adhesion and motilityis well documented in many tumors. Elevated �1,6 branching haddirect effects on �5�1-dependant HT1080 human fibrosarcoma celladhesion to FN, migration on FN and invasion through Matrigel(Guo et al., 2002). In mammary tumor cells, Lagana et al. (2006)showed the participation of Mgat-5 and branched N-glycans in�5�1-dependent cell spreading and motility. Cancer-related alter-nations in the glycosylation status of �5�1 integrin have a directeffect on cancer cell dissemination on FN (Rambaruth and Dwek,2011).

�1,6 glycosylation of �3�1 integrin plays an essential role in itsfunction. In our present study, GlcNAc �1,6 branched glycans and�3�1 integrin mediated WM1205Lu cell migration on FN (Fig. 3).Previous results has also shown the modifying effect of �1,6 gly-

cans on the interaction of �3�1 with its extracellular ligands. Janiket al. (2010a,b) concluded that glycosylation of �3�1 influences itsbinding to vitronectin in a uPAR-dependent manner. Transfecionof human gastric cancer cells with GnT-V cDNA strongly enhanced

3 al of C

�2

icwcit

A

EiCU2GJa(

A

t

R

A

A

B

C

D

D

F

F

G

G

G

G

G

G

G

H

H

J

62 E. Pochec et al. / European Journ

3�1 integrin-mediated cell migration on laminin-5 (Zhao et al.,006).

It is clear by now that both of the analyzed integrins are involvedn the migration of WM1205Lu metastatic cells on FN and that gly-ans with �1,6 branching contribute to melanoma cells’ interactionith this ECM ligand. The increasing amount of GlcNAc �1,6 gly-

ans on �5�1 and �3�1 integrins in metastatic cells plays a rolen integrin-dependent migration on fibronectin and probably con-ributes to their acquisition of metastatic ability.

cknowledgments

We thank Prof. Barbara Bilinska (Head of the Department ofndocrinology, Institute of Zoology, Jagiellonian University) for giv-ng us access to the Department’s Chemi Doc XRS system. Theonfocal Microscopy Laboratory (Institute of Zoology, Jagiellonianniversity) kindly gave us access to its LSM 510 META Axiovert00M, ConfoCor 3 confocal microscope (Carl Zeiss MicroImagingmbH, Jena, Germany). We also wish to acknowledge Michael

acobs for line-editing the manuscript. This work was supported by grant from the Polish Ministry of Science and Higher EducationGrant No. N N301 0322 34).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.ejcb.2013.10.007.

eferences

kiyama, S.K., Yamada, S.S., Yamada, K.M., 1989. Analysis of the role of glycosylationof the human fibronectin receptor. J. Biol. Chem. 264, 18011–18018.

llen, M., Jones, L.J., 2011. Jekyll and Hyde: the role of the microenvironment on theprogression of cancer. J. Pathol. 223, 162–176.

ravo-Cordero, J.J., Hodgson, L., Condeelis, J., 2012. Directed cell invasion and migra-tion during metastasis. Curr. Opin. Cell Biol. 24, 277–283.

ornil, I., Theodorescu, D., Man, S., Herlyn, M., Jambrosic, J., Kerbel, R.S., 1991. Fibro-blast cell interactions with human melanoma cells affect tumor cell growth asa function of tumor progression. Cell Biol. 88, 6028–6032.

ennis, J.W., Granovsky, M., Warren, C.E., 1999. Glycoprotein glycosylation and can-cer progression. Biochim. Biophys. Acta 1473, 21–34.

ennis, J.W., Lau, K.S., Demetriou, M., Nabi, I.R., 2009. Adaptive regulation at the cellsurface by N-glycosylation. Traffic 10, 1569–1578.

elding-Habermann, B., 2003. Integrin adhesion receptors in tumor metastasis. Clin.Exp. Metastasis 20, 203–213.

uster, M.M., Esko, J.D., 2005. The sweet and sour of cancer: glycans as novel thera-peutic targets. Nature 5, 526–542.

iancotti, F.G., Ruoslahti, E., 1990. Elevated levels of the �5�1 fibronectin receptorsuppress the transformed phenotype of Chinese hamster ovary cells. Cell 60,849–859.

il, D., Ciołczyk-Wierzbicka, D., Dulinska-Litewka, J., Zwawa, K., McCubrey, J.A., Lai-dler, P., 2011. The mechanism of contribution of integrin linked kinase (ILK) toepithelial-mesenchymal transition (EMT). Adv. Enzyme Regul. 51, 195–207.

ranovsky, M., Fata, J., Pawling, J., Muller, W.J., Khokha, R., Dennis, J.W., 2000. Sup-pression of tumor growth and metastasis in Mgat5-deficient mice. Nat. Med. 6,306–312.

u, J., Isaji, T., Sato, Y., Kariya, Y., Fukuda, T., 2009. Importance of N-glycosylation on�5�1 integrin for its biological functions. Biol. Pharm. Bull. 32, 780–785.

u, J., Isaji, T., Xu, Q., Kariya, Y., Gu, W., Fukuda, T., Du, Y., 2012. Potential roles ofN-glycosylation in cell adhesion. Glycoconj. J. 29, 599–607.

u, J., Taniguchi, N., 2008. Potential of N-glycan in cell adhesion and migration aseither a positive or negative regulator. Cell Adhes. Migrat. 2, 243–245.

uo, H.B., Lee, I., Kamar, M., Akiyama, S.K., Pierce, M., 2002. Aberrant N-glycosylationof �1 integrin causes reduced �5�1 integrin clustering and stimulates cell migra-tion. Cancer Res. 62, 6837–6845.

oja-Łukowicz, D., Link-Lenczowski, P., Carpentieri, A., Amoresano, A., Pochec, E.,Artemenko, K.A., Bergquist, J., Litynska, A., 2013. L1CAM from human melanomacarries a novel type of N-glycan with Gal�1-4Gal�1-motif. Involvement ofN-linked glycans in migratory and invasive behaviour of melanoma cells. Gly-

coconj. J. 30, 205–225.

uttenlocher, A., Horwitz, A.R., 2011. Integrins in cell migration. Cold Spring Harb.Perspect. Biol. 3, 1–16.

anik, M.E., Litynska, A., Vereecken, P., 2010a. Cell migration – the role of integringlycosylation. Biochim. Biophys. Acta 1800, 545–555.

ell Biology 92 (2013) 355–362

Janik, M.E., Przybyło, M., Pochec, E., Pokrywka, M., Litynska, A., 2010b. Effect of�3�1 and �v�3 integrin glycosylation on interaction of melanoma cells withvitronectin. Acta Biochim. Pol. 57, 55–61.

Kaplan, R.N., Riba, R.D., Zacharoulis, S., Bramley, A.H., Vincent, L., Costa, C., Mac-Donald, D.D., Jin, D.K., Shido, K., Kerns, S.A., Zhu, Z., Hicklin, D., Wu, Y., Port, J.L.,Altorki, N., Port, E.R., Ruggero, D., Shmelkov, S.V., Jensen, K.K., Rafii, S., Lyden, D.,2005. VEGFR1-positive haematopoietic bone marrow progenitors initiate thepre-metastatic niche. Nature 438, 820–827.

Labat-Robert, J., 2002. Fibronectin in malignancy. Semin. Cancer Biol. 12, 187–195.Lagana, A., Goetz, J.G., Dennis, J.W., Cheung, P., Nabi, I.R., 2006. Galectin binding to

Mgat5-modified N-glycans regulates fibronectin matrix remodeling in tumorcells. Mol. Cell. Biol. 26, 3181–3193.

Lau, K.S., Dennis, J.W., 2008. N-Glycans in cancer progression. Glycobiology 18,750–760.

Li, G., Satyamoorthy, K., Herlyn, M., 2002. Dynamics of cell interactions and commu-nications during melanoma development. Crit. Rev. Oral Biol. Med. 13, 62–70.

Link-Lenczowski, P., Litynska, A., 2011. Glycans in melanoma screening. Part 2.Towards the understanding of integrin N-glycosylation in melanoma. Biochem.Soc. Trans. 39, 374–377.

Litynska, A., Przybyło, M., Pochec, E., Hoja-Łukowicz, D., Ciołczyk, D., Laidler, P., Gil,D., 2001. Comparison of the lectin-binding pattern in different human melanomacell lines. Melanoma Res. 11, 205–212.

Litynska, A., Przybylo, M., Pochec, E., Kremser, E., Hoja-Lukowicz, D., Sulowska, U.,2006. Does glycosylation of melanoma cells influence their interactions withfibronectin? Biochimie 88, 527–534.

Meany, D.L., Chan, D.W., 2011. Aberrant glycosylation associated with enzymes ascancer biomarkers. Clin. Proteomics 8, 7.

Melchiori, A., Mortarini, R., Carlone, S., Marchisio, P.C., Anichini, A., Noonan, D.M.,Albini, A., 1995. The �3�1 integrin is involved in melanoma cell migration andinvasion. Exp. Cell Res. 219, 233–242.

Moremen, K.W., 2002. Golgi �-mannosidase II deficiency in vertebrate systems:implications for asparagine-linked oligosaccharide processing in mammals.Biochim. Biophys. Acta 1573, 225–235.

Murata, K., Miyoshi, E., Kameyama, M., Ishikawa, O., Kabuto, T., Sasaki, Y., Hiratsuka,M., Ohigashi, H., Ishiguro, S., Ito, S., Honda, H., Takemura, F., Taniguchi, N., Imaoka,S., 2000. Expression of N-acetylglucosaminyltransferase V in colorectal cancercorrelates with metastasis and poor prognosis. Clin. Cancer Res. 6, 1772–1777.

Ochwat, D., Hoja-Łukowicz, D., Litynska, A., 2004. N-glycoproteins bearing �1-6branched oligosaccharides from the A375 human melanoma cell line analysedby tandem mass spectrometry. Melanoma Res. 4, 479–485.

Pochec, E., Litynska, A., Amoresano, A., Casbarra, A., 2003. Glycosylation profileof integrin �3�1 changes with melanoma progression. Biochim. Biophys. Acta1643, 113–123.

Pochec, E., Litynska, A., Bubka, M., Amoresano, A., Casbarra, A., 2006. Characteriza-tion of the oligosaccharide component of �3�1 integrin from human bladdercarcinoma cell line T24 and its role in adhesion and migration. Eur. J. Cell Biol.85, 47–57.

Przybyło, M., Martuszewska, D., Pochec, E., Hoja-Łukowicz, D., Litynska, A., 2007.Identification of proteins bearing �1-6 branched N-glycans in human melanomacell lines from different progression stages by tandem mass spectrometry anal-ysis. Biochim. Biophys. Acta 1770, 1427–1435.

Przybyło, M., Pochec, E., Link-Lenczowski, P., Litynska, A., 2008. �1-6 branching ofcell surface glycoproteins may contribute to uveal melanoma progression byup-regulating cell motility. Mol. Vis. 14, 625–636.

Rambaruth, N.D.S., Dwek, M.V., 2011. Cell surface glycan–lectin interactions intumor metastasis. Acta Histochem. 113, 591–600.

Saito, Y., Sekine, W., Sano, R., Komatsu, S., Mizuno, H., Katabami, K., Shimada, K., Oku,T., Tsuji, T., 2010. Potentiation of cell invasion and matrix metalloproteinaseproduction by �3�1 integrin-mediated adhesion of gastric carcinoma cells tolaminin-5. Clin. Exp. Metastasis 27, 197–205.

Saravanan, C., Liu, F.T., Gipson, I.K., Panjwani, N., 2009. Galectin-3 promotes lamel-lipodia formation in epithelial cells by interacting with complex N-glycans on�3�1 integrin. J. Cell Sci. 122, 3684–3693.

Tsuji, T., Kawada, Y., Kai-Murozono, M., Komatsu, S., Han, S.A., Takeuchi, K.,Mizushima, H., Miyazaki, K., Irimura, T., 2002. Regulation of melanoma cellmigration and invasion by laminin-5 and �3�1 integrin (VLA-3). Clin. Exp.Metastasis 19, 127–134.

Wei, T., Liu, Q., He, F., Zhu, W., Hu, L., Guo, L., Zhang, J., 2012. The role of N-acetylglucosaminyltransferases V in the malignancy of human hepatocellularcarcinoma. Exp. Mol. Pathol. 93, 8–17.

Yamada, K.M., 2000. Fibronectin peptides in cell migration and wound repair. J. Clin.Invest. 105, 1507–1509.

Zhang, Y., Zhao, J.H., Zhang, X.Y., Guo, H.B., Liu, F., Chen, H.L., 2004. Relations of thetype and branch of surface N-glycans to cell adhesion, migration and integrinexpressions. Mol. Cell. Biochem. 260, 137–146.

Zhao, Y., Nakagawa, T., Itoh, S., Inamori, K., Isaji, T., Kariya, Y., Kondo,A., Miyoshi, E., Miyazaki, K., Kawasaki, N., Taniguchi, N., Gu, J.,2006. N-acetylglucosaminyltransferase III antagonizes the effect of N-acetylglucosaminyltransferase V on �3�1 integrin-mediated cell migration. J.

Biol. Chem. 281, 32122–32130.

Zheng, M., Fang, H., Hakomori, S., 1994. Functional role of N-glycosylation in �5�1integrin receptor. De-N-glycosylation induces dissociation or altered associationof �5 and �1 subunits and concomitant loss of fibronectin binding activity. J. Biol.Chem. 269, 12325–12331.