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Departments of 1 Surgery and 2 Pathology, National Taiwan University Hospital; 3 Department of Plant Pathology and Microbiology, National Taiwan University; and 4 Institute of Anatomy and Cell Biology and 5 Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan; and 6 Animal Technology Institute Taiwan, Miaoli, Taiwan
Requests for reprints: Min-Chuan Huang, Institute of Anatomy and Cell Biology, National Taiwan University College of Medicine, No. 1, Sec. 1 Jen-Ai Road, Taipei 100, Taiwan. Phone: 886-2-2312-3456, ext. 8177; Fax: 886-2-2391-5292. E-mail: mchuang{at}ntu.edu.tw
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Abstract |
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 TopNotes Abstract IntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Introduction |
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TopNotesAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Carbohydrate changes on cell surfaces play important roles in cell-cell and cell-extracellular matrix (ECM) interactions. Integrins, surface glycoprotein receptors for ECM, have been suggested to regulate cell-ECM adhesion, cell migration, invasion, and cell survival (7). Several reports show that alterations of integrin glycosylation affect cancer cell migration and invasion. ST6Gal-I has been shown to sialylate ß1 integrins and up-regulate colon cancer cell migration (8). GnT-III introduces bisecting GlcNAc into integrin 
5ß1 and down-regulates cell adhesion and migration (9). Aberrant N-glycosylation of ß1 integrin induced by GnT-V causes reduced 5ß1 integrin clustering and stimulates cell migration (10). Furthermore, overexpression of ST6GalNAc I causes a significant change in the O-glycosylation of integrin ß1 chain and leads to alterations of cell morphology and behavior (11). Recently, we found that C2GnT-M overexpression decreases colon cancer cell growth and tyrosine phosphorylation of paxillin, which is an important downstream signaling molecule of integrins (12). In addition, ß3Gn-T6 was found to decrease core 1 structure and suppress metastatic potential of colon carcinoma cells through an unclear mechanism (13). These studies suggest that glycosyltransferases can regulate cancer cell behavior through alterations of integrin glycosylation or through other mechanisms which remain to be identified.
ß4GalNAc-T3 has been shown to be significantly expressed in the gastrointestinal tract (14). In addition, the recombinant enzyme can transfer GalNAc to GlcNAc and form N,N'-diacetyllactosediamine (GalNAcß1,4GlcNAc) in both N- and O-glycans by an in vitro enzyme activity assay. However, the biological and pathologic functions of ß4GalNAc-T3 in the gastrointestinal tract remain unclear.
In the present study, we show that ß4GalNAc-T3 is frequently up-regulated in primary colon tumors when compared with their normal mucosa. To elucidate the effects of ß4GalNAc-T3 on colon cancer cell behavior, ß4GalNAc-T3 was overexpressed in HCT116 cells. Cell adhesion, migration, invasion, and anchorage-independent cell growth in vitro, as well as tumor growth and metastasis in vivo, were significantly enhanced by ß4GalNAc-T3 overexpression. The increase in malignant phenotypes was associated with activation of focal adhesion kinase (FAK) and extracellular signal–regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signalings. These results suggest that up-regulation of ß4GalNAc-T3 may contribute to the malignant behavior of colon cancer cells.
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Results |
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TopNotesAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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ß4GalNAc-T3 Increases Cell Migration
To examine whether ß4GalNAc-T3 overexpression can regulate cell migration, a monolayer wound healing assay was done. We found that the migration of ß4GalNAc-T3 transfectants was significantly faster than that of mock transfectants (Fig. 3A
). The average migration rates of mock and ß4GalNAc-T3 transfectants were 3.3 and 9.6 to 12.5 µm/h, respectively (Fig. 3B). In addition, we observed that ß4GalNAc-T3 overexpression enhanced HCT116 cell migration by Boyden chamber cell migration assays (data not shown). These stimulatory effects of ß4GalNAc-T3 on cell migration were not due to increased rates of cell proliferation because we could not detect significant changes in growth rate over a 16-h period among the two transfectants. These results suggest that ß4GalNAc-T3 expression significantly increases the motility of colon cancer cells.
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ß4GalNAc-T3 Enhances Tumor Growth in Nude Mice
To test whether ß4GalNAc-T3 overexpression can alter tumor growth in vivo, we inoculated BALB/c (nu/nu) mice subcutaneously with mock or ß4GalNAc-T3 stable transfectants. At day 20 after inoculation, mice were sacrificed and tumors were excised for analyses. The ß4GalNAc-T3 tumors showed an average wet weight of 1.64 to 1.97 g compared with 0.20 g in mock tumors (n = 5 tumors each; P < 0.001; Fig. 6A
), representing an 8- to 10-fold increase in tumor mass. The size of ß4GalNAc-T3 tumors was homogeneous and much larger than mock tumors (Fig. 6B). Macroscopically, hypervascularity was observed on the surface of all ß4GalNAc-T3 tumors (Fig. 6B). In contrast, blood vessels were rarely present on mock tumors. Microscopically, vascularization of ß4GalNAc-T3 tumors was dramatically increased compared with poorly vascularized mock tumors (data not shown). These results suggest active angiogenesis in ß4GalNAc-T3 tumors. In addition, we found that all ß4GalNAc-T3 tumors showed severe invasion into not only adjacent skeletal muscles but also the peritoneum. In sharp contrast, mock tumors showed intact capsules without local invasion. These results suggest that ß4GalNAc-T3 is a potent stimulator of tumor growth in vivo.
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For tumor metastasis analysis, nude mice were injected with mock transfectants (control, n = 5) or ß4GalNAc-T3 transfectants (n = 5 for each clone) through tail vein. We found that severe lung metastasis of colon cancer cells was observed in mice injected with ß4GalNAc-T3 transfectants (tumor nodules, n = 17-22) but not mock transfectants (tumor nodules, n = 3; Fig. 6E). In addition, the metastasis was confirmed by H&E staining of lungs (Fig. 6F). These findings suggest that ß4GalNAc-T3 overexpression can increase tumor metastasis and decrease survival.
Effects of ß4GalNAc-T3 Expression on Tyrosine Phosphorylation of FAK and Paxillin as well as MAPK Phosphorylation
Our data showed that ß4GalNAc-T3 transfectants significantly enhanced cell-ECM adhesion, migration, and invasion. Because integrin-ECM interactions have been shown to play key roles in regulating these phenotypic changes, we investigated whether integrin-mediated signalings could be regulated by ß4GalNAc-T3 overexpression. Cells were replated on fibronectin-coated culture dishes under serum-free conditions and tyrosine phosphorylation of FAK and paxillin, key signaling molecules downstream of integrins, was analyzed by Western blot. As shown in Fig. 7A
, there was no significant difference in FAK expression between ß4GalNAc-T3 and mock transfectants. However, tyrosine phosphorylation levels of FAK in ß4GalNAc-T3 stable transfectants were significantly increased compared with mock transfectants, as detected by both monoclonal antibodies 4G10 and PY20. We also found that FAK pY397 and pY576 were increased in these cells (Fig. 7B). In addition, an increase in paxillin pY118 in ß4GalNAc-T3 transfectants was observed, with total paxillin levels unchanged (Fig. 7C). Interestingly, paxillin pY31 was decreased in ß4GalNAc-T3 transfectants. Only results from clone 3 were shown. Similar results were also observed in clone 5 (data not shown).
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Discussion |
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TopNotesAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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The ability to migrate and invade through ECM proteins into surrounding tissues is essential for tumor progression and metastasis. Here, we found that ß4GalNAc-T3 overexpression significantly enhances cell adhesion, migration, and invasion. Integrins are important surface receptors to mediate these cell-ECM interactions, and integrin-mediated signalings through FAK and paxillin have been suggested to play important roles in malignant behavior of cancer cells (7, 15-19). Recently, changes in expression levels of glycosyltransferases have been found to alter integrin signaling and thereby regulate cell behavior. For example, GnT-III overexpression inhibits cell spreading and migration on fibronectin and decreases FAK phosphorylation (9). ST6Gal-I overexpression results in increased association of ß1 integrin and talin, indicating that integrin signaling is activated (8). Interestingly, we found that both integrin-mediated autophosphorylation of FAK at Y397 and Src family–dependent tyrosine phosphorylation of FAK at Y576 were increased in ß4GalNAc-T3 transfectants. Y397 is the major FAK autophosphorylation site and its phosphorylation is critical for subsequent binding of Src family kinases. Src-mediated transphosphorylation of Y576 within the kinase domain maximizes the catalytic activity of FAK (20). In addition, we found that tyrosine phosphorylation of FAK-Src substrate paxillin at Tyr118 was also increased. These results strongly suggest that ß4GalNAc-T3 is capable of inducing integrin-mediated FAK signaling pathways. Notably, we found that phosphorylation levels of paxillin Y31 were decreased in ß4GalNAc-T3 transfectants, although cell-ECM adhesion and cell migration were increased, suggesting that pY118, but not pY31, plays the predominant role in enhancing HCT116 cell migration. To our knowledge, this is the first report showing that paxillin pY31 is decreased whereas pY118 is increased. Several reports show that both Y31 and Y118 are the primary FAK-Src targets and then proteins with SH2 domains bind to pY31 and pY118, which in turn regulate cell motility (21). However, the functional difference between pY31 and pY118 is still unclear. Our findings suggest that tyrosine phosphorylation of Y31 and Y118 can be regulated separately, transmitting appropriate signals for cells to respond to particular stimuli. It will be of great interest to investigate the differential signaling roles of pY31 and pY118 by the use of the ß4GalNAc-T3 transfectants.
MAPK signalings have been suggested to play crucial roles in cell migration (22-24) as well as tumor progression and invasion (25, 26). For example, ERK regulates cell movement by phosphorylating myosin light chain kinase, calpain, FAK, or paxillin, which have been shown to modulate adhesion dynamics (23, 27-29). Furthermore, MAPKs have been shown to regulate cell invasion by modulating the proteolytic enzymes that degrade the basement membrane (25). In agreement with these findings, our data showed that enhanced cell adhesion, migration, and invasion of ß4GalNAc-T3 transfectants correlated with increased ERK phosphorylation. These results suggest that increased ERK MAPK signalings could play a critical role in the increased migration and invasion of colon cancer cells induced by ß4GalNAc-T3 overexpression.
Many reports show that glycosyltransferases can directly modify carbohydrates on cell-surface receptors or cell-adhesion molecules and then regulate signal transduction and cell behavior. GnT-V, GnT-III, ST6GalNAc I, and ST6Gal-I were found to directly modify carbohydrate structures on ß1-integrin and affect integrin activity (8-11). These changes in N-glycosylation (8, 30) or O-glycosylation (11) of ß1-integrin lead to altered cell morphology and behavior. Furthermore, GnT-III was found to modify carbohydrates on epidermal growth factor receptor and inhibit ERK-mediated neurite outgrowth in PC12 cells (31). In addition, GnT-III can also modify the glycosylation of cadherin and down-regulate tyrosine phosphorylation of ß-catenin in mouse melanoma, human colon cancer, and human hepatoma cell lines (32). Interestingly, ß4GalNAc-T3 has been shown to efficiently modify both N- and O-glycans decorated with GlcNAc in vitro (14). We therefore speculate that ß4GalNAc-T3 may directly glycosylate N- or/and O-glycans of integrins or other surface molecules such as the epidermal growth factor receptor family. The changes in glycosylation of these surface receptors then modulate integrin and MAPK signaling pathways and, in turn, regulate cell phenotyes.
In the present study, ß4GalNAc-T3 overexpression dramatically increased tumor size. However, cell proliferation in vitro was not significantly changed. These data suggest that tumor cell-host interactions, such as angiogenesis, may play a crucial role in tumor growth. Indeed, hypervascularity was found in ß4GalNAc-T3 tumors, but not mock tumors, suggesting that the increase in tumor growth induced by ß4GalNAc-T3 in vivo could be at least, in part, resulted from angiogenesis.
Taken together, we show important roles of ß4GalNAc-T3 in colon cancer cells. We present evidence that ß4GalNAc-T3 overexpression significantly promotes malignant behaviors of colon cancer cell both in vitro and in vivo. These phenotypic changes induced by ß4GalNAc-T3 are associated with enhanced integrin and MAPK signalings. The present study provides a new insight into the molecular mechanism by which glycosyltransferases may play a key role in regulating tumor malignancy.
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Materials and Methods |
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Real-time RT-PCR
Total cellular RNA was isolated from cells grown to 70% confluence by use of the Trizol reagent (Invitrogen, Life Technologies) according to the manufacturer's protocols as previously described (33). For cDNA synthesis, 2 µg of total RNA were used as template in a 25-µL reverse transcription reaction. Matched RNA pairs of colorectal cancer patients were used for real-time RT-PCR. All the clinical materials were obtained from patients at National Taiwan University Hospital after informed consent was given. For ß-actin detection, sense and antisense primers were 5'-GCTCGTCGTCGACAACGGCT-3' and 5'-AAACATGATCTGGGTCATCTTCT-3', respectively, generating a 326-bp fragment. For detection of ß4GalNAc-T3, sense and antisense primers were 5'-CTACAGCGCATTGTGAACGT-3' and 5'-TGGTTCTTCACAGGCACGAC-3', respectively, generating a 320-bp fragment.
For real-time PCR reactions, quantitative PCR System Mx3000P (Stratagene) was used according to the manufacturer's protocol. Briefly, reaction was done 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). PCRs were incubated for 15 min at 95°C followed by 40 amplification cycles with 30-s denaturation at 95°C, 50-s annealing at 54°C, and 30-s extension at 72°C. Samples were analyzed in triplicate, and product purity was checked through dissociation curves at the end of real-time PCR cycles. Relative quantity of gene expression normalized to ß-actin was analyzed with MxPro Software (Stratagene).
Cell Spreading and Morphology
The same numbers of cells were seeded onto 60-mm culture plates (Falcon). After 6 h, cells were photographed with a digital camera (Nikon COOLPIX5400). Spreading cells were calculated at three different fields and results are presented as means ± SD from three independent experiments.
Cell Adhesion Assay
Cell adhesion assays were done according to published protocols with slight modifications (12). Ninety-six-well plates were coated with bovine serum albumin (BSA) control, human collagen IV (Sigma), human fibronectin (Sigma), or murine laminin (Sigma) at concentrations of 1 or 5 µg/mL in PBS at 37°C for 4 h, and then blocked with 1% bovine serum albumin at 37°C for 2 h. Cells were trypsinized, washed with DMEM, and recovered in DMEM at 37°C for 40 min. Mock and ß4GalNAc-T3 stable transfectants (2 x 104) in 100-µL serum-free DMEM per well were allowed to attach for 30 min at 37°C in a humidified 5% CO2 incubator. Attached cells from four wells were counted manually under an inverted microscope. The number of specific adherent cells indicates the difference between the number of cells binding to ECM-coated wells and the number of cells binding to bovine serum albumin–coated wells. All data are expressed as means ± SD from three independent experiments.
Wound Healing Assay
Cells were seeded onto 12-well culture dishes and grown until confluence in DMEM containing 10% FBS. The monolayer was scratched with a 250-µL yellow tip. Migration of cells to the wounded area was observed under an inverted microscope and pictures were taken directly at the time of scratching and after scratching for 16 h. Measurements were taken from five individual microscopic fields in each experiment, and the data from three experiments are presented.
Matrigel Invasion Assay
Cell invasion assays were done in BioCoat Matrigel Invasion Chambers (Becton Dickinson) according to the manufacturer's protocols. Briefly, 500-µL DMEM containing 10% FBS was loaded in the lower part of the chamber and 3 x 105 cells in 500-µL serum-free DMEM were seeded onto the upper part. Cells were allowed to invade the Matrigel for 48 h 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 stained with 0.5% crystal violet (Sigma). The numbers of invading cells in each well were counted under a phase-contrast microscope. The means ± SD were calculated from the numbers of invading cells from three independent experiments under a microscope.
Cell Growth Analysis
Mock and ß4GalNAc-T3 stable transfectants (4 x 104) were seeded onto six-well plates. Six wells were plated for each time point, which was taken at 24-h intervals for 6 days. Viable cells were determined for each time point with a hemocytometer by trypan blue exclusion. Three independent experiments were done and the count results were used for plotting the relative growth rate with SD.
Anchorage-Independent Growth in Soft Agar
Cells (1 x 104) in 0.3% Bactoagar (Sigma) in DMEM supplemented with 10% FBS were overlaid on a base of 0.6% Bactoagar in DMEM supplemented with 10% FBS in six-well plates. Cells were incubated at 37°C, 5% CO2 atmosphere. Triplicate wells for each cell line and three independent experiments were done. The numbers of colonies with diameter >50 µm were counted at day 14.
siRNA Knockdown of ß4GalNAc-T3 Expression
SMARTpool siRNA oligonucleotides against ß4GalNAc-T3 and siCONTROL Nontargeting siRNA were synthesized by Dharmacon Research, Inc. For knockdown of ß4GalNAc-T3, cells were transfected with siRNA using Lipofectamine 2000 (InVitrogen) with a final concentration of 100 nmol siRNA. The cells were incubated for 4 h and then serum-free DMEM was replaced with complete DMEM (10% FBS). After 48-h incubation, cells were harvested for analysis. The knockdown of ß4GalNAc-T3 expression was confirmed by real-time RT-PCR.
Western Blot
Cells were resuspended in serum-free DMEM and replated onto fibronectin-coated dishes for 16 h. Equal amounts of cell lysate were electrophoresed on SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were incubated with anti–phospho-Tyr (PY20 from Transduction Laboratories and 4G10 from Upstate Biotechnology), anti-FAK pY397, pY576 polyclonal antibody (Biosources), anti-FAK polyclonal antibody (C-20, Santa Cruz Biotechnology), anti-paxillin pY118, pY31 (BD Transduction Lab), anti-paxillin polyclonal antibody (BD Transduction Lab), anti–ß-actin monoclonal antibody (BD Biosciences, CA), mouse anti–phospho-MAPK monoclonal antibodies and rabbit anti-ERK1/2 polyclonal antibody (Cell Signaling Technology), or anti–ß4GalNAc-T3 polyclonal antibody, which was generated by immunizing rabbits with keyhole limpet hemocyanin conjugated with a synthetic ß4GalNAc-T3 peptide: ARMLEGRQTPASTLEQDATD. The membrane was incubated with horseradish peroxidase–conjugated antirabbit or antimouse immunoglobulin G (Santa Cruz Biotechnology). Signals were visualized with enhanced chemiluminescence reagents (Amersham Biosciences) and images were quantified with ImageQuant 5.1 (Amersham Biosciences).
Tumor Growth, Tumor Metastasis, and Survival Rate in Nude Mice
For tumor growth analysis, 6-week-old female BALB/c nude mice were injected subcutaneously with 5 x 106 of HCT116/Mock transfectants (n = 5), ß4GalNAc-T3 transfectants clone 3 (n = 5), or ß4GalNAc-T3 transfectants clone 5 (n = 5). At day 20 after injection, tumors in each group were excised for analyses.
For tumor metastasis analysis, 6-week-old female BALB/c nude mice were injected with 2 x 106 of HCT116/Mock transfectants (control, n = 5), ß4GalNAc-T3 transfectants clone 3 (n = 5), or ß4GalNAc-T3 transfectants clone 5 (n = 5) through tail vein. The tumors in the lungs were excised after injection for 50 days and fixed with 4% paraformaldehyde for H&E staining.
For survival assays, 6-week-old female BALB/c nude mice were intraperitoneally injected with 2 x 106 of HCT116/Mock transfectants (control, n = 5), ß4GalNAc-T3 transfectants clone 3 (n = 4), or ß4GalNAc-T3 transfectants clone 5 (n = 4). All mice were monitored twice a week for body weight and other abnormalities for 50 days. Once life-threatening symptoms became markedly manifest, the mice were sacrificed. The organs, including kidneys, lungs, pancreas, intestines, and liver, were analyzed. Survival analysis was done using the Kaplan-Meier method.
Statistical Analysis
Student's t test was used for statistical analyses. Data are presented as means ± SD. Survival analysis was done using the Kaplan-Meier method with a log-rank test for comparison. P < 0.05 was considered significant.
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TopNotesAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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TopNotesAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: J. Huang and J-T. Liang contributed equally to this work.
Received 12/20/06; revised 2/20/07; accepted 2/26/07.
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5ß1 reduces ligand binding and down-regulates cell adhesion and cell migration. J Biol Chem 2004;279:19747–54.5ß1 integrin clustering and stimulates cell migration. Cancer Res 2002;62:6837–45.(1,3)-fucosyltransferase Fuc-TIV, but not Fuc-TVII, generates sialyl Lewis X-like epitopes preferentially on glycolipids. J Biol Chem 2002;277:47786–95.This article has been cited by other articles:
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  M.-K. Shyu, M.-C. Lin, C.-H. Liu, Y.-R. Fu, J.-C. Shih, C.-N. Lee, H.-Y. Chen, J. Huang, M.-C. Huang, and F.-J. Hsieh MUC1 Expression Is Increased During Human Placental Development and Suppresses Trophoblast-Like Cell Invasion In Vitro Biol Reprod, August 1, 2008; 79(2): 233 - 239. [Abstract] [Full Text] [PDF]
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 M.-K. Shyu, M.-C. Lin, J.-C. Shih, C.-N. Lee, J. Huang, C.-H. Liao, I-F. Huang, H.-Y. Chen, M.-C. Huang, and F.-J. Hsieh Mucin 15 is expressed in human placenta and suppresses invasion of trophoblast-like cells in vitro Hum. Reprod., October 1, 2007; 22(10): 2723 - 2732. [Abstract] [Full Text] [PDF]
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