1
Core3 O -glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation of
α2β1 Integrin Complex
Seung Ho Lee 1, Shingo Hatakeyama 1, Shin-Yi Yu 2, Xingfeng Bao 1, Chikara Ohyama 3,
Kai-Hooi Khoo 2, Michiko N. Fukuda 1, and Minoru Fukuda 1
1
Glycobiology Unit, Tumor Microenvironment Program, Cancer Center, Burnham Institute for Medical Research, La Jolla California 92037, 2Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan and 3Department of Urology, Hirosaki University School of
Medicine, Hirosaki, 036-2562, Japan
Running Title: Core3 O -Glycan Suppresses Prostate Tumor Metastasis
Address correspondence to: Dr. Minoru Fukuda, Burnham Institute for Medical Research, La Jolla, California 92037, USA; Phone, 1-858-646-3144; Fax, 1-858-646-3193; E-mail, minoru@https://www.doczj.com/doc/937678432.html, While there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O -glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O -glycans, we transfected PC3 and LNCaP prostate cancer cells with β3-N -acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O -glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared to mock-transfected cells. Moreover, we found that α2β1 integrin acquired core3 O -glycans in cells expressing core3 synthase with decreased maturation of β1 integrin, leading to decreased levels of the α2β1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia
formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O -glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly, LNCaP cells expressing core3 O -glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O -glycans to β1 and α2 integrin subunit in prostate cancer cells suppresses tumor formation and tumor metastasis.
Keywords : Core3 O -glycan; prostate cancer; α2β1 integrin; tumor metastasis Cancer cells often express surface carbohydrates different from normal cells {1}. One such change is expression of sialyl Lewis X and Lewis B blood group antigens in cancer cells {2, 3}. These structural elements are seen as capping oligosaccharides attached to the underlying glycan backbone where they likely function as ligands for cell adhesion molecules.
The structure of underlying glycans also changes during malignant transformation
and differentiation. In particular, there are several reports that an increase in the β1,6-N -acetylglucosaminyl branch in
N -glycans synthesized by β1,6-N -acetylglucosaminyltransferase-V
(GnT-V) is associated with oncogenic transformation {4-7}. Similar structural changes are seen in mucin-type O -glycans,
https://www.doczj.com/doc/937678432.html,/cgi/doi/10.1074/jbc.M109.010934The latest version is at JBC Papers in Press. Published on April 24, 2009 as Manuscript M109.010934
Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc. by guest, on October 9, 2010
https://www.doczj.com/doc/937678432.html, Downloaded from
2
which have N -acetylgalactosamine at the reducing end linked to polypeptide threonine or serine residues. Addition of different carbohydrate residues to N -acetylgalactosamine confers a variety of backbone structures on mucin-type O -glycans; the most abundant of those are classified as core1, core2, core3, and core4 O -glycans {8} (Fig. 1). Among these O -glycans, the synthesis of the core2-branch has been extensively studied, particularly since conversion of core1 to core2 O -glycans was observed in T cell activation {9}. Expression of core2 branch apparently represents an onco-differentiation antigen, since core2 branched O -glycans are synthesized in early stages of T cell differentiation, downregulated in mature T cells, and reappear in T cell leukemia and immune deficiencies such as AIDS and Wiskoff-Aldrich syndrome (for a review see {10}. In addition, overexpression of core2 O -glycans is seen in many cancers, including lung and breast carcinoma cells {11, 12}. By contrast, core3 and core4 O -glycans are synthesized in normal cells but apparently downregulated in gastric and colorectal carcinoma {13, 14}. Core3 O -glycans are synthesized by core3 synthase (β3GnT-6), which adds β1,3-linked N -acetylglucosamine to N -acetylgalactosamine at the reducing terminus {15} (Fig. 1). Iwai et al. showed that forced expression of core3 synthase in human fibrosarcoma HT1080 FP-10 cells resulted in significant reduction in the formation of lung tumor foci in mice after intravenous injection of tumor cells through a tail vein {16}. However, the same study did not address if the expression of core3 influences tumor metastasis since the cancer cells were intravenously injected and no primary tumor was formed to spread into the lung as metastasis in contrast to the other studies {17, 18}. Core4 O -glycan is synthesized by addition of β1,6-linked N -acetylglucosamine to a core3 acceptor by core2 β1,6-N -acetylglucosamine M type (C2GnT-M) or C2GnT-2 {18, 19} (Fig. 1). Huang et al. reported that C2GnT-M is downregulated in colonic carcinoma cells and that forced expression of C2GnT-M in HCT116 colonic carcinoma cells significantly decreased cell invasion and subcutaneous tumor formation {21}. How upregulation of core3 and core4 O -glycans influences the pathophysiology of cells expressing core3 and core4 O -glycans has not been addressed. Cell-extracelluar matrix (ECM) interaction play an essential role during acquisition of migration and invasive behavior of cancer cells. For example, α2β1 integrin is the major receptor for collagen {22}, and most abundantly expressed in prostate cancer cells {23}. Glycosylation on integrin is one of the important modulator of integrin functions and many glycan structures, mainly N -glycans, have been studied. An increase of bisecting GlcNAc structure on α5β1 integrin inhibits the cell spreading and migration {24}, and induced beta 1,6-GlcNAc sugar chains on N -glycans of β1 integrine results in stimulation of cell migration {25}. However, it has not been addressed if changes in O -glycans affect integrin maturation and functions. To determine the role of core3 O -glycans in tumor formation and metastasis, we analyzed PC3 and LNCaP human prostate cancer cells. We found these cell lines express only small amounts of detectable amounts of core3 synthase; thus we transfected the cell lines with core3 synthase. Core3 synthase-transfected PC3 and LNCaP cells expresse increased amounts of core3 O -glycans in α2β1 integrin, showed the reduced maturation of β1 integrin, and low levels of α2β1 integrin formation, and migrated less efficiently through collagen and other extracellular matrix components, and were less invasive than mock-transfected cells. Moreover, those cells exhibited decreased activation of focal adhesion kinase (FAK) compared to mock-transfected cells. by guest, on October 9, 2010
https://www.doczj.com/doc/937678432.html, Downloaded from
3
Significantly, PC3 cells expressing core3 O -glycans produced almost no primary tumors in the prostate and formed much fewer metastases in the draining lymph nodes than mock-transfected cells. Similarly, LNCaP cells expressing core3 O-glycans produced much smaller subcutaneous tumors than mock-transfected LNCaP cells. These findings indicate that addition of core3 O -glycans to the α2β1 integrin leads to decreased cell migration and invasion, resulting in decreased prostate tumor formation and metastasis.
Experimental and Procedures
Cell culture and transfection- PC3 and LNCaP prostate cancer cell lines were obtained from American Type Culture Collection and cultured in RPMI-1640 supplemented with 10% fetal bovine serum. cDNA encoding core3 synthase (β3GlcNAcT-6) {15} was amplified by reverse transcription (RT)-PCR and cloned into pcDNA 3.1(N) as described {26}. pcDNA 3.1(N) was prepared by deleting the Zeocin resistance gene and f1 origin from pcDNA 3.1/Zeo as described {26}. PC3 and LNCaP cells were transfected with pcDNA 3.1(N) harboring core3-synthase cDNA and pcDNA 3 harboring the neomycin-resistance gene using Lipofectamine. Transfected cells were selected first in 200 μg/ml Geneticin ?
(Invitrogen) and maintained in 100 μg/ml of Geneticin. Clonal transfected cells were obtained by dilution and tested for expression of carbohydrates reactive to peanut agglutinin (PNA) after neuraminidase treatment. PNA reacts with Gal β1→3GalNAc α1→R core1 structure {27}. Core3 oligosaccharides do not react with PNA. For this assay, PC3 and LNCaP cells in cloning plates were dissociated into monodispersed cells using an enzyme-free dissociation solution (Hank’s balanced saline solution-based) purchased from Cell and Molecular Technologies. Dissociated cells were incubated with fluorescein isothiocyanate (FITC)-conjugated PNA and subjected to FACS analysis using FACScan flow cytometry (BD Biosciences) as described {26}. As controls, PC3 and LNCaP cells were transfected with empty pcDNA 3.1 (N) and pcDNA 3 and selected in geneticin. Mock-transfected and core3 synthase-expressing PC3 cells cultured on glass plates were stained with phalloidin to visualize F-actin as described previously {28}.
Semi-quantitative RT-PCR analys - total RNA was isolated from PC3 and LNCaP cells using Trizol (Invitrogen). RT-PCR of core3 synthase (β3GnT-6) {15}, C2GnT-1 {29}, C2GnT-2 {19}, and C2GnT-3 {30} was
undertaken. First-strand cDNA was synthesized using Amplitag DNA polymerase (Applied Biosystems) and the following PCR
primers: C2GnT-1, 5’-tcggtggacacctgacgactatat-3’ (5’-primer) and 5’-aggtcataccgcttcttccacctt-3’ (3’-primer); C2GnT-2, 5’-agtccagggaatctcaaagccagt-3’ (5’-primer) and 5’-tgagctctggagcaagtcttccat-3’
(3’-primer); C2GnT-3, 5’-gacatccagttctctagacctctg-3’ (5’-primer) and 5’-aaggcgaggtacttagggagtact-3’ (3’-primer);
β3GnT-6, 5’-agcactgcagcagtggttc-3’ (5’-primer) and 5’-gaggaaggtgtccgcgaag-3’ (3’-primer) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH),
5’-cctggccaaggtcatccatgaca-3’ (5’-primer) and 5’-atgaggtccaccaccctgttgct-3’ (3’-primer).
The PCR reaction was carried out at 94°C for 5 min followed by 35 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 30 sec and by a single incubation at 72°C for 5 min. PCR products were separated by electrophoresis on 1% agarose gels. Similarly, the amounts of the transcripts for Cosmc {31} and core1 synthase were semi-quantitatively estimated by PCR. Twenty seven cycles of PCR reaction was done using 5-cactgtgacaaagcaga-3 and 5-ggttggggtgataagtca-3 primer for Cosmc and 29 cycles of PCR reaction using
by guest, on October 9, 2010
https://www.doczj.com/doc/937678432.html, Downloaded from
4
5-gtgggactgaaaaccaa-3 and 5-agatcagagcagcaacca-3 primers for core1 synthase. Expression levels were normalized by GAPDH expression . O-glycan structure analysis- PC3-, LNCaP-mock and PC3-, LNCaP-core3 cells were suspended in 0.1 M NH 4HCO 3, boiled for 10 min, and lyophilized. Dried samples were delipidated by chloroform-methanol (2:1 by volume) and then extracted by standard 6M-guanidine chloride protocol followed by reduction and alkylation with dithiothreitol/iodoacetic acid. After dialysis, the samples were digested with trypsin/chymotrypsin (Sigma) and then with N -glycanase F (Roche), and passed through a C18 Sep-Pak cartridge (Waters). De-N -glycosylated peptides were eluted stepwise from the C18 cartridge by 20-40 % 1-propanol in 5% acetic acid, and then treated with 0.05 M NaOH/1 M NaBH 4 at 37 °C for 3 days to release O -glycans. The samples were neutralized by acetic acid on ice until it stopped bubbling, followed by passing through Dowex 50x8 column in 5% acetic acid and dried. Borates were then removed by repeated co-evaporation with 10% acetic acid in methanol under a stream of nitrogen. An aliquot of the released and desalted O -glycans were permethylated and analyzed by MALDI-MS and MS/MS on a 4700 Proteomics Analyzer (Applied Biosystem, Farmington, MA), as described previously {32,33}. Immunoprecipitation and Western blot analysis - cells were solubilized in lysis buffer composed of 20mM Tris-HCl, pH 7.4, 150 mM NaCl, 5mM EDTA, 1% (w/v) Nonidet P-40, 5 mM sodium pyrophosphate, 10mM NaF, 1 mM sodium othovanadate, 10 mM β-glycerophophate, 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Sigma). Equal amounts of cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were incubated separately with polyclonal anti-β1 integrin antibody (Ab1952, Chemicon) {34}, rabbit anti-α2 integrin antibody (Ab1936, Chemicon), rabbit
anti-ERK antibody (Cell Signaling), mouse anti-FAK antibody (BD Biosciences), and anti-FAK [pY397] phospho-specific antibody (44-625G, BD Biosciences), and then incubated with HPR-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG. In parallel, aliquots of the lysate were treated with N -glycanase (Calbiochem) as described {33} before separation on SDS-gel electrophoresis. Alternatively, cell lysates were incubated with polyclonal anti-β1 integrin antibody followed by protein A-agarose. Immunoprecipitates were dissociated from protein A-agarose by boiling 5 min in sample buffer containing 1% SDS before electrophoresis. Solubilized proteins were separated on gels and blotted to a PVDF membrane. The blot was incubated with anti-α2-integrin antibody followed by HRP-conjugated anti-rabbit IgG. ECL reagents (Amersham Biosciences) were used to detect signals. The membrane was then stripped by incubation with 1 N NaOH for 1-2 min followed by washing three times with 10 mM Tris-HCl buffer, pH7.4 containing 0.14 M NaCl and 0.05% Tween 20 (TBS-T), and blocking with TBS-T containing 5% skim milk again. This membrane was then reacted with anti-α2 integrin antibody. The membrane was also reacted with biotin-conjugated GS-II and visualized using
a Vectastain ABC kit (Vector Laboratories Inc., Burlingame, CA) and, in parallel, with mouse monoclonal anti-β1 integrin antibody (610467, BD Biosciences) followed by HRP-conjugated anti-mouse IgG.
Flow cytometry analysis- cells in semi - confluent conditions were detached from 10-cm culture dishes using enzyme free cell dissociation solution (Chemicon) and resuspended in 50 μl of PBS. The suspended by guest, on October 9, 2010
https://www.doczj.com/doc/937678432.html, Downloaded from
5
cells (5–10 x 106 cells) were incubated with and without a primary antibody (rabbit anti-α1integrin polyclonal antibody
(Chemicon), rabbit anti-human integrin α2
polyclonal (Chemicon), and rat anti-human monoclonal antibody for α6 integrin (BD (Pharmingen) at a final concentration of 4 μg/ml for 1 h on ice. The cells were washed three times with PBS, then resuspended in PBS containing fluorescein isothiocyanate-conjugated secondary antibody,
and further incubated for 1 h on ice. After washing three times with PBS, flow cytometry analyses were performed using a FACScan instrument (BD Biosciences) operated with CELLQuest software. Migration and invasion assay - cell migration was assayed using the 3 μm pore size of Transwell ? Permeable Supports (Corning). The bottom part of the transwell membrane was coated with Human laminin mixture (Chemicon), Rat laminin-5 (Chemicon), collagen I, or fibronectin (Sigma) with the concentration of 0.5 μg/ml in PBS at 4°C overnight. 5 x 104
cells were added to the upper chamber, and 6 h later at 37°C on CO 2 incubator, cells reaching the bottom layer were stained with 0.5 % crystal violet and counted under a microscope. Cell invasion was assayed using an ECM Invasion Chamber (Chemicon) in which the upper layer of the transmembrane was coated with Matrigel. 2.8x105 cells for PC3 and 5 x105 for LNCaP were loaded in the upper chamber. After 24 h, cells reaching the bottom layer were visualized by 0.5 % crystal violet and counted. To determine the contribution of different integrins to invasion, cells were pre-incubated with 10 μg/ml of mouse anti-human integrin α1 I domain monoclonal antibody (Chemicon), mouse anti-human α2-integrin monoclonal antibody (Chemicon),
rat anti-human monoclonal antibody GoH3 for α6 blocking (BD Pharmingen), and mouse
monoclonal 4B4 anti-β1 integrin neutralizing antibody {34} (Beckman Coulter). In parallel, cells were incubated with control mouse IgG (10 μg/ml).
To exclude the possibility that clonal varian contributes to the difference in cell migration, the parent PC3 cells and LNCaP
cells were transiently transfected with core3
synthase or empty vector. Three days after the transfection, cell migration was measured in the same way as described above.
Orthotopic tumor cell inoculation- balb/c nude (nude/nude) mice (6- to 8-week old males) obtained from Tacomic were used for orthotopic tumor cell injection {35}. Mice were anesthetized with Avertin ? and laparotomy was performed; 2x106 of PC3-core3 and mock-transfected PC3 cells were suspended in 20 μl of serum-free
RPMI-1640 medium and inoculated into the posterior lobe of the prostate. The wound was then closed with surgical clips. Eight weeks later, mice were sacrificed, prostates and surrounding lymph nodes were removed, and organs were weighted. Specimens were preserved by fixation in neutral-buffered formalin.
RESULTS Core3 synthase-expressing prostate cancer cell lines exhibit abnormal lamellipodia. Previously, it was shown that core3 synthase is downregulated in colonic carcinoma cells relative to normal tissues {14}. Expression of core3 synthase in a human fibrosarcoma cell line also resulted in decreased lung tumor foci formation compared to mock-transfected cells {16}. However, these studies did not address the mechanisms how core3 synthase expression results in decreased tumor formation and decreased tumor metastasis. To address these questions, we analyzed human PC3 and
LNCaP prostate cancer cell lines, since these cells metastasize to the lymph nodes.
RT-PCR analysis of mRNAs encoding different glycosyltransferases showed that both PC3 and LNCaP cells express a by guest, on October 9, 2010
https://www.doczj.com/doc/937678432.html, Downloaded from
6
significant amount of C2GnT-1 but only negligible or small amounts of core3 synthase (β3GnT-6). PC3 cells express only a small amount of C2GnT-2, which is much less than that expressed in gastric cancer AGS cells (Fig. 2A).
Individual vectors harboring core3 synthase (β3GnT-6) cDNA {15} and the neomycin (geneticin) resistance gene were co-transfected into PC3 and LNCaP cells, and cells were selected in geneticin. Transfected cells were subjected to Arthrobacter
ureafaciens sialidase treatment and peanut
agglutinin (PNA) staining followed by flow cytometry analysis. PNA binds to core1 O -glycan, Gal β1→3GalNAc α1→Thr/Ser,
which can be formed by desialylation of sialylated core1 O -glycan. As shown in Fig. 3A, core3 synthase-transfected PC3 cell lines showed weaker PNA staining than did mock-transfected cells, since some of Gal β1→3GalNAc must be replaced by
GlcNAc β1→3GalNAc in the core3
synthase-transfected cells. Similarly, LNCaP cells (clone 2) exhibited less PNA staining after transfecting with core3 synthase, though their level of PNA staining was much higher than those of PC3 cells for both mock-transfected and core3
synthase-transfected LNCaP cells. These cell lines transfected with core3 synthase were
designated PC3-core3 and LNCaP-core3, respectively. Hereafter, we studied clones 2 of PC3-core3 and LNCaP-core3 cells.
RT-PCR analysis of PC3-core3 and LNCaP-core3 mRNAs showed that both cell lines expressed high levels of core3 synthase (Fig. 2B). By contrast, the expression level of Core1 synthase and Cosmc {31} was not changed after Core3 synthase transfection (Fig. 2C and D). We also noted that transfected LNCaP and PC3 cells differed morphologically from controls in that they showed abnormal lamellipodia (arrows in Fig.
3B). Indeed, F-actin visualized by phalloidin
was decreased in core3 synthase-expressing PC3 cells compared to mock-transfected PC3 cells (Fig. 3C). By mass spectrometry analyses we confirmed the acquisition of core3 O -glycans after transfection with core3 synthase. As shown in Fig 4A, core3-containing O -glycans (ions at m/z 779.3,
1140.5) were seen only in core3-transfected PC3 cells and absent in mock-transfected PC3
cells. The deduced structures for these molecular ion signals were further confirmed by MS/MS analyses (Fig. 4B). LNCaP-core3 cells displayed much more core3 O -glycans
than mock-transfected LNCaP, and much more than PC3-core3 cells (Fig. 4B). The latter finding is consistent with the difference in PNA labeling between PC3 and LNCaP
cells as seen in Fig. 3A. The amount of core3 O -glycans in PC3-core3 cells is almost equivalent to that in colon cells of wild-type mice {36} while that of LNCaP-core3 cells apparently represents an overexpressed level of core3 O-glycans.
PC3 and LNCaP cells expressing core3 O-glycans exhibit reduced migration and invasion. To determine how the expression of core3 synthase affects cell migration, we utilized transwell migration assay. For this assay, the bottom side of the transwell
membrane was coated with extracellular matrix components. Cells were then loaded
on the upper side of the chamber and migration to the bottom part of the membrane was determined 6 or 24 hrs later. Migration of
PC3-core3 cells was much less efficient than that mock-transfected PC3 cells (Fig. 5A).
This reduction was observed on collagen-, fibronectin-, a mixture of laminin, or laminin-5-coated membranes (Fig. 5A, lower panels). Similar results were obtained when
LNCaP-core3 cells were tested (Fig.5B).
These results indicate that forced expression of core3 results in reduced migration on various components of the extracellular
matrix. To determine how expression of core3 O -glycans influences invasion by prostate cancer cells, mock and core 3 transfected PC3
and LNCap cells were seeded on the transwell by guest, on October 9, 2010
https://www.doczj.com/doc/937678432.html, Downloaded from
7
membrane coated with Matrigel in the upper chamber of a Boyden invasion chamber, and cells reaching the bottom layer were counted 24 h later. First, we found that PC3- and LNCaP-core3 cells invade much less than mock-transfected cells. (Fig. 6A and B) Since the integrin family is well known heteromeric receptor for extracellular matrix reported to have biological functions in protection against apoptosis {37}, and malignant transformation {38, 39}, we used functional blocking antibodies for several integrins to determine the major target integrin for core3 synthase. Invasiveness of PC3 cells was completely inhibited by treatment with anti-β1 integrin-blocking 4B4 antibody {34}. Additionally, significant decrease of invasion was shown when treated with α2 blocking antibody compared with α1
or α6 blocking antibody (Fig. 6C). These results show that decreased invasion by PC3-core3 cells is likely due to decreases in α2β1-integrin-mediated adhesion and
migration.
To expand the above studies further, PC3 cells were transiently transfected with
Core3 synthase and cell migration was measured. The results showed clearly that PC3 cells migrated much slower after transfection with core3 synthase, compared to mock-transfected PC3 cells (Fig. 7A). Almost identical results were obtained on LNCaP cells (Fig. 7B). In addition, PC3-core3 and LNCaP-core3 cells shared all characteristics although two cell lines were independently obtained. These results indicate that the results obtained after core3 synthase transfection is not due to clonal variation of the transfected cells, and that core3 synthase is responsible for decreased migration and invasion, and impaired tumor formation Forced expression of core3 synthase decreases prostate cancer formation and lymph node metastasis. To determine how core3 expression influences tumor formation, PC3-mock and PC3-core3 cells were orthotopically inoculated into the prostate of nude mice as described {35}. The prostate and the surrounding lymph nodes were isolated 8 weeks later and analyzed for tumor formation. Mice receiving mock-transfected cells showed larger prostate tumors, and the surrounding lymph nodes contained metastatic tumors (Fig. 8A). By contrast, mice inoculated with PC3-core3 cells showed much smaller prostate tumors, and the tumor metastasis to the surrounding lymph nodes was not noticed. These results indicate that core3 synthase expression results in reduction of both primary tumor and metastasis to the lymph node. Similar results were obtained on subcutaneously inoculated LNCaP-Core3 cells. Compared to robust subcutaneous
tumor formation by mock-transfected LNCaP cells, LNCaP-core3 cells barely formed subcutaneous tumor (Fig. 8B). These results demonstrate that expression of core3
O-glycans suppress tumor formation and metastasis.
Maturation and cell surface expression of β1 integrin is attenuated in PC3-core3 cells. Among different integrin subunits, we examined glycosylation status of β1 integrin, since this protein has been shown to have
mucin-type
O -glycans {40}. The N -acetylglucosaminyl terminus of core3 O -glycan, GlcNAc β1→3GalNAc α1→Thr/Ser, can be detected by Griffonia simplifolia lectin II (GS-II) {41}. Western blot analysis of β1-integrin immunoprecipitated from PC3- and LNCaP-core3 cells showed a strong band detected by GS-II. By contrast, β1 integrin from mock-transfected cells did not react with GS-II, although β1-integrin levels did not differ between the two cell types (Fig. 9A). α2 integrin of LNCaP cells but not PC3 cells apparently acquired core3 O -glycan while α2
integrin from PC3 cells barely acquired it. In support of this finding, PNA-binding to β1 integrin was slightly decreased in PC3-core3 cells compared to the mock-transfected PC3 by guest, on October 9, 2010
https://www.doczj.com/doc/937678432.html, Downloaded from
cells. By contrast, PNA binding to β1 integrin and α2 integrin was significantly decreased in LNCaP-core3 cells. (Fig. 9B). The results confirmed that β1 integrin from PC3-core3 cells and β1 and α2 integrin from LNCaP-core3 cells express core3 O-glycans. The results are consistent with the conclusions obtained by mass spectrometric analysis (Fig.
4). It is possible that Arthrobacter ureafaciens sialidase treatment did not efficiently remove sialic acid from core1 O-glycans of PC3 cells yielding weak signals by PNA staining (Figs. 3A), while the same treatment efficiently removed sialic acid from LNCaP cells.
The immunoblotting using anti-β1 integrin antibody detected two forms of β1 integrin, and core3 expressing PC3 cells expressed lower levels of β1 integrin with the higher molecular weight (arrowhead in Fig. 9C) than did the mock transfected cells, while the amount of α2 integrin was equivalent in both cell types (Fig. 9C). It was reported that the higher molecular weight form represents the mature form of β1 integrin {42}. Two differently glycosylated forms of β1 integrin resolved to one lower molecular weight band after N-glycanase treatment (Fig. 9C).
We then examined the cell surface expression of several integrin subunits. Interestingly, surface expression of β1 integrin is significantly reduced without changing that of α2 integrin expression in PC3-core3 cells (Fig. 9D). For LNCaP cells, reduced surface expression of α2 and α6 integrins as well as β1 integrin was detected. These results suggest that adding the core3 O-glycan could affect the maturation and surface expression for β1 integrin for PC3 cells and β1-, α2-integrin, and possibly for α6-integrin for LNCaP cells.
Association of α2 and β1 integrin is attenuated in PC3-core3 and LNCaP-Core3 cells. Previously, it was reported that PC3 cells express primarily α2β1 integrin {23},
which is consistent to our data (Fig. 9B).
The assays described in Fig. 6 indicate that invasion of PC3 cells is largely dependent on
α2β1-integrin. Since the above results suggest
that α2β1 integrin may not function well in
PC3-core3 and LNCaP-core3 cells, we analyzed the amount of β1 integrin complexed with α2 integrin in those cells and compared them with mock-transfected cells.
We thus immunoprecipitated β1 integrin from
two cell types using rabbit anti-β1 integrin
and reacted immunoprecipitates sequentially
with anti-α2 integrin and anti-β1 integrin antibodies. As a complimentary experiment,
α2 integrin was immunoprecipitated followed
by blotting with anti-β1 antibody and
α2-antibody. Levels of α2-integrin
co-immunoprecipitated with β1 integrin were significantly decreased in PC3 cells expressing core3 O-glycans compared to
mock-transfected PC3 cells (Fig. 10A).
Almost identical results were obtained for
LNCaP cells (Fig. 10B). These results suggest that expression of core3 O-glycans in
α2β1 integrin led to decreased heterodimerization. In this experiment, β1
integrin was immunoprecipitated by rabbit
anti-β1 integrin antibody, and then immunoblotted with the monoclonal anti-β1
integrin antibody or anti-α2 integrin antibody. Consistent with the previous report {42}, the polyclonal anti-β1 antibody can detect two
forms of β1 integrin by immunoblotting (Fig.
9C), while the same antibody and the monoclonal antibody immunoprecipitates
mostly a major β1 integrin with lower molecular weight (Fig. 10) as shown previously {43}.
Reduction in integrin-mediated activation of PC3-core3 cells. The above
results suggested that PC3 and LNCaP cells expressing core3 synthase express lower
levels of functional α2β1 integrin than do
mock transfected PC3 cells. To support this
by guest, on October 9, https://www.doczj.com/doc/937678432.html,
Downloaded from
8
9
conclusion, we examined the downstream signaling mediated by integrin. Integrin-mediated cell adhesion activates various protein tyrosine kinases, such as focal adhesion kinase (FAK), which is directly activated by integrin signaling and leads to association of integrins with the cytoskeleton {38}. In PC3-core3 cells, the levels of phosphorylated FAK on collagen coated plate were significantly decreased relative to mock-transfected cells, although both cell types expressed similar levels of FAK (Fig. 11). These results suggest that expression of core3 O -glycan is associated with decreased levels of functional α2 and β1 integrin complexes, leading to decreased levels of activation of downstream signaling as indicated by FAK phosphorylation. DISCUSSION This study shows that forced expression of core3 oligosaccharides in PC3 prostate cancer cell line significantly reduces both primary prostate tumor formation and tumor metastasis to the draining lymph nodes. In the previous work on core3 O -glycans, tumors
were directly formed in the lung without
forming a primary tumor {16}. This is
different from metastasis where metastatic
tumor is formed by cells migrated from the
primary tumor {17, 44}. By contrast, it has
been reported that lung tumor formation after intravenous injection is called as lung tumor colonization as seen in many studies {for example refs. 45, 46}. In our present study, we inoculated prostate cancer cells in the prostate and assayed the formation of tumor in the prostate (primary tumor) and in the draining lymph node (metastasis). As a variation of this, we also measured the tumor
formation of LNCaP cells after subcutaneous injection. In this case, the formed tumor is mostly primary tumor but some of them invaded into surrounding tissues. Our assay therefore measured the tumor formation in the primary site and metastatic sites. Thus, we can definitely say that the increased core3 structure can attenuate the prostate tumor formation as well as metastasis. We also show that PC3 and LNCaP cells expressing increased amounts of core3 oligosaccharides display incomplete maturation of β1 integrin, which contains mucin-type O -glycans, resulting in formation of fewer α2β1 integrin complexes and possibly α6β1 integrin complexes. Reduced amounts of α2β1 integrin complex lead to impaired cell migration by PC3 and LNCaP cells expressing core3 O -glycans. Reduced cell migration then results into reduced tumor cell invasion, tumor formation, and tumor metastasis. Our work is the first report that modulation of mucin-type O -glycans in α2β1 integrin decreases tumorigenicity and tumor metastasis.
Recently, it was reported that mouse embryonic fibroblasts from GnT-V null mice
exhibit increased α5β1 integrin activation due to elevated protein kinase C signaling, which increases cell surface expression of α5β1 integrins. These results suggest that decreases in N -acetylglucosamine in N -glycans increase cell surface expression of α5β1 integrin {25}.
The 1,6-GlcNAc-branched N -glycans on α3
subunit of integrin was reduced by GnT-III,
and this leads to reduced migration {47}.
Moreover, it was discovered that N -glycans of
the β-propeller domain of the integrin α5 subunit is essential for α5β1 heterodimerization. As the same time, loss of the same N -glycan resulted in the decreased cell surface expression of α5β1 {48}. These reports are consistent with our findings that incomplete N -glycosylation leads to the reduced cell surface expression of α2β1.
In our study, the α2 and β1 integrin of LNCaP and β1 integrin of PC3 cells increased core3 structure with decreased core1 structure, and these cells showed decreased cell surface expression of α2β1 integrin (Fig. 9A, B and D). Although it is not clear how these different glycosylation modulate the cell by guest, on October 9, 2010
https://www.doczj.com/doc/937678432.html, Downloaded from
10
surface expression, there is a possibility that increased core3 structure hindered Ν-glycosylation of α2β1 integrin, resulting in reduced cell surface expression.
We also showed that core3 synthase expression affects β1 integrin glycosylation and that much more amount of β1 integrin remains incompletely N -glycosylated in PC3-core3 and LNCaP-core3 cells compared to the mock-transfected cells (Fig 9C). Thus it
is likely that addition of core3 O -glycan may hinder glycosyltransferases that form complex-type N -glycans, due to hydrophobic nature of neighboring core3 O -glycans.
Incompletely N -glycosylated β1 integrin apparently associates less efficiently with α2
integrin than mature β1 integrin. Although it
is not formally proven yet, it is likely that the mature form of β1 integrin contains more N -glycan chains due to increased number of N -glycans per molecule since N -glycanase
treatment converted two forms to one form with a lower molecular weight. Alternatively,
N -glycans are not fully processed when core3 O -glycan are attached. It is tempting to speculate that β1 integrin containing less
N -glycans may have different conformation
than the completely N -glycosylated β1
integrin, leading to the decreased α2-β1 complex comformation. The decrease in α2β1-integrin complex-formation led to decreased FAK phosphorylation, resulting in reduced lamellipodia formation. Conversely, forced expression of C2GnT-M resulted in decreases in paxillin but not FAK phosphorylation {21}. Since paxillin phosphorylation likely occurs downstream of FAK phosphorylation in integrin signaling {38}, C2GnT-M may affect other molecules in addition to integrin, thereby modulating paxillin phosphorylation. Core3 synthase is expressed in normal intestine and colon but its activity is downregulated in cancer cells {13, 14}. It was also shown that forced expression of core3 synthase in fibrosarcoma cells reduces lung tumor focus formation when cells are injected intravenously {16}. These results are consistent with our findings that core3-synthase expression suppresses tumor
formation. Our studies extend previous studies by showing that PC3 and LNCaP cells
expressing core3 O-glycans form much less metastasis and subcutaneous tumor,
respectively than mock-transfected cells.
Moreover, our studies show that PC3 and
LNCaP cells expressing core3 synthase exhibits impaired β1-integrin maturation, resulting in impaired cytoskeletal organization as assayed by stress fiber formation,
decreased FAK phosphorylation, decreased cell migration, leading to decreased tumor
metastasis spread from primary tumors. These results are striking since only small increase of core3 O -glycans in PC3 cells resulted in the dramatic change in these phenotypes. The difference in the phenotypes of these
cells was as dramatic as LNCaP cells that significantly increased core3 O -glycans.
More recently, formation of colonic carcinoma after treatment with azoxymethane and dextran sodium sulfate was shown to be
much greater in core3 synthase-deficient mice
than in wild-type mice {36}. This was
associated with reduction in Muc2 protein and
increased permeability of the intestinal barrier.
Our findings suggest that one of the tumor-suppressing functions of core3 O -glycan is to attenuate the integrin/extracellular matrix interaction, making cells prone to growth control and rendering them less motile and invasive. These findings suggest that forced expression of core3 synthase suppresses tumor formation. Further studies should address development of therapies via ectopic expression of core3 synthase.
by guest, on October 9, 2010
https://www.doczj.com/doc/937678432.html, Downloaded from
REFERENCES
1. Hakomori, S. (2002) Proc Natl Acad Sci U S A 99, 10231-10233
2. Fukushima, K., Hirota, M., Terasaki, P. I., Wakisaka, A., Togashi, H., Chia, D., Suyama,
N., Fukushi, Y., Nudelman, E., and Hakomori, S. (1984) Cancer Res 44, 5279-5285
3. Nakamori, S., Kameyama, M., Imaoka, S., Furukawa, H., Ishikawa, O., Sasaki, Y.,
Kabuto, T., Iwanaga, T., Matsushita, Y., and Irimura, T. (1993) Cancer Res 53, 3632-3637
4. Yamashita, K., Ohkura, T., Tachibana, Y., Takasaki, S., and Kobata, A. (1984) J Biol
Chem 259, 10834-10840
5. Pierce, M., and Arango, J. (1986) J Biol Chem 261, 10772-10777
6. Dennis, J. W., Laferte, S., Waghorne, C., Breitman, M. L., and Kerbel, R. S. (1987)
Science 236, 582-585
7. Hubbard, S. C. (1987) J Biol Chem 262, 16403-16411
8. Schachter, H., and Brockhausen, I. (1992) The biosynthesis of serine
(threonine)-N-acetylgalactosamine-linked carbohydrate moieties, Marcel Dekker, New York
9. Piller, F., Piller, V., Fox, R. I., and Fukuda, M. (1988) J Biol Chem 263, 15146-15150
10. Fukuda, M. (1996) Cancer Res 56, 2237-2244
11. Machida, E., Nakayama, J., Amano, J., and Fukuda, M. (2001) Cancer Res 61,
2226-2231
12. Dalziel, M., Whitehouse, C., McFarlane, I., Brockhausen, I., Gschmeissner, S.,
Schwientek, T., Clausen, H., Burchell, J. M., and Taylor-Papadimitriou, J. (2001) J Biol Chem 276, 11007-11015
13. Vavasseur, F., Dole, K., Yang, J., Matta, K. L., Myerscough, N., Corfield, A., Paraskeva,
C., and Brockhausen, I. (1994) Eur J Biochem 222, 415-424
14. Vavasseur, F., Yang, J. M., Dole, K., Paulsen, H., and Brockhausen, I. (1995)
Glycobiology 5, 351-357
15. Iwai, T., Inaba, N., Naundorf, A., Zhang, Y., Gotoh, M., Iwasaki, H., Kudo, T.,
Togayachi, A., Ishizuka, Y., Nakanishi, H., and Narimatsu, H. (2002) J Biol Chem 277, 12802-12809
16. Iwai, T., Kudo, T., Kawamoto, R., Kubota, T., Togayachi, A., Hiruma, T., Okada, T.,
Kawamoto, T., Morozumi, K., and Narimatsu, H. (2005) Proc Natl Acad Sci U S A 102, 4572-4577
17. Olsson, L., and Forchhammer, J. (1984) Proc Natl Acad Sci U S A 81, 3389-3393.
18. Gupta, G., Perk, J., Acharyya, S., de Candia, P., Mittal, V., Todorova-Manova, K.,
Gerald, W., Brogi, E., Benezra, R, and Massague, J. (2007) Proc Natl Acad Sci U S A 104, 19506-19511.
19. Yeh, J. C., Ong, E., and Fukuda, M. (1999) J Biol Chem 274, 3215-3221
20. Schwientek, T., Nomoto, M., Levery, S. B., Merkx, G., van Kessel, A. G., Bennett, E. P.,
Hollingsworth, M. A., and Clausen, H. (1999) J Biol Chem 274, 4504-4512
21. Huang, M. C., Chen, H. Y., Huang, H. C., Huang, J., Liang, J. T., Shen, T. L., Lin, N. Y.,
Ho, C. C., Cho, I. M., and Hsu, S. M. (2006) Oncogene 25, 3267-3276
22. Emsley, J., Knight, C. G., Farndale, R. W., Barnes, M. J., and Liddington, R. C. (2000)
Cell, 101, 47-56 by guest, on October 9, https://www.doczj.com/doc/937678432.html, Downloaded from
11
23. Bonaccorsi, L., Carloni, V., Muratori, M., Salvadori, A., Giannini, A., Carini, M., Serio,
M., Forti, G., and Baldi, E. (2000) Endocrinology 141, 3172-3182
24. Jsaji, T., Gu, J., Nishiuchi, R., Zhao, Y., Takahashi, M., Miyoshi, E., Honke, K.,
Sekiguchi, K., and Taniguchi, N. (2004) J Bio chem 279, 19747-19754
25. Guo, H. B., Lee, I., Bryan, B. T., and Pierce, M. (2005) J Biol Chem 280, 8332-8342
26. Mitoma, J., Petryniak, B., Hiraoka, N., Yeh, J. C., Lowe, J. B., and Fukuda, M. (2003) J
Biol Chem 278, 9953-9961
27. Lotan, R., Skutelsky, E., Danon, D., and Sharon, N. (1975) J Biol Chem 250, 8518-8523
28. Sugihara, K., Sugiyama, D., Byrne, J., Wolf, D. P., Lowitz, K. P., Kobayashi, Y.,
Kabir-Salmani, M., Nadano, D., Aoki, D., Nozawa, S., Nakayama, J., Mustelin, T., Ruoslahti, E., Yamaguchi, N., and Fukuda, M. N. (2007) Proc Natl Acad Sci U S A 104, 3799-3804
29. Bierhuizen, M. F., and Fukuda, M. (1992) Proc Natl Acad Sci U S A 89, 9326-9330
30. Schwientek, T., Yeh, J. C., Levery, S. B., Keck, B., Merkx, G., van Kessel, A. G.,
Fukuda, M., and Clausen, H. (2000) J Biol Chem 275, 11106-11113
31. Ju, T. and Cummings, R.D. (2002) Proc Natl Acad Sci U S A 99, 16613-16618
32. Yu, S. Y., Wu, S. W., and Khoo, K. H. (2006) Glycoconj J 23, 355-369
33. Mitoma, J., Bao, X., Petryanik, B., Schaerli, P., Gauguet, J. M., Yu, S. Y., Kawashima,
H., Saito, H., Ohtsubo, K., Marth, J. D., Khoo, K. H., von Andrian, U. H., Lowe, J. B.,
and Fukuda, M. (2007) Nat Immunol 8, 409-418
34. Takada, Y., and Puzon, W. (1993) J Biol Chem 268, 17597-17601
35. Inaba, Y., Ohyama, C., Kato, T., Satoh, M., Saito, H., Hagisawa, S., Takahashi, T.,
Endoh, M., Fukuda, M. N., Arai, Y., and Fukuda, M. (2003) Int J Cancer 107, 949-957 36. An, G., Wei, B., Xia, B., McDaniel, J. M., Ju, T., Cummings, R. D., Braun, J., and Xia, L.
(2007) J Exp Med 204, 1417-1429
37. Cardone, M. H., Salvesen, G. S., Widmann, C., Johnson, G., and Frisch, S. M. (1997)
Cell 90, 315-323
38. Giancotti, F. G., and Ruoslahti, E. (1999) Science 285, 1028-1032
39. Hynes, R. O. (2002) Cell 110, 673-687
40. Clement, M., Rocher, J., Loirand, G., and Le Pendu, J. (2004) J Cell Sci 117, 5059-5069
41. Lyer, P. N., Wilkinson, K. D., and Goldstein, L. J. (1976) Arch Biochem Biophys 177,
330-333
42. Bellis, S. L., Newman, E., and Friedman, E. A. (1999) J Cell Physiol 181, 33-44
43. Dulabon, L., Olson, E. C., Taglienti, M. G., Eisenhuth, S., McGrath, B., Walsh, C. A.,
Kreidberg, J. A., and Anton, E. S. (2000) Neuron 27, 33-44
44. Chen, S., Kawashima, H., Lowe, J. B., Lanier, L. L., and Fukuda M. (2005) J Exp Med
19, 1679-89
45. Meromsky, L., Lotan, R., and Raz, A. (1986) Cancer Res 46, 5270-5275
46. Hatakeyama, S., Sugihara, K., Nakayama, J., Akama, O., Wong, S. M., Kawashima, H.,
Zhang, J., Smith, D. F., Ohyama, C., Fukuda, M., and Fukuda, M. N. (2009) Proc Natl Acad Sci U S A 106, 3959-3100
47. Zhao, Y., Nakagawa, T., Itoh, S., Inamori, K., Isaji, T., Kariya, Y., Kondo, A., Miyoshi,
E., Miyazaki, K., Kawasaki, N., Taniguchi, N., and Gu, J. (2006) J Biol Chem 281,
32122-32130
48. Isaji, T., Sato, Y., Zhao, Y., Miyoshi, E., Wada, Y., Taniguchi, N., and Gu, J. (2006) J
Biol Chem 281, 33258-33267 by guest, on October 9, https://www.doczj.com/doc/937678432.html, Downloaded from
12
FOOT NOTES
*The authors thank Dr. Kiyohiko Angata and the rest of the laboratory staff members of Drs. Minoru and Michiko Fukuda for useful discussion, Dr. Elise Lamar for critical reading of the manuscript, and Ms. Aleli Morse for organizing the manuscript. The work is supported by NIH grants CA33000 and CA48737 (to M.F.) and in part by P01 CA71932 (to M.N.F. and M.F.) Abbreviations used are: Core3 synthase, β3-N-acetylglucosaminyltransferase-6; C2GnT, core2β1,6-N-acetylglucosaminyltransferase; GnT-V, N-acetylglucosaminyltransferase-V; FAK, focal adhesion kinase; MS, mass spectrometry; TBS-T, Tris-Hcl buffer, pH 7.4 containing 0.14 M NaCl and 0.05% Tween 20.
FIGURE LEGENDS
Figure 1.Biosynthetic pathways of mucin-type O-glycans.N-acetylgalactosamine is transferred to a serine or threonine residue in a polypeptide. Resultant GalNAcα1→Ser/Thr is converted by core3 synthase (β3GnT-6) to GlcNAcβ1→3GalNAcα1→Ser/Thr (core3). Core3 is then converted to core4 by C2GnT-2 (C2GnT-M). GalNAcα1→Ser/Thr is also converted to core1, Galβ1→3GalNAcα1→Ser/Thr by core1 synthase. Core1 is then converted to core2 by C2GnT-1, C2GnT-2, and C2GnT-3.
Figure 2.Expression of enzymes forming mucin-type O-glycans.Total RNA was isolated
from gastric cancer AGS cells, prostate cancer LNCaP and PC3 cell lines, and LNCaP-core3 and PC3-core3 cells. After reverse transcription, amounts of cDNAs for the enzymes were semi-quantitatively estimated by PCR. β3GnT-6 and GAPDH refer to core3 synthase and glyceraldehyde phosphate dehydrogenase, respectively. The parental cell lines (A), transfected PC3 and LNCaP cells (B, C, D) were subjected to the analysis. In C and D, PC3-core3 clone 2 and LNCaP-core3 clone 2 (see Fig. 3) were analyzed
Figure 3.Establishment of PC3 and LNCaP cells expressing core3 synthase.A, PC3 and LNCaP cells selected after transfection of core3 synthase and geneticin-resistance markers were subjected to FACS analysis after neuraminidase treatment and PNA staining. Mock-transfected clone 1 and clone 3-1 served as PC3 and LNCaP controls, respectively. B, Micrography of mock-transfected LNCaP (a), LNCaP-core3 (b), mock-transfected PC3 (c), and PC3-core3 (d) are shown. Lamellipodia formation is impaired as shown by arrows. C, F-actin was visualized by phalloidin. Scale bar=0.025 mm for B and 0.05 mm for C.
Figure 4. Core3 O-glycans are revealed by mass spectrometric analyses in PC3-core3 and LNCaP-core3 cells but barely in mock-transfected PC3 and LNCaP cells. MALDI-MS analysis of permethylated O-glycans in positive ion mode from PC3 (A) and LNCaP cells (B). The m/z values of the labeled peaks correspond to monoisotopic mass and assignment of the molecular compositions is shown in the figure. Ions at m/z 895.4, 1256.5, 1344.6 and 1705.7 correspond to sialicacid→Gal→GalNAcitol, (Sialicacid)2→Gal→GalNAcitol, Sialic acid→[Gal→GlcNAc→(Gal→)GalNAcitol] and (Sialic acid)2→[Gal→GlcNAc→(Gal→) GalNAcitol], respectively. Ions at m/z 779.3 and 1140.5 correspond to core3 O-glycans, Gal→GlcNAc→GalNAcitol and silaic acid→Gal→GlcNAc→GalNAcitol, respectively. Ion at by guest, on October 9, https://www.doczj.com/doc/937678432.html, Downloaded from
13
m/z 825.3 of PC3 cells corresponds to in source prompt fragment ion of sialic acid→Gal→GlcNAc. In B, ms/ms spectrum of m/z 779 in the middle panel is shown at the bottom.
Figure 5.Core3 synthase inhibits cell migration of PC3 (A) and LNCaP (B) cells. Mock-transfected PC3 (a, c), PC3-core3 (b, d), mock-transfected LNCaP (a, c), and
LNCaP-core3 (b, d) cells were seeded in a chamber containing different extracellular matrix
components in the bottom layer of the transwell membrane. Cells migrating to the bottom part of the transwell membrane were fixed, stained with 0.5 % crystal violet, and counted. Represent figure of three independent experiments was shown in upper panels. M igration through type 1 collagen matrix (a and b are enlarged figures of c and d, respectively). Bar = 0.1 mm. The
number of cells was counted in 4 different fields and tabulated in the bottom panel. *, P<0.05; **,
P<0.01 compared with mock-transfectants.
Figure 6.Core3 synthase inhibits PC3 and LNCaP cell invasion. (A), mock-transfected and
core 3-expressing LNCaP cells (5 x 105 cells) and PC3 cells (2.8x105 cells) were seeded in the
upper part of the transmembrane. The upper part of the transmembrane was pre-coated with
Matrigel. Before seeding, PC3-mock and PC3-core3 transfectant cells were incubated with either control mouse IgG (10 μg/ml) or each functional blocking antibody (10 μg/ml) against integrin for 10 min at 37 o C in a CO2 incubator. Cells reaching the bottom part of the filter were stained and counted. Invasive activity of mock and core 3 transfectants was inhibited mostly by β1 integrin functional blocking antibody. (B) and (C), Results obtained by calculating 4 different fields are shown and repeated two times. Error bars represent s. d. of the mean. *, P<0.01; scale
bar=0.2 mm.
Figure 7. Transient transfection of core3 synthase attenuates cell migration.PC3 (A) and LNCaP (B) cells were transiently transfected with core3 synthase or empty vector. Three days after the transfection, 5x104 of PC3 or 2x105 LNCaP cells were seeded on the upper side of the membrane coated with or without collagen 1 in the same way in Fig. 6. The number of cells at the bottom side of the transwell membrane was counted after 6h (for PC3) or 17h (for LNCaP) of incubation. The results obtained by counting the migrated cell number and tabulated in right side. scale bar = 0.1mm in PC3 and 0.2 mm in LNCaP.
Figure 8.Core3 synthase suppresses tumor formation and metastasis. Mock-transfected and core3-expressing PC3 cells (2x106 cells) and LNCaP cells (5x107 cells) were inoculated into nude mice. (A) PC3-core3 and mock-transfected PC3 cells were orthopically inoculated into the prostate of nude mice, and animals were sacrificed 8 weeks later. (B) LNCaP-core3 and mock-transfected cells were subcutaneously injected with matrigel (50:50, v/v), and measured the weight of tumor after 3 month. Mock-transfected PC3 cells produced a large prostate tumor (arrow), while tumors produced by core3-expressing PC3 were not observed in the prostate (arrow). Seminal vesicles are also seen (arrowhead). Four representative prostate and lymph nodes with tumors are shown (left panel). Normal prostate represent those of the nude mice without any treatment. Wet weight of 8 prostate and lymph nodes is shown (right panel). PC3 cells expressing core3 O-glycans formed almost no prostate tumors and no metastasis to the draining lymph node. In B, representative tumors are shown in the upper panel and wet weight of six to seven tumors is shown in the lower panel. Core3 synthase transfected LNCaP cells by guest, on October 9, https://www.doczj.com/doc/937678432.html, Downloaded from
14
showed almost no tumor. *, P<0.05 compared with mock-transfectants using Mann-Whitney U-test. Bar = 0.5 cm
Figure 9. α2β1 integrin acquired β-N-acetylglucosaminyl residues and surface expression of α2β1 integrin is decreased in core3-expressing cells.(A) β1 integrin was immunoprecipitated with rabbit anti-β1 integrin at 4°C overnight, and blotted to a PVDF membrane. The membrane was reacted with GlcNAc-specific GS-II or the same anti-β1 integrin antibody. Similar experiments were carried out after immunoprecipitation of α2 integrin. (B), Binding of PNA to α2 and β1 integrins in PC3 and LNCaP cells. The experiment was carried out in the same way as in panel A, except the PNA was used instead of GS-II. (C), Immunoblot analysis using rabbit anti-β1 integrin antibody shows decreased levels of mature, higher molecular weight β1 integrin indicated by arrowhead. The amount of α2 integrin and control t-ERK protein is equivalent in both cell types. (D), Mock and core3-transfected PC3 and LNCaP cells were incubated with anti-β1, anti-α2, and anti-α6 antibody and then stained with fluorescein isothiocyanate-conjugated secondary antibody for flow cytometry analysis.
Figure 10. α2β1 integrin association in core3-expressing PC3 (A) and LNCaP (B) cells is impaired.α2 integrin was immunoprecipitated with rabbit anti-α2 integrin antibody and the blot was reacted with mouse monoclonal anti-β1 integrin antibody. The membrane was then stripped by incubation with 1 N NaOH for 1-2 min and then reacted with anti-α2 integrin antibody. In parallel, β1 integrin was first immunoprecipitated by polyclonal anti-β1 integrin antibody and the immunoprecipitates were sequentially reacted with anti-α2 antibody and anti-β1 antibody. The experiments were repeated three times and a representative result is shown.
Heterodimerization rate (α2/β1 or β1/α2) was estimated by scanning the gel and tabulated in the lower panel.
Figure 11. FAK phosphorylation is impaired in PC3-core3 cells. (A), Cells were seeded on collagen I (1 μg/ml) -coated plate and harvested with various times as indicated. The blot was incubated with mouse anti-FAK [pY397]-phospho-specific antibody (P-FAK). This membrane was stripped by incubation with 1 N NaOH for 1-2 min at room temperature, and washed tree times with TBS-T and then used for detection of total FAK. Total FAK amounts were detected using mouse anti-FAK-antibody. (B), Activation (phosphorylation) rate of FAK (p-FAK/t-FAK) was estimated using Image J program. by guest, on October 9, https://www.doczj.com/doc/937678432.html, Downloaded from
15
N-acetylgalactosamine N-acetylglucosamine galactose
Sialic acid
A)
B)
Mock
Core 3 synthase transfected
MS/MS