Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, accounting for 8% to 10% of all pediatric malignancies.
1- Gurney J.G.
- Severson R.K.
- Davis S.
- Robison L.L.
Incidence of cancer in children in the United States: sex-, race-, and 1-year age-specific rates by histologic type.
This tumor arises from primitive neuroepithelial cells of the neural crest.
2Molecular biology of neuroblastoma.
The behavior of NB is markedly heterogeneous, ranging from highly undifferentiated tumors with very poor outcomes to well-differentiated benign ganglioneuroma or NB that may spontaneously regress with favorable prognosis.
3Neuroblastoma: biological insights into a clinical enigma.
Half of all patients with newly diagnosed NB are in a high-risk subset with poor overall survival despite intensive therapy. Therefore, it is important to develop useful prognostic tools and to understand NB pathogenesis to help design improved NB therapies.
Glycosylation is regulated spatiotemporally during development of the nervous system.
4- Breen K.C.
- Coughlan C.M.
- Hayes F.D.
The role of glycoproteins in neural development function, and disease.
Altered carbohydrate structures on tumors are often associated with tumor metastasis and progression. Tumor-associated carbohydrate epitopes commonly found in cancers include GM2, GD2, GD3, T, Tn, Globo H, Lewis y, sialyl Lewis x, sialyl Lewis a, and polysialic acid.
5The sweet and sour of cancer: glycans as novel therapeutic targets.
, 6Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines.
Among these, GM2, GD2, and GD3 are expressed in NB.
6Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines.
Changes in expression levels of glycogenes may play an important role in alterations of carbohydrate structures in tumors. However, the differential expression of these molecules in NB and their effects on tumor cell behavior are poorly understood.
β1,4-
N-acetylgalactosaminyltransferase III (B4GALNT3) has been cloned and is expressed in various tissues.
7- Sato T.
- Gotoh M.
- Kiyohara K.
- Kameyama A.
- Kubota T.
- Kikuchi N.
- Ishizuka Y.
- Iwasaki H.
- Togayachi A.
- Kudo T.
- Ohkura T.
- Nakanishi H.
- Narimatsu H.
Molecular cloning and characterization of a novel human β1,4-N-acetylgalactosaminyltransferase, β4GalNAc-T3, responsible for the synthesis of N,N′-diacetyllactosediamine, galNAc β1-4GlcNAc.
This enzyme can transfer GalNAc to any nonreducing terminal GlcNAc-β
in vitro, resulting in synthesis of GalNAcβ1,4GlcNAc (LacdiNAc or LDN). The terminal β1,4–linked GalNAc of LDN can be recognized by a lectin,
Wisteria floribunda agglutinin (WFA). This special terminal β1,4GalNAc structure is found in certain glycoproteins and glycohormones, including lutropin,
8- Green E.D.
- van Halbeek H.
- Boime I.
- Baenziger J.U.
Structural elucidation of the disulfated oligosaccharide from bovine lutropin.
thyrotropin,
8- Green E.D.
- van Halbeek H.
- Boime I.
- Baenziger J.U.
Structural elucidation of the disulfated oligosaccharide from bovine lutropin.
, 9- Green E.D.
- Baenziger J.U.
Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin, II: distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones.
, 10- Green E.D.
- Baenziger J.U.
Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin, I: structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones.
proopiomelanocortin,
11- Skelton T.P.
- Kumar S.
- Smith P.L.
- Beranek M.C.
- Baenziger J.U.
Pro-opiomelanocortin synthesized by corticotrophs bears asparagine-linked oligosaccharides terminating with SO4-4GalNAcβ1,4GlcNAcβ1,2Manα.
, 12- Siciliano R.A.
- Morris H.R.
- Bennett H.P.
- Dell A.
O-glycosylation mimics N-glycosylation in the 16-kDa fragment of bovine pro-opiomelanocortin: the major O-glycan attached to Thr-45 carries SO4-4GalNAcβ1-4GlcNAc β1-, which is the archetypal non-reducing epitope in the N-glycans of pituitary glycohormones.
, 13- Siciliano R.A.
- Morris H.R.
- McDowell R.A.
- Azadi P.
- Rogers M.E.
- Bennett H.P.
- Dell A.
The Lewis x epitope is a major non-reducing structure in the sulphated N-glycans attached to Asn-65 of bovine pro-opiomelanocortin.
and the sorting protein–related receptor SorLA/LR11.
14- Fiete D.
- Mi Y.
- Oats E.L.
- Beranek M.C.
- Baenziger J.U.
N-linked oligosaccharides on the low density lipoprotein receptor homolog SorLA/LR11 are modified with terminal GalNAc-4-SO4 in kidney and brain.
SorLA/LR11, highly expressed by neurons in the central and peripheral nervous systems, bears
N-linked oligosaccharides modified with terminal β1,4–linked GalNAc-4-SO
4 that can be synthesized by B4GALNT3 in CHO cells.
14- Fiete D.
- Mi Y.
- Oats E.L.
- Beranek M.C.
- Baenziger J.U.
N-linked oligosaccharides on the low density lipoprotein receptor homolog SorLA/LR11 are modified with terminal GalNAc-4-SO4 in kidney and brain.
In
Drosophila, β1,4-
N-acetylgalactosaminyltransferase A (B4GALNTA) catalyzes synthesis of LacdiNAc on glycoconjugates.
15- Stolz A.
- Haines N.
- Pich A.
- Irvine K.D.
- Hokke C.H.
- Deelder A.M.
- Gerardy-Schahn R.
- Wuhrer M.
- Bakker H.
Distinct contributions of β4GalNAcTA and β4GalNAcTB to Drosophila glycosphingolipid biosynthesis.
Drosophila mutants deficient in
B4GALNTA have defects in behavior and in the neuromuscular system.
16Functional roles for β,4-N-acetlygalactosaminyltransferase-A in Drosophila larval neurons and muscles.
In the present study, we hypothesized that B4GALNT3 may be involved in the pathogenesis of NB development.
Materials and Methods
Patients and Treatment
Between December 1, 1990, and December 31, 2007, 102 patients with NB were treated at National Taiwan University Hospital. The use of human tissues for this study was approved by the National Taiwan University Hospital Ethics Committee, and written consent was obtained from patients before the collection of samples. Eighty-seven of these 102 patients had complete follow-up and sufficient prechemotherapy tumor tissues for evaluation and were enrolled in this study. The median age at diagnosis was 2.5 years (range, 0 to 11.5 years). Male patients were slightly predominant, with a male/female ratio of 49:38. Most tumors (52 cases) originated primarily from the adrenal gland, with others from the retroperitoneum (19 cases), mediastinum (7 cases), neck (5 cases), and pelvis (4 cases). According to the criteria of the International Neuroblastoma Pathology Classification,
17- Shimada H.
- Ambros I.M.
- Dehner L.P.
- Hata J.
- Joshi V.V.
- Roald B.
Terminology and morphologic criteria of neuroblastic tumors: recommendations by the International Neuroblastoma Pathology Committee.
, 18- Shimada H.
- Ambros I.M.
- Dehner L.P.
- Hata J.
- Joshi V.V.
- Roald B.
- Stram D.O.
- Gerbing R.B.
- Lukens J.N.
- Matthay K.K.
- Castleberry R.P.
The International Neuroblastoma Pathology Classification (the Shimada system).
, 19- Hsu W.M.
- Hsieh F.J.
- Jeng Y.M.
- Kuo M.L.
- Tsao P.N.
- Lee H.
- Lin M.T.
- Lai H.S.
- Chen C.N.
- Lai D.M.
- Chen W.J.
GRP78 expression correlates with histologic differentiation and favorable prognosis in neuroblastic tumors.
the histologic morphologic features of the tumors were categorized into four types: i) NB (including undifferentiated, poorly differentiated, and differentiating subtypes); ii) ganglioneuroblastoma (GNB), intermixed; iii) GNB, nodular; and iv) GNB, maturing subtype. Because GNB, maturing subtype is a benign lesion, it is not included in this study. For a simpler description of the relationship between B4GALNT3 expression and the histologic grades of tumor differentiation, we defined undifferentiated NB (UNB), poorly differentiated and differentiating NB (DNB), and GNB, intermixed for tumor histologic statement in this study. The GNB, nodular subtype was classified into either UNB or DNB according to the morphologic features of their NB nodules because the tumor behavior of this subtype of tumors depends mainly on their NB nodules.
18- Shimada H.
- Ambros I.M.
- Dehner L.P.
- Hata J.
- Joshi V.V.
- Roald B.
- Stram D.O.
- Gerbing R.B.
- Lukens J.N.
- Matthay K.K.
- Castleberry R.P.
The International Neuroblastoma Pathology Classification (the Shimada system).
For prognostic analysis, GNB, intermixed was classified as a favorable histologic type, and UNB and DNB were classified as either a favorable or an unfavorable histologic type according to the mitosis-karyorrhexis index and patient age at diagnosis based on the criteria of the International Neuroblastoma Pathology Classification.
17- Shimada H.
- Ambros I.M.
- Dehner L.P.
- Hata J.
- Joshi V.V.
- Roald B.
Terminology and morphologic criteria of neuroblastic tumors: recommendations by the International Neuroblastoma Pathology Committee.
, 18- Shimada H.
- Ambros I.M.
- Dehner L.P.
- Hata J.
- Joshi V.V.
- Roald B.
- Stram D.O.
- Gerbing R.B.
- Lukens J.N.
- Matthay K.K.
- Castleberry R.P.
The International Neuroblastoma Pathology Classification (the Shimada system).
Tumors were staged according to the International Neuroblastoma Staging System.
20- Brodeur G.M.
- Pritchard J.
- Berthold F.
- et al.
Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment.
The MYCN status of the tumor tissue was evaluated by fluorescence
in situ hybridization analysis of formalin-fixed, paraffin-embedded tissues or single fresh tumor cells.
21- Tajiri T.
- Shono K.
- Fujii Y.
- Noguchi S.
- Kinoshita Y.
- Tsuneyoshi M.
- Suita S.
Highly sensitive analysis for N-myc amplification in neuroblastoma based on fluorescence in situ hybridization.
, 22- Kopf I.
- Hanson C.
- Delle U.
- Verbiene I.
- Weimarck A.
A rapid and simplified technique for analysis of archival formalin-fixed, paraffin-embedded tissue by fluorescence in situ hybridization (FISH).
Based on the risk classifications of the Children's Cancer Group, patients were classified into low-, intermediate-, and high-risk groups and were treated with either surgery alone or a combination of multiple modalities, including chemotherapy, radiotherapy, and/or autologous bone marrow transplantation.
Mean follow-up after diagnosis was 70.6 months (range, 1 to 204 months), and overall predictive 5-year survival in this cohort was 48.7%.
IHC Analysis
Human NB tissue sections were deparaffinized in xylene and rehydrated in a series of graded alcohols. After quenching the activity of endogenous peroxidase with 1% H2O2 in PBS for 10 minutes, the sections were rinsed three times with PBS and then were incubated with 5% nonfat milk/PBS for 30 minutes to reduce nonspecific binding. Sections were incubated with an anti-B4GALNT3 polyclonal antibody (1:400; Sigma-Aldrich, St. Louis, MO) diluted with 5% nonfat milk/PBS for 16 hours at 4°C. After rinsing twice with PBS, the Super Sensitive link-label immunohistochemical (IHC) detection system (BioGenex, San Ramon, CA) was used, and specific immunostaining was visualized using 3,3-diaminobenzidine liquid substrate system (Sigma-Aldrich). All the sections were counterstained with hematoxylin for 1 minute and were mounted with UltraKitt (J.T. Baker, Deventer, Holland). Negative controls were performed by replacing primary antibodies with a control nonimmune IgG at the same concentration. To further confirm the specificity of staining, B4GALNT3 peptide (10 μg/mL) was used to block binding of anti-B4GALNT3 antibody to tissues.
Cell Culture and Transfection
NB cell lines SK-N-SH and SH-SY5Y, from American Type Culture Collection (Manassas, VA), were maintained using Dulbecco's modified Eagle's medium (DMEM; JRH Biosciences, Lenexa, KS) containing 10% fetal bovine serum (FBS) (PAA Laboratories GmbH, Pasching, Austria) in a humidified tissue culture incubator at 37°C, in a 5% CO
2 atmosphere. For stable transfection, 4 μg of
B4GALNT3/pcDNA3.1 (a gift from Dr. Hisashi Narimatsu, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan) or pcDNA3.1/myc-His (Invitrogen, Life Technologies Inc., Grand Island, NY) was transfected into 5 × 10
5 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After transfection for 24 hours, cells were trypsinized and plated on three 100-mm dishes with 10% FBS-DMEM containing 400 μg/mL of G418 (Calbiochem, Darmstadt, Germany). After 10 days of selection, G418-resistant clones were pooled and transferred to 6-well plates. Expression levels of B4GALNT3 were analyzed by RT-PCR and immunoblotting. For transient expression of constitutively active Akt and MEK, pCIS2/Akt and pCMV/MEK plasmids were used. The pCIS2/Akt plasmids were constructed as previously described.
24- Montagnani M.
- Ravichandran L.V.
- Chen H.
- Esposito D.L.
- Quon M.J.
Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells.
The pCMV/MEK plasmids were a gift from Dr. Zee-Fen Chang (Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University).
25- Chen C.H.
- Wang W.J.
- Kuo J.C.
- Tsai H.C.
- Lin J.R.
- Chang Z.F.
- Chen R.H.
Bidirectional signals transduced by DAPK-ERK interaction promote the apoptotic effect of DAPK.
The empty pCIS2 and pCMV vectors were used as control vectors, respectively.
RT-PCR and Real-Time PCR
Total cellular RNA was isolated from cells grown to 70% confluence using TRIzol reagent (Invitrogen) according to manufacturer protocols as previously described.
26- Huang M.
- Laskowska A.
- Vestweber D.
- Wild M.
The α(1,3)-fucosyltransferase Fuc-TIV, but not Fuc-TVII, generates sialyl Lewis X-like epitopes preferentially on glycolipids.
For cDNA synthesis, 2 μg of total RNA was used as template in a 25-μL reverse transcription reaction. PCR reactions were incubated for 5 minutes at 95°C, followed by 35 amplification cycles with 30 seconds of denaturation at 94°C, 30 seconds of annealing at 60°C, and 30 seconds of extension at 72°C. For real-time PCR, the quantitative PCR system Mx3000P (Stratagene, La Jolla, CA) was used to analyze gene expression according to the manufacturer's protocol. Briefly, reactions were performed in a 25-μL volume with 2 μL of cDNA, 400 nmol/L each of sense and antisense primers, and 12.5 μL of Brilliant SYBR Green QPCR Master Mix (Stratagene). For
GAPDH detection, sense and antisense primers were 5′-ACAGTCAGCCGCATCTTCTT-3′ and 5′-GACAAGCTTCCCGTTCTCAG-3′, respectively, generating a 259-bp fragment. For detection of
B4GALNT3, sense and antisense primers were 5′-CTACAGCGCATTGTGAACGT-3′ and 5′-TGGTTCTTCACAGGCACGAC-3′, respectively, generating a 320-bp fragment. The PCR products were confirmed to be correct by DNA sequencing.
Immunoblotting and Immunoprecipitation
To confirm expression of B4GALNT3 in differentiated NB cells, SK-N-SH cells were treated with dimethyl sulfoxide (0.1%) or all-trans retinoic acid (ATRA, 10 μmol/L; Sigma-Aldrich) for 3 days. Equal amounts of cell lysates (20 μg of total protein) were subjected to SDS-PAGE, and the gel contents were transferred to polyvinylidene difluoride membrane. Membranes were incubated with mouse anti–glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody (BIODESIGN International, Saco, MA) or anti-B4GALNT3 polyclonal antibody, whose specificity has been demonstrated. Neuronal differentiation was detected by mouse monoclonal antibodies, anti-GAP-43 (clone GAP-7B10; Sigma-Aldrich) and anti–neuron-specific enolase (Chemicon, Billerica, MA). A biotinylated lectin, WFA (Vector Laboratories, Burlingame, CA), was used to detect the LacdiNAc structure in primary tumors. For detecting the LacdiNAc structure on cell surface proteins, intact cells were surface biotinylated with sulfosuccinimidyl biotin (Pierce Chemical Co., Rockford, IL), lysed, and then pulled down with WFA agarose beads (Vector Laboratories). The pulled-down proteins were detected using horseradish peroxidase–conjugated streptavidin (Santa Cruz Biotechnology). For cell signaling analyses, we used rabbit anti–β1 integrin antibody (BD Transduction Laboratories, San Jose, CA), mouse anti-phosphotyrosine (pY) antibody (4G10; Upstate Biotechnology Inc., Lake Placid, NY), rabbit anti-FAK pY397 polyclonal antibody (Biosource, Nivelles, Belgium), rabbit anti-FAK polyclonal antibody (C-20; Santa Cruz Biotechnology), anti-paxillin pY118 (BD Transduction Laboratories, Heidelberg, Germany), anti-paxillin polyclonal antibody (BD Transduction Laboratories, Heidelberg), rabbit anti-pERK1/2 monoclonal antibody, rabbit anti-pAkt, and mouse anti-pan Akt monoclonal antibody (Cell Signaling Technology Inc., Beverly, MA), rabbit anti-ERK1/2 antibody (Santa Cruz Biotechnology), rabbit anti-Src pY418 antibody (Invitrogen), and rabbit anti-c-Src antibody (Invitrogen). Immunoblotted membranes were then incubated with horseradish peroxidase–conjugated streptavidin, horseradish peroxidase–conjugated anti-rabbit IgG, or anti-mouse IgG (Santa Cruz Biotechnology). Signals were visualized using ECL reagents (Amersham Biosciences, Piscataway, NJ), and images were quantified using ImageQuant 5.1 (Amersham Biosciences). For immunoprecipitation, 1 mg of cell extracts were incubated with WFA agarose beads (Vector Laboratories) or protein G sepharose 4 Fast Flow (GE Healthcare UK Ltd., Little Chalfont, UK) conjugated with 2 μg of antibody overnight at 4°C.
Cell Growth Analysis
Cells were plated in triplicate wells of 96-well plates at a density of 3 × 103 cells per well. After incubation for 24 or 48 hours, 3-(4,5-dimethyl-2 thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide solution (MTT; Sigma-Aldrich) was added to each well at a final concentration of 0.5 mg/mL and was incubated for 4 hours to allow MTT reduction. Formazan crystals were dissolved in a solution containing 0.01 mol/L HCl and 10% (w/v) SDS, and absorbance was measured at the dual wavelengths of 570 and 630 nm using a spectrophotometer.
Anchorage-Independent Growth in Soft Agar
Cells (1 × 104) in 0.3% (w/v) Bacto Agar (Sigma-Aldrich) in DMEM supplemented with 10% FBS were overlaid on a base of 0.6% Bacto Agar in DMEM supplemented with 10% FBS in 6-well plates. Cells were incubated at 37°C, 5% CO2 atmosphere. Triplicate wells were used for each cell line, and three independent experiments were performed. The number of colonies with a diameter >30 μm was counted on day 7.
Migration and Invasion Assay
Transwell motility assays were performed using 8-μm pore, 6.5-mm polycarbonate Transwell filters (Corning Costar Corp., Cambridge, MA). Single-cell suspensions (3 × 105 cells) were seeded in serum-free DMEM medium onto the upper surface of the filters and were allowed to migrate toward 10% fetal calf serum/DMEM or 1 μg/mL of extracellular matrix proteins (collagen IV, fibronectin, and laminin) (Sigma-Aldrich). After a 48-hour incubation period, cells that had migrated to the underside of the filter were fixed, stained with 0.5% crystal violet (Sigma-Aldrich), and counted under a microscope at ×100 in five random fields.
Cell invasion assays were performed in BioCoat Matrigel invasion chambers (Becton-Dickinson, Bedford, MA) according to manufacturer protocols. Briefly, 500 μL of DMEM containing 10% FBS was loaded in the lower part of the chamber, and 3 × 105 cells in 500 μL of serum-free DMEM were seeded to the upper part. Cells were allowed to invade the Matrigel for 48 hours in a humidified tissue culture incubator at 37°C, 5% CO2 atmosphere. Noninvading cells on the upper surface of the membrane were removed from the chamber, and the invading cells on the lower surface of the membrane were fixed with 100% methanol and were stained with 0.5% crystal violet. Invading cells were counted in each well under a phase contrast microscope. The mean ± SD values were calculated from the numbers of invading cells from three independent experiments under the microscope.
To determine the contribution of β1 integrin to migration and invasion, cells were preincubated with 10 μg/mL of β1 integrin blocking antibody (clone P4C10; Millipore Corp., Billerica, MA) or 10 μg/mL of control mouse nonimmune IgG antibody (SouthernBiotech, Birmingham, AL) for 30 minutes at 37°C.
Statistical Analysis
SPSS 10.0 for Windows software (SPSS Inc., Chicago, IL) was used to perform the statistical analyses. The comparison between pairs of categorical variables was evaluated using Pearson's χ2 test. The significance of the variations between the data resulting from different treatments was assessed by Student's t-test. Kaplan-Meier method was used for estimation of survival probabilities in various subgroups, and the significant differences in each group were analyzed by log-rank tests. Each variable possibly affecting patient survival was further evaluated by univariate and multivariate Cox proportional hazards model analysis. All statistical tests were two-sided, and those with a P < 0.05 were considered significant.
Discussion
B4GALNT3 has
in vitro activity to promote the synthesis of LacdiNAc, an important structure found on glycoproteins expressed by neurons.
14- Fiete D.
- Mi Y.
- Oats E.L.
- Beranek M.C.
- Baenziger J.U.
N-linked oligosaccharides on the low density lipoprotein receptor homolog SorLA/LR11 are modified with terminal GalNAc-4-SO4 in kidney and brain.
Herein, we demonstrated that B4GALNT3 was highly expressed in either differentiated NB or mature ganglion cells. However, its expression was markedly decreased in UNB cells. B4GALNT3 expression correlates well with early clinical stages and, hence, predicts a favorable outcome in patients with NB. Reexpression of B4GALNT3 in NB cells resulted in reduction of malignant phenotypes, including cell growth, colony formation, migration, and invasion. Moreover, these data show that these phenotypic changes caused by B4GALNT3 expression are likely mediated through suppression of β
1 integrin–mediated signaling and attenuation of Akt and ERK activation. Thus, B4GALNT3 may play a critical role in suppressing the malignant properties of NB, and its altered expression may contribute to the pathogenesis of NB.
NB is a heterogeneous tumor. The treatment of NB is based on patient risk grouping according to various clinicopathologic and biologic factors.
Detailed evaluation of biologic markers of NB is crucial for tailored therapy for each patient. Of the clinical and biologic prognostic factors of NB, clinical stage and MYCN status are the two most important factors for risk grouping of patients.
However, the prognosis of patients with NB with the same clinical stage or MYCN status still varies. The present study revealed that B4GALNT3 expression could discriminate the prognostic difference among patients with NB and any clinical stages and patients with NB and a normal MYCN status (
Figure 2). The result suggested that assessing B4GALNT3 by simple IHC staining may provide additional prognostic information complementary to clinical stage and MYCN status, which, in turn, may allow the physician to determine the most appropriate therapy for each patient with NB.
The present result demonstrated that B4GALNT3 expression strongly correlated with a favorable tumor histologic profile. However, in multivariate survival analysis, only B4GALNT3 expression but not tumor histologic profile predicted prognosis independently. This finding suggests that B4GALNT3 expression may inhibit the NB cell malignant phenotype by alternative mechanisms in addition to affecting cell differentiation. The present cell line studies demonstrated that B4GALNT3 expression significantly suppressed cell growth, colony formation, migration, and invasion.
Another important finding is that B4GALNT3 reexpression significantly inhibited phosphorylation of β
1 integrin and its downstream signaling molecules FAK, Src, and paxillin. Integrins play a crucial role in cell survival, differentiation, migration, and invasion through the recruitment and activation of signaling proteins such as FAK and c-Src.
28- Mitra S.K.
- Schlaepfer D.D.
Integrin-regulated FAK-Src signaling in normal and cancer cells.
The FAK-Src complex binds to and phosphorylates various adaptor proteins, including paxillin, which serves as a platform for the recruitment of numerous proteins that together control the cytoskeletal reorganization and gene expression that are necessary for cell migration and survival.
Activated FAK-Src complex in many tumor cells generates signals leading to tumor growth and metastasis.
28- Mitra S.K.
- Schlaepfer D.D.
Integrin-regulated FAK-Src signaling in normal and cancer cells.
Moreover, the paxillin-ERK complex plays a role in cell survival and motility.
30- Ishibe S.
- Joly D.
- Liu Z.X.
- Cantley L.G.
Paxillin serves as an ERK-regulated scaffold for coordinating FAK and Rac activation in epithelial morphogenesis.
Furthermore, ERK can regulate cell invasion by modulating the proteolytic enzymes that degrade the basement membrane.
31- Reddy K.
- Nabha S.
- Atanaskova N.
Role of MAP kinase in tumor progression and invasion.
We showed that B4GALNT3 suppresses ERK phosphorylation. In addition, we found that Akt activation is significantly suppressed by B4GALNT3. Activation of Akt predicts poor outcome in NB and inhibits TRAIL (tumor necrosis factor–related apoptosis-inducing ligand)- or chemotherapy-induced apoptosis of NB cells.
32- Opel D.
- Poremba C.
- Simon T.
- Debatin K.M.
- Fulda S.
Activation of Akt predicts poor outcome in neuroblastoma.
The present data also showed that constitutively active Akt or MEK can significantly reverse the suppressive effect of B4GALNT3 on migration and invasion. Thus, decreased ERK and Akt signaling in NB may be essential for the phenotypic changes caused by B4GALNT3 expression. Because ERK and Akt are important downstream signaling molecules for integrins and receptor tyrosine kinases, it is possible that B4GALNT3 affects signaling through both types of receptors on the cell surface. Although a detailed mechanism explaining how these signaling pathways affect NB requires further investigation, the present data strongly suggest that B4GALNT3 is an effective suppressor of NB cell malignancy.
There are several possible mechanisms that determine how B4GALNT3 regulates cell signaling. One possibility is that B4GALNT3 modifies carbohydrates on integrins, followed by conformational changes that, in turn, modulate cell signaling and cellular properties. For example, GnT-V, GnT-III, ST6GalNAc I, and ST6Gal-I directly modify carbohydrate structures on β
1 integrin and affect integrin activity.
33- Seales E.
- Jurado G.
- Brunson B.
- Wakefield J.
- Frost A.
- Bellis S.
Hypersialylation of beta1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility.
, 34- Isaji T.
- Gu J.
- Nishiuchi R.
- Takahashi M.
- Miyoshi E.
- Honke K.
- Honke K.
- Sekiguchi K.
- Taniguchi N.
Introduction of bisecting GlcNAc into integrin α5β1 reduces ligand binding and down-regulates cell adhesion and cell migration.
, 35- Guo H.
- Lee I.
- Kamar M.
- Akiyama S.
- Pierce M.
Aberrant N-glycosylation of β1 integrin causes reduced α5β1 integrin clustering and stimulates cell migration.
, 36- Clement M.
- Rocher J.
- Loirand G.
- Le Pendu J.
Expression of sialyl-Tn epitopes on β1 integrin alters epithelial cell phenotype, proliferation and haptotaxis.
These changes in
N-glycosylation
33- Seales E.
- Jurado G.
- Brunson B.
- Wakefield J.
- Frost A.
- Bellis S.
Hypersialylation of beta1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility.
, 37- Seales E.
- Jurado G.
- Singhal A.
- Bellis S.
Ras oncogene directs expression of a differentially sialylated, functionally altered β1 integrin.
or
O-glycosylation
36- Clement M.
- Rocher J.
- Loirand G.
- Le Pendu J.
Expression of sialyl-Tn epitopes on β1 integrin alters epithelial cell phenotype, proliferation and haptotaxis.
of β
1 integrin lead to altered cell morphologic features and behavior. B4GALNT3 modifies
N- and
O-glycans decorated with GlcNAc
in vitro.
7- Sato T.
- Gotoh M.
- Kiyohara K.
- Kameyama A.
- Kubota T.
- Kikuchi N.
- Ishizuka Y.
- Iwasaki H.
- Togayachi A.
- Kudo T.
- Ohkura T.
- Nakanishi H.
- Narimatsu H.
Molecular cloning and characterization of a novel human β1,4-N-acetylgalactosaminyltransferase, β4GalNAc-T3, responsible for the synthesis of N,N′-diacetyllactosediamine, galNAc β1-4GlcNAc.
In the present study, we found that B4GALNT3 adds LacdiNAc to β
1 integrin and, thereby, suppresses its downstream signaling. Glycolipids on the cell surface may interact with functional membrane proteins, such as integrins, growth factor receptors, and Src family kinases to form glycosynaptic microdomains that control cell adhesion, growth, and motility.
38- Regina Todeschini A.
- Hakomori S.I.
Functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains.
Because B4GALNT3 also exhibits enzymatic activity to modify glycolipids with the LacdiNAc structure, it is possible that B4GALNT3 modulates integrin signaling and tumor cell behavior via changing the carbohydrate structures of glycolipids at the glycosynapse.
In summary, we demonstrate, for the first time, that B4GALNT3 may play a critical role in regulating NB malignancy. Expression of B4GALNT3 positively correlates with the differentiation status of NB. We present evidence that B4GALNT3 expression suppresses malignant properties of NB cells by modifying β1 integrin with LacdiNAc to inhibit downstream signaling through FAK, Src, and paxillin. The present findings suggest that B4GALNT3 inhibits cell migration and invasion primarily through β1 integrin. These effects of B4GALNT3 on migration and invasion are dependent on Akt and ERK signaling pathways.
Article info
Publication history
Published online: July 11, 2011
Accepted:
May 9,
2011
Footnotes
Supported by the Frontier and Innovative Research Program of National Taiwan University (grant 95R0101 to M.-C.H.), by the National Health Research Institute (grants NHRI-EX97-9410BC to M.-C.H. and NHRI-EX96-9620NI to W.-M.H.), by the National Science Council (R.O.C. grants NSC98-2320-B-002-032-MY3 to M.-C.H. and NSC99-2628-B-002-056-MY3 to W.-M.H.), and, in part, by the Intramural Research Program of the National Center for Complementary and Alternative Medicine, National Institutes of Health (M.J.Q.).
W.-M.H. and M.-I.C. contributed equally to this work.
Copyright
© 2011 American Society for Investigative Pathology. Published by Elsevier Inc.