b4-Integrin/PI3K Signaling Promotes Tumor Progression ... · b4 subunit only pairs with the a6...

Oncogenes and Tumor Suppressors

b4-Integrin/PI3K Signaling Promotes TumorProgression through the Galectin-3–N-GlycanComplexYukiko Kariya1, Midori Oyama1,Yasuhiro Hashimoto1, Jianguo Gu2, and Yoshinobu Kariya1

Abstract

Malignant transformation is associated with aberrant N-glyco-sylation, but the role of protein N-glycosylation in cancer pro-gression remains poorly defined. b4-integrin is a major carrierof N-glycans and is associated with poor prognosis, tumorigen-esis, and metastasis. Here, N-glycosylation of b4-integrin contri-butes to the activation of signaling pathways that promote b4-dependent tumor development and progression. Increasedexpression of b1,6GlcNAc-branched N-glycans was found to becolocalized with b4-integrin in human cutaneous squamous cellcarcinoma tissues, and that the b1,6GlcNAc residuewas abundanton b4-integrin in transformed keratinocytes. Interruption ofb1,6GlcNAc-branching formation on b4-integrin with the intro-duction of bisecting GlcNAc by N-acetylglucosaminyltransferaseIII overexpression was correlated with suppression of cancer cellmigration and tumorigenesis. N-Glycan deletion on b4-integrinimpaired b4-dependent cancer cell migration, invasion, andgrowth in vitro and diminished tumorigenesis and proliferationin vivo. The reduced abilities of b4-integrin were accompanied

with decreased phosphoinositol-3 kinase (PI3K)/Akt signals andwere restored by the overexpression of the constitutively activep110 PI3K subunit. Binding of galectin-3 to b4-integrin viab1,6GlcNAc-branched N-glycans promoted b4-integrin–mediat-ed cancer cell adhesion and migration. In contrast, a neutralizingantibody against galectin-3 attenuated b4-integrin N-glycan–mediated PI3K activation and inhibited the ability of b4-integrinto promote cell motility. Furthermore, galectin-3 knockdown byshRNA suppressed b4-integrin N-glycan–mediated tumorigene-sis. These findings provide a novel role for N-glycosylation ofb4-integrin in tumor development and progression, and theregulatory mechanism for b4-integrin/PI3K signaling via thegalectin-3–N-glycan complex.

Implications: N-Glycosylation of b4-integrin plays a functionalrole in promoting tumor development and progression throughPI3K activation via the galectin-3–N-glycan complex. Mol CancerRes; 16(6); 1024–34. �2018 AACR.

IntroductionIntegrins are a large family of heterodimeric transmembrane

receptors comprising a and b subunits. The extracellular domainsof integrin subunits bind to extracellular matrix proteins, such ascollagen, fibronectin, and laminin, and the cytoplasmic domainsinteract with the actin cytoskeleton and signaling molecules. Theinteraction of integrin with its ligand can activate intracellularsignaling and reorganize the actin cytoskeleton (1). Such integrinsignaling regulates various cellular functions, such as cell adhe-sion, migration, and proliferation, not only in normal tissues butalso in tumor tissues (1, 2).

Thea6b4-integrin (referred to herein as b4-integrin because theb4 subunit only pairs with the a6 subunit) is a receptor for

laminin-332 and is an essential component of the hemidesmo-some, an anchoring structure in the basal membrane of stratifiedepithelial cells (2, 3). In contrast to such a static role in normalepithelial cells, b4-integrin was originally identified as a tumor-associated antigen (4, 5).b4-integrin overexpressionwas found inseveral types of metastatic cancers, which correlates with poorprognosis and reduced survival (6, 7). Recent studies have shownthat b4-integrin is required for tumorigenesis in several in vivomouse models (8–10). In addition, b4-integrin promotes cellmotility, invasion, and proliferation through activation of thephosphoinositol-3-kinase (PI3K) and ERK pathways (10–14).

Glycosylation is the most common posttranscriptional mod-ification of proteins, and it modulates the folding, stability,and function of glycoproteins (15). Malignant transformationis associated with aberrant glycosylation of cell surface pro-teins, and the structural change of glycans is mainly dueto the altered activity and expression of multiple glycosyltrans-ferases in cancer cells. In addition, some glycans are used astumor markers for cancer diagnosis (16). Overexpressionof b1,6GlcNAc-branched N-glycans, which is due to increasedactivity of N-acetylglucosaminyltransferase V (GnT-V; Fig. 1A),is often found in tumor tissues, and the increase inb1,6GlcNAc-branched N-glycans is directly associated withmalignancy and poor prognosis (17, 18). GnT-V knockoutmice exhibit reduced mammary tumor growth and metastasisinduced by the polyomavirus middle T oncogene (19). Fur-thermore, introduction of bisecting GlcNAc residues catalyzed

1Department of Biochemistry, FukushimaMedical University School of Medicine,Fukushima, Japan. 2Division of Regulatory Glycobiology, Institute of MolecularBiomembrane and Glycobiology, Tohoku Medical and PharmaceuticalUniversity, Miyagi, Japan.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

CorrespondingAuthor:YoshinobuKariya, FukushimaMedical University Schoolof Medicine, 1 Hikarigaoka, Fukushima City, Fukushima 960-1295, Japan. Phone:81-24-547-1144; Fax: 81-24-548-8641; E-mail: kariya@fmu.ac.jp

doi: 10.1158/1541-7786.MCR-17-0365

�2018 American Association for Cancer Research.

MolecularCancerResearch

Mol Cancer Res; 16(6) June 20181024

Published OnlineFirst March 16, 2018; DOI: 10.1158/1541-7786.MCR-17-0365

by N-acetylglucosaminyltransferase III (GnT-III) into N-gly-cans, which can disturb further processing and elongation ofN-glycans, such as the formation of b1,6GlcNAc-branchedstructures (Fig. 1A), suppresses cell migration and cancermetastasis (20).

b1,6GlcNAc-branchedN-glycans are often further modified byadditional sugars to form poly-N-acetyllactosamine (polylacto-samine) for the elongation of N-glycans consisting of b-galacto-side sugars (Fig. 1A). This polylactosamine structure is a preferredligand for one of the galectin isoforms, galectin-3 (21). Galectin-3is widely expressed in epithelial and immune cells, and itsexpression is associated with cancer aggressiveness andmetastasis(22–24). Cross-linking between glycoproteins by the binding ofgalectin-3 to polylactosamine on proteins regulates diverse cel-lular functions in cancer cells (25–27). We previously reportedthat N-glycosylation of b4-integrin plays a crucial role in celladhesion, migration, and galectin-3 binding in human keratino-cyte cells (28). However, the contribution of N-glycosylation ofb4-integrin to tumor progression remains poorly defined. In thepresent study, we investigated the contribution ofN-glycosylationto b4-integrin-dependent tumor progression.

Materials and MethodsAntibodies and reagents

The following antibodies were used in this study: rat mono-clonal antibodies specific for galectin-3 (M3/38; #sc-2393; SantaCruz Biotechnology), a6-integrin (GoH3; #sc-19622; Santa Cruz

Biotechnology), and b4-integrin (439-9B; #555719; BD Trans-duction Laboratories); rabbit polyclonal antibodies againsthuman b4-integrin (H101; #sc-9090; Santa Cruz Biotechnology),phospho-Akt (pSer473; #9271; Cell Signaling Technology), andAkt (#9272; Cell Signaling Technology); and mouse monoclonalantibodies against Ki-67 (#610968; BD Transduction Laborato-ries), and b4-integrin (3E1; #MAB1964; Merck Millipore). AlexaFluor 488-conjugated leukoagglutinating phytohemagglutinin(L4-PHA; #L-11270), Alexa Fluor 546-conjugated goat anti-rabbitIgG secondary antibody (#A11035) and Alexa Fluor 546-conju-gated goat anti-rat IgG secondary antibody (#A11081) werepurchased from Thermo Fisher Scientific. Biotinylated L4-PHA(#B-1115) was obtained from Vector Laboratories. Hoechst33342 (#382065) was obtained from Merck Millipore. Biotiny-lated erythroagglutinating phytohemagglutinin (E4-PHA;#300425) and biotinylated Sambucus sieboldiana agglutinin (SSA;#300442) were purchased from J-OIL MILLS. Purified humanlaminin-332 and galectin-3were prepared as described previously(27, 29).

Cell cultureThe human cancer MDA-MB435S cell line, which lacks b4-

integrin, was purchased from the American Type Culture Col-lection. Modified human 293 phoenix cells were received as agift from Dr. M. Peter Marinkovich (Stanford University, Stan-ford, CA). The human epidermoid carcinoma cell line A431(RCB0202) was provided by the RIKEN BRC through theNational Bio-Resource Project of the MEXT, Japan. These cells

Figure 1.

Increased expression of b1,6GlcNAcresidues on b4-integrin in humancarcinoma cells. A, Glycosylationreactions catalyzed byglycosyltransferases, GnT-III andGnT-V. b1,6GlcNAc-branchedN-glycans can be preferentiallymodified by polylactosamine N-glycancontaining b-galactoside. BisectingGlcNAc N-glycans inhibit b1,6GlcNAc-branching formation catalyzed by GnT-V. B, Immunohistochemical analysis ofbisecting GlcNAc and b1,6GlcNAcexpression in human cutaneous SCCusing E4-PHA and L4-PHA lectins.C, Immunofluorescence microscopicanalysis of b4-integrin (red) andb1,6GlcNAc (L4-PHA, green) in paraffin-embedded sections of humancutaneous SCC. Merged images areshown with nuclear Hoechst staining(blue). Normal human skin was stainedas comparison.D,Western blot analysisof b4-integrin immunoprecipitatesfrom normal human keratinocytes(NHK) and Ras/IkB-transformed NHK(transformed NHK) with L4-PHA andE4-PHA lectins, and anti–b4-integrinpolyclonal antibody. Results of thedensitometric analysis are shown as theintegrated density of lectin-recognizedb4 to total b4 bands, which was1.0 for NHK cells.

N-Glycosylation of b4-Integrin Promotes Tumor Progression

www.aacrjournals.org Mol Cancer Res; 16(6) June 2018 1025

were cultured in DMEM supplemented with 10% FBS, penicil-lin and streptomycin sulfate at 37�C in a humidified 5% CO2

incubator. Primary human keratinocytes isolated fromnormal skin (NHK) and Ras/IkB-transformed NHK (a gift fromDr. M. Peter Marinkovich; ref. 8) were cultured in a 50:50mixture of defined keratinocyte serum-free medium (#10744-019; Life Technologies) and medium 154 (#M-154-500; LifeTechnologies) with human keratinocyte growth supplement(#S-001-5; Life Technologies) containing penicillin and strep-tomycin sulfate at 37�C in a humidified 5% CO2 incubator. Allcells were passaged fewer than 6 months after purchasing orreceiving them for all the experiments and tested formycoplasmacontamination.

Immunohistochemistry/immunofluorescenceHuman formalin-fixed paraffin-embedded sections were pur-

chased from BioChain and US Biomax. Specimens were depar-affinized, rehydrated, and immersed in 0.3% hydrogen perox-idase-containing methanol for 20 minutes at room temperatureto inactivate the intrinsic peroxidase. After blocking with 5%skim milk for 1 hour at room temperature, the sections wereincubated with each biotinylated lectin overnight at 4�C, fol-lowed by peroxidase-labeled streptavidin (#426061; NichireiCorp.) and 3,30-diaminobenzidine detection using the Histo-fine DAB substrate kit (#425011; Nichirei Corp.). The slideswere then counterstained with Mayer's hematoxylin solution(#131-09665; Wako). Images were obtained using a PROVISAX-80 microscope (Olympus). For immunofluorescent stain-ing, sections were deparaffinized, rehydrated, and treated with0.05% protease XXIV in 50 mmol/L Tris-HCl (pH 7.5) for 20minutes at room temperature. After blocking with 2% BSA inPBS for 1 hour at room temperature, the sections were visual-ized using a b4-integrin antibody (H101) and Alexa Fluor 488-conjugated L4-PHA. Fluorescence images were obtained usingan IX71 fluorescent microscope (Olympus). Lectin reactivityand b4-integrin expression were assessed as follows: positive,�10% positive tumor cells; negative, <10% positive tumorcells. For cross-linking inhibitory assay, glass-bottom dishes(#3971-035; IWAKI) were coated with laminin-332 proteinsand blocked with 1% BSA in PBS. Cells (5 � 104 cells) wereincubated in serum-free medium in the presence of IgG or afunctional blocking antibody against galectin-3 for 20 minutes.Then, the cells were plated to the glass-bottom dish andincubated for 1.5 hours at 37�C in a humidified 5% CO2

incubator. The cells were fixed with 4% (w/v) paraformalde-hyde in PBS for 10 minutes and blocked with 2% BSA in PBS for1 hour at room temperature. The fixed cells were stained with ab4-integrin antibody (H101) and Alexa Fluor 488-conjugatedanti-rabbit IgG antibody. Fluorescence images were obtainedusing an A1 confocal microscope (Nikon).

Preparation of cell lysates and immunoprecipitationCell lysates were prepared as follows. The cells were washed

twicewith coldPBS and then lysedwith a lysis buffer [1%TritonX-100, 20mmol/L Tris-HCl (pH 7.4), 150mmol/L NaCl, 5mmol/LEDTA] containing a protease inhibitor cocktail (#25955; Nacalaitesque) and a phosphatase inhibitor cocktail (#07575; Nacalaitesque). After incubation for 10 minutes on ice, the cell lysateswere clarified by centrifugation at 15,000 rpm for 10 minutes at4�C. The resulting supernatant was used in the following experi-

ments. The protein concentration was determined using a proteinassay kit (#29449-44; Nacalai tesque). For immunoprecipitation,the primary antibody was added to the supernatant and rotatedfor 2 hours at 4�C. Then, protein G-Sepharose was added, fol-lowed by 3 hours of incubation at 4�C. Immunoprecipitates werewashedfive timeswith awashing buffer [50mmol/L Tris-HCl (pH7.5), 150 mmol/L NaCl, 2 mmol/L EDTA, 0.2% NP40 (v/v)],suspended in a reducing sample buffer, and heated at 95�C for 5minutes.

Western blot analysesProtein samples were resolved by SDS-PAGE under reducing

conditions and then transferred to nitrocellulose membranes.The blots were probed with each specific antibody or biotiny-lated lectin. Immunoreactive bands were detected using anImmunoStar Zeta (#297-72403; WAKO) or Trident femto-ECL(#GTX14698; GeneTex). Band intensity was calculated usingNIH ImageJ software.

Expression vectorsRetroviral expression vectors encoding b4-integrin were pre-

pared as previously described (28). Retroviral expression vectorencoding active PI3K p110-CAAX was received as a gift from Dr.M. Peter Marinkovich. The cDNA encoding human GnT-III wasamplified by PCR using a specific primer set and KOD Pluspolymerase (#KOD-201; TOYOBO) for cloning into pENTR-D-TOPO (#K2400-20; Life Technologies) for the Gateway Conver-sion System according to the manufacturer's instructions. Thefinal constructwas recombined frompENTR-D-TOPO to the LZRSblast retroviral vector, including a Gateway cassette, using the LRclonase II Enzyme mix (#11791-020; Life Technologies) by arecombination reaction. The cDNA sequence was verified bysequencing.

Retrovirus infectionRetrovirus vectors were transfected into 293 phoenix cells

using FuGENE6 transfection reagent (#11814443001; Roche).After transfection, cells were selected with 5 mg/mL puromycin(#P8833; Sigma-Aldrich). The retrovirus was then produced in293 phoenix cells. One day before infection, 4 � 105 cells wereplated in 6-well plates. After incubation with 5 mg/mL poly-brene (#10768-9; Sigma-Aldrich) for 15 minutes, media wereexchanged to 3 mL retroviral supernatant and another 5 mg/mLpolybrene was added. Plates were centrifuged at 1,200 rpm for1 hour at 32�C using a Hitachi CR22N centrifuge machine.After centrifuge, the retroviral supernatant was replaced withgrowth medium and the cells were maintained. To establish acell line, cells were selected with 5 mg/mL blasticidin S(#203350; Calbiochem).

Flow cytometry analysisCells were detached from a 10-cm dish using trypsin with 1

mmol/L EDTA. After quenching trypsinization with a mediumthat contained 10%FBS, the cells werewashed twicewith PBS thatcontained 1 mmol/L EDTA and incubated with a primary anti-body or control IgG on ice for 30 minutes. The cells were thenwashed thrice with PBS that contained 1mmol/L EDTA, followedby incubation for 15 minutes with the appropriate Alexa Fluor-conjugated secondary antibodies. After washing thrice with PBScontaining 1 mmol/L EDTA, the cells were analyzed by flow

Kariya et al.

Mol Cancer Res; 16(6) June 2018 Molecular Cancer Research1026

cytometry using a FACSCalibur and aCellQuest software program(BD Biosciences).

Cell migration assayCell migration was assayed using a 24-well chemotaxis cham-

ber (BD Falcon cell culture companion plates; #353504 and 8.0-mminsets; #353097; BDBiosciences). Two hundredmicroliters of2.5 � 105 cells/mL (5 � 104 cells in serum-free DMEM) wasinoculated into each upper well of a 24-well chemotaxis chamber,and 750 mL of 10% FBS containing DMEM was placed in thebottom chamber to act as a chemoattractant. After incubating for22 hours, the cells on the upper side of the membrane wereremovedwith a cotton swab, and the cells on the lower side of themembrane were fixed with 4% paraformaldehyde and stainedwith 0.5% crystal violet in 20% methanol. Random fields werephotographed using a phase contrastmicroscope and the numberof migrated cells was counted.

In vivo tumorigenicity assayAll animal studies were performed in accordance with proto-

cols approved by the Fukushima Medical University Animal Careand Use Committee. MDA-MB435S transfectants (1 � 106 cells/mouse) were injected subcutaneously along with Matrigel(#354234; BD Biosciences) into 6-week-old female nude mice.Tumor volume was measured weekly for a total of 6 weeks.Proliferating cells in 5-mm frozen sections were detected withKi-67 immunofluorescent staining. Proliferation was quantifiedas the ratio of Ki-67 staining to total nuclear staining.

Cell adhesion assayCell adhesion assays were performed as described previously

(29). In brief, the wells of a 96-well ELISA plate (#3590; Corning)were coatedwith 50mL of indicated concentration of laminin-332proteins and blocked with 1% BSA in PBS. Cell suspensions inserum-free medium were plated to each well (5 � 104 cells/well)and incubated at 37�C for 20 minutes in a 5% CO2 incubator.After removing the non-adherent cells, the adherent cells werefixed with 25% (w/v) glutaraldehyde for 10 minutes and stainedwith 0.5% crystal violet in 20% (v/v) methanol for 10 minutes.Random fields were photographed using a phase contrast micro-scope, and the number of adherent cells was counted.

Cell proliferation assayCell suspensions in 1% serum-containingmediumwere plated

to each well of a 96-well ELISA plate (5 � 103 cells/well) andincubated at 37�C for 45 hours in a 5% CO2 incubator. Afterincubation, the number of growing cellswasmeasuredusing a cellcounting kit-8 (#347-07621; DOJINDO) according to the man-ufacturer's instructions.

Cell invasion assayOne hundred microliters of Matrigel, diluted to a final con-

centration of 1.6 mg/mL, was added to the upper chamber of 24-well Transwell plates and dried for 24 hours in a hood. The wellswere reconstituted by incubation with 200 mL of serum-freeDMEM at 37�C for 1 hour. After removing the medium, 200 mLof 5� 105 cells/mL (1� 104 cells in 0.1% BSA containing serum-free DMEM) were inoculated into each upper well of a 24-wellchemotaxis chamber, and 750 mL of 10% FBS containing DMEMwas placed in the bottom chamber to act as a chemoattractant.After 6 hours of incubation, the cells on the upper side of themembrane were removed with a cotton swab, and the cells on the

lower side of the membrane were fixed with 4% paraformalde-hyde and stained with 0.5% crystal violet in 20% methanol.Random fields were photographed using a phase contrast micro-scope and the number of invaded cells was counted.

Lentiviral short hairpin RNA (shRNA)Lentiviral shRNA clones (Dharmacon RNAi consortium Lenti-

viral shRNA) targeting galectin-3 (#RHS3979-201759611, cloneID: TRCN0000029304) 50-ATTGTACTGCAACAAGTGAGC-30

and control shRNA (#RHS4459) 50-TACAACAGCCACAACGTC-TAT-30 were purchased from GE Healthcare. These vectors werecotransfectedwith the packaging vectors intoHEK293T cells usingthe Trans-Lentiviral shRNA Packaging kit (#TLP5912; GE Health-care) according to the manufacturer's instructions. After incuba-tion for 15 hours, media were exchanged to 5% FBS containingDMEM. After further incubation for 48 hours, viral supernatantwas harvested and then centrifuged at 1,600 g for 10 minutes at4�C. The supernatant was filtrated through a 0.45-mm filter.Lentivirus was infected into MDA-MB435S cells using the samemethod as retrovirus infection. To establish a cell line, cells wereselected with 5 mg/mL puromycin.

Statistical analysisResults are given as mean � SEM and are representative of two

or three independent experiments. Statistical comparisons werecalculated between two groups using unpaired Student t test andamong the groups using one-wayor two-wayANOVA followed bya Bonferroni posttest, with GraphPad Prism Version 5.0a soft-ware. A P value of <0.05 was considered statistically significant.

Resultsb1,6GlcNAc residue on b4-integrin was associated with cellmigration and tumorigenesis

Expression of b1,6GlcNAc is associated with metastasis andpoor prognosis, whereas bisectingGlcNAc suppresses the effect byinhibition of b1,6GlcNAc-branching formation (20). Humancutaneous squamous cell carcinoma (SCC) showed positiveexpression of b1,6GlcNAc (52 out of 75) and negative expressionof bisecting GlcNAc (72 out of 75) by immunohistochemistryusing L4-PHA and E4-PHA lectins (Fig. 1B, n ¼ 75), which isconsistent with previous observations that b1,6GlcNAc was morelikely to be associated with tumor malignancy (17). As over-expression of b4-integrin in SCC has been reported (6, 30), wenext examined the expression patterns of b4-integrin andb1,6GlcNAc in human cutaneous SCC. In skin cancer specimens(n ¼ 37), b4-integrin was abundantly present and colocalizedwith b1,6GlcNAc-branched N-glycans. Thirty-three b4-integrinpositive tumor cells, 33 (100%) colocalized b1,6GlcNAc-branched N-glycans (Fig. 1C). In contrast, b4-integrin was clearlylocalized in the basement membrane, while the expression ofb1,6GlcNAc was almost undetectable in all tested normal humanskin samples (n ¼ 7). Taking these results into consideration, wehypothesized that the amount of b1,6GlcNAc-branched N-gly-cans on b4-integrin increases in cancer cells. To test this hypoth-esis, we examined whether b1,6GlcNAc was attached to b4-integ-rin upon transformation of NHK cells. Ras/IkB-mediated trans-formation of NHK cells increased attachment of b1,6GlcNAc tob4-integrin while the extent of the bisecting GlcNAc modificationof b4-integrin was decreased after transformation (Fig. 1D). These

results suggest that the b1,6GlcNAcmodification of b4-integrin isincreased in cancer cells.

GnT-III inhibits the formation of b1,6GlcNAc branching onN-glycans by the introduction of bisecting GlcNAc (Fig. 1A). Toaddress the role of b1,6GlcNAc modification of b4-integrin intumor malignancy, we used MDA-MB435S cells, which do notexpress b4-integrin, for preparing stable transfectants retrovirallytransduced with control lacZ, human b4-integrin, lacZ with GnT-III, or b4 with GnT-III. The FACS analysis exhibited that GnT-IIIexpression did not affect the cell surface expression of b4-integrin(Fig. 2A, lacZ versus lacZþGnT-III and b4 versus b4þGnT-III).Overexpression of GnT-III in cells expressing b4-integrin signif-icantly reduced b1,6GlcNAc-branched N-glycans on b4-integrinaccompanied with increased bisecting GlcNAc (Fig. 2B). Expres-sion of b4-integrin exhibited enhanced cell migration (Fig. 2C)and tumor formation (Fig. 2D) in the MDA-MB435S cells com-pared with the control cells, which were impaired by GnT-IIIexpression. These results suggest that the b1,6GlcNAc residue inb4-integrin may be associated with primary tumor growth andmetastatic cancer cell behavior.

N-Glycosylation of b4-integrin drives proliferation, migration,and invasion through PI3K/Akt activation

GnT-III overexpression modifies not only b4-integrin butalso other glycoproteins (Supplementary Fig. S1). b4-integrincontains five potential N-glycosylation sites in its extracellulardomain (Fig. 3A). To directly address the biological role of N-glycosylation of b4-integrin in cancer cell behavior, we pre-pared two b4-integrin constructs; a full-length wild-type (WT)and a mutant lacking all five N-glycosylation sites (DN; ref. Fig.3A). These constructs were retrovirally expressed in MDA-MB435S cells that endogenously expressed no b4-integrin (Fig.2A, lacZ). To confirm the loss of N-glycans on DN, we per-formed lectin blot analysis using L4-PHA, E4-PHA, and SSAlectins, which recognize b1,6GlcNAc, bisecting GlcNAc, and

a2,6 sialic acid in N-glycans, respectively. The WT reacted withall tested lectins, but the DN did not react (Fig. 3B), suggestingthe loss of N-glycans in the DN. Similar to our previous reportsusing keratinocytes (28), the deletion of N-glycans had noeffect on either cell surface expression (Supplementary Fig.S2A) or heterodimer formation of b4-integrin with a6-integrin(Supplementary Fig. S2B) in the MDA-MB435S cells. Theseresults suggest that N-glycans on b4-integrin were not requiredfor either expression or heterodimer formation of a6b4-integ-rin. We next assessed whether N-glycosylation on b4-integrincan affect cell adhesion and spreading, which are generallyassociated with cancer progression, using the MDA-MB435Stransfectants. Typically, b4-integrin causes cellular signalingthrough cell adhesion to the extracellular matrix protein lami-nins in cancer progression (2, 31). Compared with the WT cells,the DN cells showed decreased cell adhesion and spreading on alaminin-332 substrate, which was comparable to those in thecontrol lacZ cells (Fig. 3C), suggesting that N-glycans on b4-integrin are related to b4-integrin-dependent cell adhesion andspreading in MDA-MB435S cancer cells.

b4-integrin regulates cell proliferation,migration, and invasionin cancer cell lines (2, 6). Therefore, we examined the cellularfunctions of MDA-MB435S cancer cells expressing WT and DN.The expression of b4-integrin markedly enhanced cell prolifera-tion,migration, and invasion of theMDA-MB435S cells (Fig. 3D–

F,WT) comparedwith the control lacZ cells. The enhanced cellularfunctions were significantly impaired by the loss of N-glycans onb4-integrin (Fig. 3D–F,DN). Previous studies have shown that b4-integrin–dependent cell proliferation, migration, and invasionare closely associated with the PI3K pathway in several carcinomacell lines (9, 13). To address whether the decreased cellularfunctions due to the deletion of N-glycosylation were correlatedto the downregulation of PI3K signaling, we examined the acti-vation of the PI3K signaling pathway by Western blot analysiswith an anti-phosphorylated Akt antibody. The DN cells, as well

Figure 2.

Decreased b1,6GlcNAc residues in b4-integrin by GnT-III correlates withsuppression of cancer cell migrationand tumorigenesis. A, Cell surfaceexpression of b4-integrin in MDA-MB435S cells expressing control lacZ,lacZ with GnT-III (lacZþGnT-III), b4,and b4 with GnT-III (b4þGnT-III).B, Lectin blot analysis of b4-integrinimmunoprecipitates from MDA-MB435S cells expressing b4 and b4with GnT-III using L4-PHA(b1,6GlcNAc) and E4-PHA (bisectingGlcNAc) lectins. Data arerepresentative of three independentexperiments. C, Effect of GnT-IIIoverexpression on b4-integrin–mediated cell migration. Results arethe mean� SEM of three independentexperiments conducted in triplicate.�� , P < 0.001. ns, not significant (two-way ANOVA, Bonferroni posttest). D,Tumor formation in nudemice injectedsubcutaneously with the indicatedcells. Results are the mean � SEM of5mice per group. � , P < 0.05 (two-wayANOVA, Bonferroni posttest).

Kariya et al.

as the lacZ cells, showed decreased phosphorylation of Akt com-pared with the WT cells (Fig. 3G). We also found that theimpairment of cellular functions in the DN cells was completelyrestored through overexpression of the constitutively active PI3Kp110a subunit (Fig. 3D–F, DNþPI3K). In contrast, overexpres-sion of the active PI3K subunit in WT cells did not significantlyaffect the WTb4-dependent cell proliferation, migration, andinvasion (Fig. 3D–F, WTþPI3K), suggesting that the WTb4-medi-ated PI3K/AKT activation is sufficient for such functions in the

cells and higher PI3K/AKT activation does not always promoteactivities of cancer cells. Together, these findings demonstrate thatthe N-glycosylation of b4-integrin plays a fundamental role inactivating the PI3Kpathway, thereby promoting cell proliferation,migration, and invasion.

N-Glycans of b4-integrin promote in vivo tumor growth.To determine whether N-glycosylation of b4-integrin is related

to tumorigenesis in vivo, we subcutaneously injected MDA-

Figure 3.

N-Glycosylation of b4-integrin drives tumor cell proliferation, migration, and invasion through PI3K/Akt activation. A, Schematic diagram of the human b4-integrincDNA constructs in this study. WT, wild-type b4-integrin; DN,N-glycosylation–defective b4-integrin mutant. The sites corresponding to theN-glycosylation sites onb4-integrin subunit (Asn327, Asn491, Asn579, Asn617, and Asn695) are shown by flags. Numbers indicate the number of amino acid residues. TM, transmembraneregion. B, Lectin blot analysis of b4-integrin immunoprecipitates from MDA-MB435S cells expressing lacZ, WT, and DN with L4-PHA, E4-PHA, and SSA lectins.C,Cell adhesion and spreading ofMDA-MB435S transfectants to laminin-332. Note thatMDA-MB435S cells expressing nob4-integrin (lacZ) orDNshoweddecreasedcell adhesion and spreading compared with the cells expressing WT. Cell spreading morphology was examined under a phase-contrast microscope afterincubation for 20 minutes. D–F, Effect of the N-glycosylation defect in b4-integrin on cell proliferation (D), migration (E), and matrigel invasion (F). Note that cellsexpressing DN showed decreased cell proliferation, migration, and matrigel invasion, which were rescued by overexpression of constitutively active PI3K p110asubunit (DNþPI3K). In contrast, overexpression of the active PI3K subunit had no significant effect on WT-b4–dependent cell proliferation, migration, or matrigelinvasion (WTþPI3K). Results are the mean� SEM for three independent experiments conducted in triplicate. �� , P < 0.01; �� , P < 0.001; ns, not significant (one-wayANOVA, Bonferroni posttest) versusWT.G,Western blot analysis of cell lysates fromMDA-MB435S transfectantswith phospho-Akt and total Akt antibody. Note thatcells expressing lacZ or DN showed decreased PI3K/Akt pathway activation, which was rescued by overexpression of constitutively active p110a PI3Ksubunit (DNþPI3K). Results of the densitometric analysis are shown as the integrated density of the ratio of phosphorylated protein to total protein bands. All blotsare representative of at least three independent experiments.

MB435S transfectants intonudemice andanalyzed tumor growth.Compared with the WT cells, the DN cells showed significantlyimpaired tumorigenesis, which was comparable with the b4null (lacZ) cells (Fig. 4A and B). Impairment of the tumorigenesisin the DN cells was fully restored through overexpression of theconstitutively active PI3K p110a subunit (Fig. 4A, DNþPI3K). Incomparison with the WT tumors, staining with anti–Ki-67 anti-body demonstrated that the DN tumors included notably fewerproliferating cells, whichwas fully increased toWT tumor levels byoverexpression of the constitutively active PI3K p110a subunit(Fig. 4C and D). These results demonstrate that the ability of b4-integrin to drive tumorigenesis, and tumor proliferation is asso-ciated with activation of PI3K pathway via N-glycans.

b4-integrin–dependent tumor progression is associatedwith interaction between galectin-3 and b1,6GlcNAcresidue on b4-integrin

Galectin-3 is known to be associated with cancer aggres-siveness and metastasis (22–24). To examine the role ofgalectin-3 in b4-integrin–mediated tumor progression, weperformed inhibition assays using a functional blocking anti-body against galectin-3 (M3/38). The functional blockingantibody against galectin-3 suppressed b4-integrin clusteringprobably through inhibition of galectin-3 multimerization,suggesting that galectin-3 actually cross-links b4-integrin (Fig.5A). The antibody suppressed WT cell motility, whereas noeffect was observed in the DN cells (Fig. 5B). Correspondingwith the decreased cell motility of the WT cells, the galectin-3antibody reduced Akt phosphorylation compared with controlIgG in the WT cells (Fig. 5C). Furthermore, we tested thetumorigenicity of the WT cells expressing control shRNA orgalectin-3 shRNA to confirm the importance of galectin-3

expression on tumor formation (Supplementary Fig. S3). Theresult showed that reduced expression of galectin-3 in WT cellssuppressed tumor formation, suggesting that galectin-3 playsan important role in b4-integrin–promoting tumor formation(Fig. 5D).

Galectin-3 has a high affinity for b1,6GlcNAc-branched N-glycans modified by polylactosamine consisting of b-galactosidesugars. The oligomerization of integrins by galectin-3–mediatedcross-linking between the b1,6GlcNAc-branched N-glycans onintegrins promotes integrin function (25, 27, 32). To understandwhy deletion of N-glycosylation suppressed the ability of b4-integrin to promote cancer cell progression, we examined therelationship between galectin-3 binding to b4-integrin and cel-lular functions of b4-integrin. b4-integrin immunoprecipitatesfrom theWTcells containedhigh amounts of galectin-3 comparedwith those from the lacZ and DN cells, indicating that N-glycanson b4-integrin promote complex formation by the twomolecules(Fig. 5E). The association of b4-integrin with galectin-3 wasinhibited by a competitive inhibitor of galectin binding, b-lac-tose but not by control sucrose, suggesting that galectin-3 isbound to b1,6GlcNAc-branched N-glycans modified by poly-lactosamine on b4-integrin (Fig. 5F). Similar results wereobtained from the squamous cell carcinoma cell line, A431cells, which endogenously express b4-integrin. The addition ofgalectin-3 significantly enhanced cell adhesion of the WT cellsbut not the lacZ or DN cells to a laminin-332 substrate (Fig.5G). Furthermore, the addition of galectin-3 to the cell culturesignificantly promoted cell motility of the WT cells but not thatof either the WT with GnT-III cells or DN cells (Fig. 5H). Thesefindings suggest that galectin-3 binding to b4-integrin throughb1,6GlcNAc-branched N-glycans promotes the ability of b4-integrin to drive cell adhesion and motility.

Figure 4.

N-Glycosylation of b4-integrinfunctionally contributes totumorigenesis in vivo. A, Tumorgrowth after subcutaneous injectionof MDA-MB435S cells expressing theindicated proteins into mice (n ¼ 5mice per group � SEM). �� , P < 0.01;�� , P < 0.001 (one-way ANOVA,Bonferroni posttest). B, Hematoxylin/eosin staining of the indicated tumors.C, Immunofluorescent analysis ofKi-67 (green) and b4-integrin (red)expression in the indicated tumors.Nuclei were stained with Hoechst33342 (blue). D, Quantifiedpercentage of Ki-67-positive cells tototal cells in C. The bar graphrepresents the mean � SEM forthree independent experiments.�� , P < 0.001; ns, not significant(one-way ANOVA, Bonferroniposttest) versus WT.

Kariya et al.

Figure 5.

Galectin-3 is a keymodulator of b4-integrin function throughN-glycan.A, Inhibitory effect of a functional blocking antibody against galectin-3 (Ab) or control IgG onb4-integrin clustering. Note that MDA-MB435S cells expressing WT showed decreased b4-integrin clustering by the treatment of a functional blocking antibodyagainst galectin-3. B, Inhibitory effect of a functional blocking antibody against galectin-3 on N-glycosylation of b4-integrin–mediated cell migration. Note thatMDA-MB435S cells expressing WT but not DN showed decreased cell migration by the treatment of a functional blocking antibody against galectin-3.C,MDA-MB435S cells expressingWT andA431 cells were incubated in serum-freemedium in the presence of IgG or a functional blocking antibody against galectin-3(Ab) for 20minutes. Cell lysateswere analyzed byWestern blot with phospho-Akt and total Akt antibody.D, Effect of galectin-3 (gal3) knockdown on tumor growthinMDA-MB435S cells expressingWT. Results are themean�SEMof sixmiceper group. �� ,P<0.001 (unpairedStudent t test).E,Western blot analysis ofb4-integrinimmunoprecipitates from MDA-MB435S transfectants and A431 cells. F, MDA-MB435S cells expressing WT and A431 cells were serum-starved for 2 hoursand were then incubated in serum-free medium in the presence of conditioned medium of MDA-MB435S cells and saccharides (0.2 mol/L) for 30 minutes.The b4-integrin immunoprecipitates from the cells were analyzed byWestern blot. Note that the binding of galectin-3 to b4-integrin was inhibited by a competitivedisaccharide, b-lactose, but not a noncompetitive disaccharide, sucrose. Results of the densitometric analysis are shown as the integrated density of the ratio ofgalectin-3 protein to b4-integrin protein bands. G, Effect of galectin-3 on N-glycans of b4-integrin–mediated cell adhesion. Note that galectin-3 enhancedcell adhesion of MDA-MB435S cells expressing WT but not either lacZ or DN to laminin-332. H, Effect of galectin-3 on N-glycosylation in b4-integrin–mediated cellmigration. Note that MDA-MB435S cells expressing WT, but not either WT with GnT-III or DN, showed increased cell migration in the presence of galectin-3.Results are the mean � SEM for two or three independent experiments conducted in triplicate. � , P < 0.05; ��, P < 0.001; ns, not significant (one-way ANOVA,Bonferroni posttest).

DiscussionIn the current study, b4-integrin was colocalized with

b1,6GlcNAc-branched N-glycans in tumor tissues. The suppres-sion of the modification of b4-integrin by GnT-III was associatedwith reduced cancer cell migration and tumorigenesis in MDA-MB435S cells expressing b4-integrin. Deletion ofN-glycosylationsites inb4-integrin,whichwas accompaniedwithdownregulationof the PI3K signaling pathway, inhibited b4-integrin–dependentcancer cell migration, invasion, proliferation, and tumor forma-tion. Furthermore, loss of association between galectin-3 and b4-integrin via b1,6GlcNAc-branched N-glycans abolished galectin-3–promoting cancer cell adhesion and migration. These resultsprovide evidence that N-glycosylation of b4-integrin plays afunctional role in promoting tumor development and progres-sion through the PI3K activation.

b4-integrin promotes cell proliferation, migration, and inva-sion (31, 33), and plays pivotal roles in tumorigenesis (8). In thepresent study, deletion of N-glycosylation sites in b4-integrinsuppressed those cellular functions in vitro as well as cell prolif-eration and tumorigenesis in vivo. GnT-V knockout mice havebeen reported to suppress polyomavirus middle T oncogene-induced mammary tumor growth and metastasis (19). Previousstudies have shown that an increase ofb1,6GlcNAc in thea3-,a5-,or b1-integrin subunit resulted in increased cancer cell migration(34, 35). Our findings indicate that modification of b4-integrin

withb1,6GlcNAcmaybeupregulated in tumor tissue. In addition,suppression of the b1,6GlcNAc modification in b4-integrin byoverexpression of GnT-III reduced b4-integrin–dependent cancercell migration and tumor formation. Therefore, this loss of b4-integrin functionby theN-glycosylation defectmaybemainly dueto a lack of b1,6GlcNAc modification in b4-integrin.

Galectin-3 has a high affinity for b1,6GlcNAc-branched N-glycans through multivalent binding, and thereby cross-linksglycoproteins on the cell surface and in the extracellular matrixto form molecular complexes (24). The formation of the macro-molecular complexes on the cell surface by galectin-3 affects thedistribution of glycoproteins and cellular signaling. Furthermore,integrin clustering mediated by galectin-3 promotes integrinactivation (25, 27). In our study, we found that the deletion ofN-glycans of b4-integrin, resulting in impaired galectin-3 bindingto b4, suppressed b4-integrin functions mainly through down-regulation of the PI3K pathway. These results support the hypoth-esis that the cross-linking between b1,6GlcNAc-branched N-gly-cans of b4-integrins by galectin-3 directly or indirectly affects PI3Kactivation and cellular functions such as cell motility and tumor-igenesis (Fig. 6); however, further investigation is required.

The association of b4-integrin with laminin-332 is known toinduce PI3K activation, thereby promoting cell adhesion andmigration (2). Our previous study showed that a decreased levelof b1,6GlcNAc-branched N-glycans on laminin-332 by

Figure 6.

Hypothetical model of N-glycosylation–mediated b4-integrin–dependent tumorprogression. Galectin-3 cross-linksbetween b1,6GlcNAc-branchedN-glycans, which in turn promotesb4-integrin clustering. The complexformation activates PI3K/Aktsignaling, thereby promoting tumorprogression by stimulating cellmigration, invasion, proliferation, andtumorigenesis.

Kariya et al.

introduction of bisecting GlcNAc suppressed its cell adhesion andmigration activity as well as galectin-3–mediated b4-integrinclustering (27). The present study showed that a defect of N-glycosylation in b4-integrin suppressed the association with lam-inin-332 and PI3K activation, suggesting that N-glycosylation ofb4-integrin played a critical role in PI3K activation through theinteraction with laminin-332. These results suggest that the asso-ciation between laminin-332 and b4-integrin throughb1,6GlcNAc-branched N-glycans may also be important for b4-integrin–dependent tumor progression.

In the present study, we cannot exclude the potential effect ofthe lack of N-glycosylation on b4-integrin protein folding. N-Glycosylation on integrins is important for such protein folding,which is required for heterodimer formation. In fact, the lack ofN-glycans on a5-integrin causes its misfolding and loss of hetero-dimer formationwithb1-integrin (36). In contrast,DNb4-integrincould form a heterodimer with a6-integrin (Supplementary Fig.S2B). Furthermore, FACS analysis showed that DNb4 expressedon the cell surface, which is comparable with WTb4 (Supplemen-tary Fig. S2A). In essence, noncomplexed integrin is degradedimmediately or remains in the endoplasmic reticulum (37).Therefore, the lack of N-glycans on b4-integrin seems to affectits function and the association with other molecules, rather thanb4-integrin folding.

In conclusion, our study suggests that N-glycosylation of b4-integrin is associated with tumor development and progressionthrough b4-integrin/PI3K signaling via the galectin-3–N-glycancomplex. N-Glycosylation of b4-integrin may therefore representa potential therapeutic target for cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Gu, Y. KariyaDevelopment of methodology: Y. Kariya, Y. KariyaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Hashimoto, Y. KariyaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Kariya, J. Gu, Y. KariyaWriting, review, and/or revision of the manuscript: Y. Kariya, J. Gu, Y. KariyaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Kariya, M. Oyama, Y. KariyaStudy supervision: J. Gu, Y. Kariya

AcknowledgmentsThis work was supported by the Japan Society for Promotion of Science

KAKENHI grant number 25860243, 17K08665 (Y. Kariya), 15H04354(J. Gu), and the Fukushima Medical University Research Project fromFukushima Medical University No. KKI28001 (Y. Kariya).

The authors are grateful to Dr. M. Peter Marinkovich (Stanford University,Standford, CA) for providing the cells. We thank the Fukushima MedicalUniversity English Editing Service for their English language review.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 11, 2017; revised February 7, 2018; accepted March 12, 2018;published first March 16, 2018.

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2018;16:1024-1034. Published OnlineFirst March 16, 2018.Mol Cancer Res Yukiko Kariya, Midori Oyama, Yasuhiro Hashimoto, et al.

-Glycan ComplexN−the Galectin-3 4-Integrin/PI3K Signaling Promotes Tumor Progression throughβ

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