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Tumor Biology |
(1,2) Fucosyltransferase Activity Decreases Adhesive and Metastatic Properties of Human Pancreatic Cancer Cells1
Institut National de la Santé et de la Recherche Médicale Unité 260, Faculté de Médecine, 13385 Marseille cedex 5 [M. A., L. P., C. C., D. L., M. O. S., E. M.], and Institut National de la Santé et de la Recherche Médicale Unité 119, 13009 Marseille [P. G.], France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(1,2) fucosyltransferases that catalyze the fucose
transfer to galactose of the N-acetyl(iso)lactosamine
chain is decreased in human metastatic pancreatic cancer cells.
(2,3) Sialyltransferases catalyze the transfer of sialic acid to the
same substrate to form, with (1,3/1,4) fucosyltransferases,
sialyl-Lewis a and sialyl-Lewis x determinants on cell surface that are
involved in pancreatic metastatic invasion. The aim of this study was
to determine whether this decrease of (1,2) fucosyltransferase
expression can favor the (2,3) sialyltransferase activity to form
metastatic sialyl-Lewis antigens. Restoration of (1,2)
fucosyltransferase activity in the human pancreatic cancer cell line
BxPC-3 was obtained by selecting stable transfectants expressing
FUT1. Overexpression of FUT1 in BxPC-3
cells resulted in a substantial reduction of sialyl-Lewis antigen
expression that correlated with an increase of expression of Lewis y
and H-type antigens on cell surface. The modified oligosaccharide
structures were preferentially restricted to three major glycoproteins,
which could in part be related to mucin-type glycoproteins. The
reduction of sialyl-Lewis antigen expression was associated with an
inhibition of adhesive properties to E-selectin and a decrease of
gastrointestinal metastatic power of BxPC-3 cells after xenograft
transplantation into nude mice. This study provides evidence that the
expression level of (1,2) fucosyltransferase may regulate the
expression of sialyl-Lewis a and sialyl-Lewis x antigens and
consequently could play an important role in metastatic properties of
human pancreatic cancer cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The key glycosyltransferases that regulate the synthesis of
sialyl-Lewis antigens are either (2,3)
STs,3
which add sialic acid onto the galactose of type I (Galß1,3GlcNAc-R)
or type II (Galß1,4GlcNAc-R) disaccharide, or (1,3) Fuc-Ts
and (1,4) fucosyltransferase, which transfer fucose onto the GlcNAc
for sialyl-Lewis x and sialyl-Lewis a synthesis, respectively (Fig. 1)
. The genes encoding the (2,3) ST family, ST3Gal I(14)
, ST3Gal II (15)
,
ST3Gal III (16)
and ST3Gal IV
(14
, 17)
, have been cloned to date. We have
detected the presence of ST3Gal I, ST3Gal III,
and ST3Gal IV in pancreatic cancer
cells.4
To date, five human (1,3) Fuc-Ts—FUT3 (18)
,
FUT4 (19
, 20)
, FUT5 (21)
,
FUT6 (22
, 23)
, and FUT7 (24
, 25)
—have been cloned and characterized. FUT3, which
corresponds to the Lewis type Fuc-T, is also an (1,4) Fuc-T
(18)
. In a previous study, we demonstrated that these five
Fuc-Ts were expressed in normal and tumoral human pancreas
(26)
.
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(1,2) linkage to form H-type I
or H-type II structures, has also been characterized (Fig. 1)
. Two
human (1,2) Fuc-T genes, FUT1 and FUT2,
which encoded the H and Secretor enzymes, respectively, have been
cloned (27
, 28)
. Lewis b, Lewis y, and H antigens were
expressed in normal pancreas, whereas Lewis x, sialyl-Lewis x, and
sialyl-Lewis a were detected principally in pancreatic cancer tissues
(29, 30, 31, 32)
.
(2,3) ST and (1,2) Fuc-T use the same substrate (Fig. 1)
, and
their expression levels in tumor cells could be an important factor in
the formation of Lewis antigens. Some investigators have reported that
the gene transfection of (1,2) Fuc-T results in a decrease of
(2,3) sialylation of (poly)N-acetyl(iso)lactosamine
structures (33, 34, 35)
, suggesting a competition between
(1,2) Fuc-T and (2,3) ST for their common acceptor substrate,
N-acetyl(iso)lactosamine of glycoproteins and glycolipids.
However, to date no study has been performed on human cancer
cells in which sialyl-Lewis expression was controlled by a still
unknown sophisticated system involving many glycosyltransferases. We
recently observed a significant decrease in (1,2) Fuc-T activity in
tumoral pancreatic cell lines compared with normal tissue
(26)
. These data suggested that decreased expression of
(1,2) Fuc-T activity in correlation with (2,3) ST, (1,3)
Fuc-T, and (1,4) Fuc-T activities could favor the expression of
sialyl-Lewis x and sialyl-Lewis a antigens on the cell surface and
consecutively cell adhesion and metastasis.
To resolve this problem, we chose a reverse approach by overexpressing
FUT1 in the human pancreatic tumor cell line BxPC-3, which
expresses very low (1,2) Fuc-T activity and harbors the sialyl-Lewis
a and sialyl-Lewis x antigens on cell surface (26)
. We
have shown that the restoration of FUT1 expression in human
pancreatic tumor cells resulted in the decrease of sialyl-Lewis a and
sialyl-Lewis x antigen expression, which correlated to an increase of
Lewis y and H-type antigen expression. Consequently, a
significant inhibition of E-selectin-mediated cell adhesion and a
decrease of metastatic properties were observed in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western Blotting
Cells were harvested with a rubber policeman, washed in PBS, and
pelleted by centrifugation. Pellets were washed twice with PBS and
lysed for 1 h at 4°C in 100 µl of lysis buffer [10
mM Tris-HCl (pH 7.0), 150 mM NaCl, 1% Triton
X-100] containing a mixture of protease inhibitors (Complete-EDTA
free; Boehringer Mannheim, Germany). These homogenates
were clarified by centrifugation at 14,000 x g for 30 min at 4°C. The concentration of protein was
determined using the bicinchoninic acid assay (Pierce, Rockford, IL).
(Glyco)proteins in reduced SDS buffer were loaded onto 7.5%
polyacrylamide gels, separated electrophoretically, and transferred
onto nitrocellulose membrane. (Glyco)proteins were stained with Ponceau
red, detected by lectin, or immunodetected using mAbs to specific
oligosaccharide structures as primary antibody. Detection and
development were performed as described previously (37)
.
To remove sialic acid residues, cell lysates (15 µg of proteins) were
treated with 2.5 milliunits of Arthrobacter
ureafaciens sialidase (Calbiochem, San Diego, CA) for 6 h at
37°C in 50 mM sodium acetate buffer (pH 5.5).
Fucosyltransferase Assays
Details of the fucosyltransferase assays using
N-acetyllactosamine, lacto-N-biose, and phenyl
ß-D-galactoside (Sigma, St. Louis, MO) as
acceptor substrates have been described elsewhere (26)
.
The different radiolabeled products were subjected to HPLC analysis on
a Supelcosil LC-NH2 (4.6 x 250
mm; 5 µm) column (Supelco, Bellefonte, PA) equilibrated with
acetonitrile/water (75:25, v/v) at a flow rate of 1 ml/min (LC 200
binary system; Perkin-Elmer, Norwalk, CT).
Antibodies
Anti-sialyl-Lewis x (clone KM-93) was from Seikagaku (Tokyo, Japan),
anti-sialyl-Lewis x (clone 2H5) was from Becton Dickinson (Le Pont de
Claix, France), and anti-sialyl-Lewis a (clone C241) was a generous
gift from Dr. Ke Zhang. Anti-sialyl-Lewis a (clone 121SLE), anti-Lewis
b (clone 2-25LE), anti-Lewis a (clone 7LE), anti-Lewis y (clone
12-4LE), and anti-H-type II (clone 19-0LE) were generously donated by
Dr. Jacques Bara. Anti-Lewis x (clone SH1) was a gift from Dr. Else K.
Philipsen. Anti-H-type (clone HMS2 1101A4) was obtained from Sanofi
Diagnostic Pasteur (Marne-la-Coquette, France). Anti-CA19/9 (clone
NCL-CA19/9) was from Novacastra (Newcastle, United Kingdom), and
anti-MUC-1 (clone 4058) was from Euromedex (Souffelmeyersheim, France).
cDNA Probes and Northern Blots
The cDNA probes for FUT1 and actin were obtained by reverse
transcription and PCR, using specific primers as described
previously (26)
. Purified cDNA probes were
[32P]-labeled by random priming, using
[-32P]-dCTP (NEN, Les Ullis,
France) and the random primed DNA labeling kit (Life Technologies,
Inc.) to a specific radioactivity of 
4 x 108 cpm/µg. Total RNA from cultured cells was
isolated following a standard protocol (38)
. mRNA
quantitation was performed as described by Sbarra et al.
(39)
The amount of specific mRNA was estimated by scanning
the autoradiogram on a Macintosh power PC computer, using the public
domain NIH Image program. The slope of each regression line was
obtained from the densitometric data. It was then possible to
appreciate the relative abundance of mRNA specific for FUT1
(in arbitrary units) in a mixture of total RNA.
Transfection
The cDNA coding for the sequence of FUT1 was obtained by PCR
from pCDM7 plasmid containing human (1,2) Fuc-T cDNA
(FUT1; Ref. 27
) and was cloned into
EcoRI/XhoI sites of pCDNA3-neo vector
(Invitrogen, Leek, The Netherlands) to obtain pCDNA3-FUT1 plasmid. The
BxPC-3 cell line was transfected with the pCDNA3-FUT1, using DAC 30
reagent (Eurogentec, Seraing, Belgium). The latter plasmid confers
neomycin resistance to transfected cells. Cells were dispensed into
6-well culture plates and grown to 50% confluence. Growth medium
was removed, and the cells were washed twice with OptiMEM (Life
Technologies, Cergy-Pontoise, France) and then incubated 6 h in 1
ml of OptiMEM with 5 µg of DAC 30 reagent and 1–5 µg of plasmid
DNA. Then transfection medium was replaced for 48 h with RPMI
medium (Life Technologies) and with fresh medium containing 1 mg/ml of
neomycin (Life Technologies). After 5–6 weeks, individual colonies
were isolated using cloning cylinders. Selected clones were referred to
as BxPC-3 FUT1 cells. Control cells (BxPC-3-neo) were obtained by
transfection of BxPC-3 cells with the empty vector
pCDNA3-neo. The cDNA for human E-selectin cloned into pCDM8
vector and obtained from R&D Systems (Abingdon, United Kingdom) was
cotransfected with pCDNA3-neo vector into CHO-K1 cells, and
the selected transfectant was designated as CHO-ES cells. The CHO-K1
cell line transfected with the empty pCDNA3-neo was also
selected and termed CHO-neo.
Flow Cytometry
Detection of fucosylated oligosaccharide epitopes on the surface
of BxPC-3 cells was carried out by indirect fluorescence under the
following conditions. Cells were released from culture plates by
treatment with nonenzymatic cell dissociation solution (Sigma) for 15
min at 37°C. All subsequent steps were carried out at 4°C. The
cells were washed three times in PBS, fixed with 2% paraformaldehyde
in PBS for 10 min, and washed extensively with 1% BSA in PBS.
Oligosaccharide epitopes were exposed for 1 h to specific
antibodies (see above), washed three times with PBS, and finally
incubated for 30 min with fluorescein-labeled antimouse IgG as
secondary antibody (Sigma). Alternatively, cells were incubated with
FITC-conjugated lectin for 1 h on ice. The cells were then washed,
resuspended in Isoflow buffer, and analyzed on an EPICS Profile II flow
cytometer (Coulter, Hialeah, FL).
Cell Adhesion Assays
The cell adhesion assays were performed using a BrdUrd labeling and
detection kit (Roche Diagnostic, Meylan, France) with the following
modifications. Control BxPC-3-neo and BxPC-3 FUT1 cells were labeled
24 h with BrdUrd. Excess label was removed with PBS buffer, and
cells were harvested by cell dissociation solution, washed twice with
PBS, and resuspended in PBS buffer supplemented with 1 mM
CaCl2. Labeled cells were added to CHO-ES and
control CHO-neo cells grown in 96-well plates and saturated previously
with 1% BSA in PBS. After a 30-min incubation at 4°C on a rocking
platform, the plates were washed three times with PBS to remove
nonadherent cells. After cell fixation and denaturation with the
buffers of manufacturer, BrdUrd-labeled DNA was detected using
anti-BrdUrd-POD antibody according to the manufacturer’s instructions.
Specific binding was determined by the measure of absorbance at 370 nm
using an MR 5000 microplate spectrophotometer (Dynatech, Billingohurst,
United Kingdom).
Metastasis Formation Assays in Nude Mice
Female NMRI nu/nu mice (8 weeks old) were obtained from Janvier
(le Genest-St-Isle, France) and were kept in pathogen-free conditions.
All surgical procedures and animal care were carried out according to
accreditation number 04333 given by the French Ministère de
l’Agriculture. Details of orthotopic transplantation in
nude mice, which is the appropriate method for the in vivo
metastasis assays, have been described elsewhere (40
, 41)
.
Briefly, the human pancreatic cancer cell line BxPC-3 was cultured to
90% confluence, washed twice with cold PBS buffer, and harvested with
the cell-dissociation solution. The cells were washed three times with
PBS buffer and kept on ice until injection. The mice were anesthetized
with i.p. injections of a mixture of xylazine (Bayer, Sens, France) and
ketamine (Rhône-Mérieux, Lyon, France), the peritoneal
cavity was opened, and the tumor cell suspension
(106 cells in 10 µl of PBS) was injected into
the pancreas, taking care to ensure that all tumor cells remained
within the pancreas. Four weeks after tumor implantation, the mice were
sacrificed and examined for the pancreatic primary tumor and the
metastatic foci observed mainly in the peritoneum, mesenteric lymphatic
duct, small intestine, mesentery, stomach, and liver. The colonization
of these different tissues by pancreatic tumoral cells have been
described by several investigators (13
, 40, 41, 42, 43)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(1,2)
Fuc-T, FUT1(1,2) Fuc-T activity and high levels of
endogenous (1,3) and (1,4) Fuc-T activities as well as
sialyl-Lewis a and sialyl-Lewis x antigens on its cell surface. The
cDNA encoding the human H-type (1,2) Fuc-T (FUT1) was
transfected into the BxPC-3 cells, and several stable cell clones were
selected in the presence of neomycin. One of these clones, named BxPC-3
FUT1-A, was selected for further studies for the higher expression of
FUT1 mRNA and (1,2) Fuc-T activities. Control cells
(BxPC-3-neo) were concomitantly transfected with the empty pCDNA3.
Northern blot analyses were used to assess the relative mRNA abundance
in the BxPC-3-neo and BxPC-3 FUT1-A clones. As shown in Fig. 2
, quantitation of the Northern blot using FUT1-specific probe
indicated that the abundance of mRNA of FUT1 was 4-fold
higher in BxPC-3 FUT1-A than in BxPC-3-neo cells. The amount of dotted
RNA was normalized with the actin probe. Northern blot analysis with
actin probe indicated no mRNA degradation (data not shown).
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(1,2) Fuc-T Activities in BxPC-3 FUT1-A and
BxPC-3-neo Cells(1,2) Fuc-T was measured using three different
acceptors: phenyl ß-D-galactoside,
N-acetyllactosamine, and lacto-N-biose. As shown
in Fig. 3A
, the amount of [14C]fucose
transferred to phenyl ß-D-galactoside was
significantly increased in BxPC-3 FUT1-A cells compared with BxPC-3-neo
cells. N-Acetyllactosamine may also be the acceptor for
fucose transfer catalyzed by an (1,2) Fuc-T, which allows the
formation of H-type II structures, or by an (1,3) Fuc-T, which leads
to the formation of Lewis x structures (Fig. 1)
. As shown in Fig. 3B
, we observed a significant increase of H-type II antigens
(Fig. 3B
, open columns) in BxPC-3 FUT1-A
compared with BxPC-3-neo cell values. A comparable increase of the
(1,2) Fuc-T activity was also observed in BxPC-3 FUT1-A cells when
we used lacto-N-biose as acceptor that can be fucosylated by
(1,2) Fuc-T to generate H-type I structures (Fig. 3C
,
open columns) or by (1,4) Fuc-T to generate Lewis a
structures (Fig. 3C
, hatched columns). The
formation of Lewis x and Lewis a structures (Fig. 3, B and C
,
hatched columns) was not significantly modified between
these two cell clones.
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(1,2) Fuc-T can transfer a fucose residue onto the terminal
galactose of type I and type II disaccharide (Galß1,3/1,4GlcNAc-R)
and can block the accessibility of other sugar residues to these
galactoside structures. In particular, sialic acid residue, as fucose
residue, is known to terminate elongation of the carbohydrate chain
(Fig. 1)
. Thus, flow cytometric analyses with anticarbohydrate
antibodies and lectin were performed to compare fucosylated and
sialylated oligosaccharides expressed by BxPC-3 FUT1-A and BxPC-3-neo
cells. As shown in Fig. 4, B and C
, a strong expression of H-type antigens
was observed on the cell surface of BxPC-3 FUT1-A cells, which
correlated with the increased activity of (1,2) Fuc-T. These
antigens were undetectable or poorly detectable in parental BxPC-3
cells and in control BxPC-3-neo cells. Similar profiles were obtained
with two different anti-H antibodies. The same result was also obtained
with UEA I lectin, which recognizes the terminal fucose of
Fuc1,2Gal-R motif (Fig. 4D)
. Lewis y epitopes were more
or less detected in two subpopulations of BxPC-3-neo cells. However, an
homogeneous increased binding of anti-Lewis y was observed in the
BxPC-3 FUT1-A clone (Fig. 4E)
. On the other hand, a weak
modification of Lewis b expression between these two cell clones was
observed (Fig. 4F)
.
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, we observed a substantial decrease in expression of these epitopes on
BxPC-3 FUT1-A compared with BxPC-3-neo cell surface. The same results
were obtained with two different anti-sialyl-Lewis x antibodies (Fig. 5, B and C)
, two different anti-sialyl-Lewis a
antibodies (Fig. 5, D and E)
, and with CA19/9
antibody, which recognizes an oncofetal gastrointestinal marker
associated with sialyl-Lewis a antigen (Fig. 5F)
. These data
indicate that the cell surface expression of
NeuAc2,3Galß1,3/1,4[Fuc1,3/1,4]GlcNAc-R structure was
inversely related to that of the
Fuc1,2Galß1,3/1,4[Fuc1,3/1,4]GlcNAc-R structure. This most
likely results from a competition between the (1,2) Fuc-T and the
(2,3) ST for the same acceptor substrate. Interestingly, the
expression of Lewis x and Lewis a was not modified, indicating an
unchanged level of (1,3)- and (1,4)-linked fucose (Fig. 5, G and H)
.
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. These bands, detected by the antibodies 121SLE and CA19/9, exhibited
an apparent molecular size of 195 and 175 kDa (Fig. 6, B and C)
. Another band, migrating at 155 kDa, was detected with
CA19/9 antibody (Fig. 6C)
. On the other hand, we observed
the lack of reactivity of sialyl-Lewis a antibodies in the BxPC-3
FUT1-A cells, whereas the Ponceau red staining of (glyco)proteins
electrotransferred onto nitrocellulose membrane from BxPC-3 FUT1-A and
BxPC-3-neo cell lysates did not show differences (Fig. 6A)
.
Treatment of BxPC-3-neo cell lysate with sialidase abolished the
sialyl-Lewis a antibody reactivity, thus confirming the specificity of
antibodies against sialic acid residues. Interestingly, these
glycoproteins were not detected by Lewis y antibodies and UEA I lectin
in BxPC-3-neo cell lysate (Fig. 6, D and E)
. On
the other hand, these glycoproteins were recognized by Lewis y mAb and
UEA I lectin in BxPC-3 FUT1-A cellular lysate (Fig. 6, D and E)
. These results corroborate the decreased binding of
sialyl-Lewis a antibodies and the increased binding of Lewis y
antibodies and UEA I lectin on BxPC-3 FUT1-A cells observed by
cytofluorometry (see Fig. 5
). These data suggested that the (1,2)
Fuc-T and (2,3) ST compete for sugar transfer on the same
glycoproteins of BxPC-3 cells.
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, probably corresponding to the glycoproteins detected with
sialyl-Lewis a antibodies (Fig. 6, B and C)
. On
the other hand, no reaction was observed with BxPC-3 FUT1-A cells (Fig. 7)
. Because the reactivity of mAb MUC-1 could be increased by sialic
acid residues, the lysates were treated with sialidase. As also shown
in Fig. 7
, this treatment abolished the reactivity of the 195-kDa
protein and promoted a substantial decrease in the reactivity of the
155-kDa protein in the lysate of BxPC-3-neo cells. These data strongly
suggest that glycoproteins carrying modified oligosaccharide structures
could be, in part in BxPC-3 cells, related to MUC-1.
|
(1,2) Fuc-T on adhesion to E-selectin, we recorded
the binding of BxPC-3 FUT1-A and BxPC-3-neo cells to
E-selectin-expressing CHO cells (CHO-ES). As shown in Fig. 8
, we observed a significant increase of adhesion of BxPC-3-neo cells
(Fig. 8
, open columns) to CHO-ES cells compared with control
CHO-neo cells. On the other hand, a significant inhibition of adhesion
of BxPC-3 FUT1-A cells (Fig. 8
, hatched columns) to CHO-ES
cells was observed when compared with BxPC-3-neo cells (Fig. 8
,
open columns), demonstrating that adhesion of these
pancreatic cancer cells to E-selectin depends on the terminal sialic
acid residue linked to (poly)N-acetyl(iso)lactosamine
structures.
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, the number of mice with metastatic foci in the lymph mesentery, small
intestine, and stomach was reduced in the experimental group injected
with BxPC-3 FUT1-A cells compared with the control group of mice
injected with BxPC-3-neo cells. None of the mice in the experimental
group developed tumor nodules in the liver and mesentery. Associated
with the decrease in the number of foci detected, we also noticed a
reduced size of these secondary tumors, in particular the peritoneal
tumors.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(2,3) ST and Fuc-T were the key
glycosyltransferases that regulated the Lewis antigen expression. In a
previous study, we showed a significant decrease in (1,2) Fuc-T
activity in pancreatic cell lines compared with normal tissue. Two
(1,2) Fuc-Ts, FUT1 and FUT2, which are expressed in pancreatic
tissue, can be responsible for this decrease. Related to this decrease
is a reduced expression of Lewis b, Lewis y, and H-type antigens on the
cell surface. On the other hand, tumoral pancreatic cells presented an
enhanced expression of sialyl-Lewis a and sialyl-Lewis x antigens,
which are essential factors in adhesion and metastasis. This decrease
of (1,2) Fuc-T activity in cell lines, in correlation with increased
(2,3) ST and (1,3/1,4) Fuc-T activities, could favor the
expression of sialyl-Lewis x and sialyl-Lewis a determinants on the
cell surface (26)
. To determine whether the low level of
(1,2) Fuc-T expression can promote (2,3) sialylation of
N-acetyl(iso)lactosamine to form the precursor of the
sialyl-Lewis antigens, we overexpressed FUT1 cDNA in the
human metastatic pancreatic cell line, BxPC-3.
The data presented here show that the restoration of (1,2) Fuc-T
activity in BxPC-3 cells results in increased (1,2) fucosylation
associated with decreased (2,3) sialylation on terminal
galactosyl residues of cell surface carbohydrate structures. Indeed,
our results showed, for the first time in human cancer cells, a marked
reduction of sialyl-Lewis a and sialyl-Lewis x antigens that correlated
to an increase of Lewis y and H-type antigens, suggesting a competition
between FUT1 and (2,3) ST for the same acceptor substrate. This
phenomenon has been observed in animal models by some investigators. A
study comparing the types of glycans synthesized by CHO cells
expressing FUT1 with those synthesized by parental CHO cells
lacking FUT1 showed that FUT1 preferentially fucosylates
polylactosamine structures expressed by these cells, resulting in
decreased (2,3) sialylation of these structures (33)
.
Gorelik et al. (34)
and Goupille et
al. (35)
transfected mouse BL6 melanoma cells and rat
REGb colon carcinoma cells, respectively, with FUT1 cDNA and
observed a decrease of N-acetyllactosamine sialylation.
The expression of sialyl-Lewis antigens in human cancer tissues
and cell lines has been studied exhaustively. However, few studies
showed a direct reciprocal relation between Lewis b, Lewis y, and H
antigens, and sialyl-Lewis a and sialyl-Lewis x antigens in the same
tissues or tumoral cell lines. It has been shown that the Lewis b and H
antigens were present in normal human gastric cells (46)
,
whereas sialyl-Lewis antigens were detected in the primary tumor in
patients with advanced gastric cancer (47)
. Bryne et
al. (48)
and Kurahara et al.
(49)
showed, respectively, that the loss of expression of
H antigen and the high expression of sialyl-Lewis a is associated with
the metastatic potential of oral squamous cell carcinomas. Conversely,
an increase of Lewis y expression associated with an increase of
sialyl-Lewis a were often observed in human colorectal cancer tissues (50
, 51)
. On the other hand, a recent study showed
that Lewis b antigen expression was inversely related to Lewis a and
sialyl-Lewis a expression in human colon cancer tissues, suggesting
that FUT2 could compete with (2,3) ST for the type I acceptor
substrates (52)
. Our results suggest that in pancreatic
cells, FUT1 competes preferentially with (2,3) ST for the type II
acceptor substrate because we observed a substantial increase of Lewis
y antigen associated with a weak increase of Lewis b antigen expression
on the cell surface (Fig. 4)
. This corroborates with the FUT1
specificity, which efficiently uses type II chain oligosaccharides as
acceptors (27)
. Interestingly, the expression of Lewis a
and Lewis x was not modified, indicating an unchanged level of
(1,3)- and (1,4)-linked fucose but also that the glycoconjugates
carrying these structures are not acceptor substrates for (1,2)
Fuc-T or (2,3) ST.
The Golgi subcompartmentation of these glycosyltransferases,
which is difficult to examine, and their levels of expression
seem to be an important factor in the aberrant expression of cell
surface carbohydrates during tumor formation (53)
.
Interestingly, our results showed that the competition between (1,2)
Fuc-T and (2,3) ST is highly selective in terminal fucosylation and
sialylation during glycoprotein biosynthesis. Indeed, (1,2)
fucosylation and (2,3) sialylation in BxPC-3 cells are
preferentially restricted to three major glycoproteins (Fig. 6)
. This
selective (1,2) fucosylation was also observed on only two
glycoproteins, lysosome-associated membrane protein-1 and -2, in
CHO cells expressing FUT1 (33)
and on a variant
of the CD44 adhesion molecule in rat colon carcinoma cells expressing
FUT1 (35)
. In our study, we tentatively
identified these glycoproteins as mucin-type glycoprotein MUC-1.
Immunodetection with the mAb for MUC-1 that we have used seems to be
affected by terminal sialic acid and fucose residues. This observation
was supported by Sawada et al. (54)
, and Ho
et al. (55)
, who have shown that the
accessibility of different antibodies against MUC-1 could be altered by
terminal sialic acid. It has been shown that sialyl-Lewis a and
sialyl-Lewis x antigens are present on MUC-1 expressed by human
pancreatic cancer cells, SW1990 (55)
, and could be
implicated in tumor cell binding to the endothelial cell adhesion
molecule E-selectin and in cellular extravasion during metastasis
(56)
. Purified SW1990 mucin as well as pancreatic cancer
sera rich in sialylated Lewis antigens can also inhibit the binding
SW1990 cells to E-selectin (10)
. The modifications of
sialyl-Lewis antigens, specifically on MUC-1, could be very useful in
the development of antiadhesion therapy for cancer metastatic cells. In
addition, these modifications of the glycosylation of MUC-1, which now
is an attractive target for immunotherapy, could enhance the generation
of both anti-MUC-1 antibodies and CTL response (57)
. Our
results suggested a complete inhibition of adhesion of BxPC-3 FUT1-A
cells on a CHO-ES cell monolayer compared with control cells (Fig. 8)
.
These data confirm studies that have shown (a) that the
expression of sialyl-Lewis a on cell surface proteins and/or lipids
could be essential for the adhesion of human pancreatic cancer cells to
activated endothelium mediated by E-selectin (9
, 12)
, and
(b) that the level of surface sialyl-Lewis a expression of
pancreatic cancer cells correlates with the number of metastatic
colonies in the liver (13)
.
In analogy to these in vitro experiments, we observed a decrease of the number and mass of metastatic foci in gastrointestinal tissues when BxPC-3 FUT1-A cells were xenografted in nude mice. Moreover, no difference was observed in the development of pancreatic tumors between mice xenotransplanted with BxPC-3 FUT1-A cells and BxPC-3-neo cells, suggesting that the decrease of sialyl-Lewis antigens could target only the invasiveness throughout activated endothelium mediated by E-selectin. These data corroborate results reported by Gorelik et al. (34) , who reported that transfection of mouse BL6 melanoma cells with FUT1 cDNA resulted in a reduction of sialylation of N-acetyllactosamine structures and a decrease in the metastatic ability of the transfected cells. The impairment of sialyl-Lewis antigen expression could become a good challenge in the development of antiadhesion treatments of pancreatic cancer metastasis (58 , 59) .
It has been shown that the (1,2) fucosylation of CD44v in rat colon
carcinoma contributes to the tumorigenicity (35
, 52
, 60) and that the increase of Lewis b, Lewis y, and H antigens may be
associated with metastatic and invasive properties of lung tumor cells
(61)
and bladder carcinoma cells (62)
. On the
other hand, these same antigens were expressed in normal pancreas.
Thus, Lewis b, Lewis y, and H-antigen expression could vary with
tissues and with the differentiation and pathophysiological states of
these tissues (29
, 52
, 63
, 64)
. The amounts of specific
messages for FUT1 and FUT2 are certainly a
determinant factor in the regulation of sialyl-Lewis antigen
expression. A recent study has identified several forms of the
FUT1 transcript, generated by two transcription start codons
and alternative splicing of 5'-untranslated exons in several tumor cell
lines (64)
. These authors suggested that dual promotors
regulated the stage- and tissue-specific expression of the
FUT1 transcript and thus the expression of Lewis-related
antigens in many human tissues. Hakomori (65)
proposed a
scheme for the involvement of glycosylation in tumor cell metastasis
where the mechanism at each step is greatly influenced by a different
glycosylation associated with expression of key adhesion molecules,
such as CD44, integrin, and E-cadherin. The aberrant
glycosylation may alter tumor cell adhesion or motility. In particular,
the (1,2) fucosylation of CD44 could be implicated in extracellular
matrix-dependent cell adhesion and motility of tumoral cells, whereas
sialyl-Lewis antigens could be implicated in the E-selectin-mediated
adhesion of tumoral cells to activated endothelium (65)
.
It has also been shown that sialyl-Lewis a and integrin mediate the
process from adhesion to implantation of human pancreatic cancer SW1990
cells to endothelial cells and that CD44 and integrin play important
roles in the initial attachment of these cells to mesothelium cells
(66)
.
To modify the metastatic phenotype of pancreatic carcinoma cells, it is
important to understand the mechanisms that regulate the biosynthesis
of sialyl-Lewis a and sialyl-Lewis x. The expression of these
structures in pancreas could be controlled by a sophisticated system
involving (1,2) Fuc-T and (2,3) ST associated with (1,3) Fuc-T
and/or (1,4) Fuc-T expression. For the first time, we have shown
that the decrease of (1,2) Fuc-T expression in human cancer cells
could favor the (2,3) ST activity to form metastatic sialyl-Lewis
antigens. An investigation of the expression level of (2,3) ST and
(1,3) and (1,4) Fuc-T gene products in normal and neoplastic human
pancreatic tissues (and model cell lines) would be required to gain
more information concerning the regulation of the expression of
sialyl-Lewis determinants on the cell surface, which is implicated in
cell adhesion and metastasis, and to develop antiadhesion cancer
therapy.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by Grants 9506 from the Association
pour la Recherche sur le Cancer (Villejuif, France) and 930/25LC/64
from the Ligue Nationale Contre le Cancer (Comité des
Régions PACA et Corse, France; to E. M.). M. A., and
L. P. are recipients of doctoral fellowships from Ministère
de l’Enseignement Supérieur, de la Recherche et de la
Technologie. 
2 To whom requests for reprints should be
addressed, at INSERM Unité 260, Faculté de Médecine,
27, Boulevard Jean Moulin, 13385 Marseille Cedex 05 France. Phone:
330-491-32-4400; Fax: 330-491-83-0187; E-mail: Eric.Mas{at}medecine.univ-mrs.fr
3 The abbreviations used are: ST,
sialyltransferase; GlcNAc, N-acetylglucosamine; Fuc-T,
fucosyltransferase; mAb, monoclonal antibody;
N-acetyllactosamine, type II precursor,
Galß1,4GlcNAc; lacto-N-biose,
N-acetyl(iso)lactosamine, type I precursor,
Galß1,3GlcNAc; HPLC, high-pressure liquid chromatography; CHO,
Chinese hamster ovary; BrdUrd, 5-bromo-2'-deoxyuridine.
Received 9/27/99. Accepted 1/ 5/00.
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) and BxPC-3 FUT1-A (b, 
).
Intensity (pixels) in arbitrary units plotted versus
µg of RNA.
