C2-O-sLeXGlycoproteins Are E-Selectin Ligands that
Regulate Invasion of Human Colon and Hepatic
Catherine A. St. Hill.*, Dahabo Baharo-Hassan, Mariya Farooqui.
Department of Veterinary Clinical Sciences, University of Minnesota, St. Paul, Minnesota, United States of America
Similar to mechanisms of recruitment of activated leukocytes to inflamed tissues, selectins mediate adhesion and
extravasation of circulating cancer cells. Our objective was to determine whether sialyl Lewis X modified core 2 O-glycans
(C2-O-sLeX) present on colon and hepatic carcinoma cells promote their adhesion and invasion. We examined membrane
expression of C2-O-sLeX, selectin binding, invasion of human colon and hepatic carcinoma cell lines, and mRNA levels of
alpha-2,3 fucosyltransferase (FucT-III) and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1) genes, necessary for C2-
O-sLeXsynthesis, by quantitative reverse-transcriptase (RT) PCR. Synthesis of core 2 branched O-glycans decorated by sLeXis
dependent on C2GnT1 function and thus we determined enzyme activity of C2GnT1. The cell lines that expressed C2GnT1
and FucT-III mRNA by quantitative RT-PCR were highly positive for C2-O-sLeXby flow cytometry, and colon carcinoma cells
possessed highly active C2GnT1 enzyme. Cells bound avidly to E-selection but not to P- and L-selectin. Gene knock-down of
C2GnT1 in colon and hepatic carcinoma cells using short hairpin RNAs (shRNA) resulted in a 40–90% decrease in C2-O-sLeX
and a 30–50% decrease in E-selectin binding compared to control cells. Invasion of hepatic and colon carcinoma cells
containing C2GnT1 shRNA was significantly reduced compared to control cells in Matrigel assays and C2GnT1 activity was
down-regulated in the latter cells. The sLeXepitope was predominantly distributed on core 2 O-glycans on colon and
hepatic carcinoma cells. Our findings indicate that C2GnT1 gene expression and the resulting C2-O-sLeXcarbohydrates
produced mediate the adhesive and invasive behaviors of human carcinomas which may influence their metastatic
Citation: St. Hill CA, Baharo-Hassan D, Farooqui M (2011) C2-O-sLeXGlycoproteins Are E-Selectin Ligands that Regulate Invasion of Human Colon and Hepatic
Carcinoma Cells. PLoS ONE 6(1): e16281. doi:10.1371/journal.pone.0016281
Editor: Donald Gullberg, University of Bergen, Norway
Received October 4, 2010; Accepted December 9, 2010; Published January 19, 2011
Copyright: ? 2011 St. Hill et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by funds from the National Institutes of Health, National Cancer Institute grant 5KO8CA111829-04. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Recognition and binding of selectins to sialyl Lewis X (sLeX)
and related oligosaccharides are crucial interactions that regulate
leukocyte adhesion to blood vessels and extravasation into tissues
in an inflammatory response . Similar mechanisms are used by
circulating tumor cells during metastasis to enter target organs
[2,3]. Selectin adhesion molecules: L-selectin constitutively
expressed on most leukocytes , P-selectin expressed in activated
platelets and endothelial cells [5,6], and E-selectin induced on
cytokine stimulated endothelial cells , mediate these processes.
High affinity binding of selectins to sLeXon human leukocytes is
greatly enhanced when sLeXis terminally displayed on core 2
based O-linked glycans (C2-O-sLeX) [8,9,10]. On leukocytes, C2-
O-sLeXdecorates the mucin P-selectin glycoprotein ligand-1
(PSGL1, CD162) and is particularly important for high-strength
binding interactions with P-selectin [11,12,13,14].
Synthesis of carbohydrates with sialyl Lewis structures increases
upon neoplastic transformation and are useful markers for cancers
. Several carcinomas including colon, gastric, lung, pancreatic,
prostate, and urinary bladder highly express the selectin ligand sLeX
poor prognosis [16,17,18,19,20,21]. Up-regulation of vascular E-
selectin in cancers with high expression of sialyl Lewis structures is
thought to be a risk factor for hematogenous metastasis . In
colon and hepatocellular carcinomas, a high content of sLeX
antigens is associated with increased metastatic potential [17,23] but
the molecular mechanisms involving sLeX that regulate metastasis
are not well understood. It is important to understand the role of
glycans in cancer progression because the altered N- or O-
glycosylation status of tumors may predict their metastatic potential
and promote invasion and metastasis . We have previously
demonstrated that a coloncarcinoma celllineexpressing C2-O-sLeX
carbohydrates binds to E-selectin , and that C2-O-sLeXis a
tumor-associated antigen in colon cancer tissues . In this report,
we investigated the impact of disrupting C2-O-sLeXsynthesis on the
distribution of sLeXon core 2 O-glycans versus N-glycans on colon
and hepatic carcinoma cell lines, their E-selectin binding capacity,
and the influence on their invasive properties as initial steps to
elucidate the function of these carbohydrates in metastasis.
C2-O-sLeXbiosynthesis is complex and occurs by a series of
enzymatic steps involving activities of glycosyltransferases (Fig. 1).
Briefly, N-acetylgalactosamine is added to serine or threonine
residues at the protein backbone, followed by galactose in a b1,3
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linkage, creating the core 1 extension. The enzyme core 2 b1,6-N-
acetylglucosaminyltransferase (C2GnT1) initiates the core 2
extension by adding N-acetylglucosamine to N-acetylgalactosa-
mine in a b1,6 linkage and is the key branching enzyme in core 2
O-glycan biosynthesis. Core 2 branches are elongated by several
glycosyltransferases and terminated by the addition of sialic acid in
an a2,3 linkage to galactose. Finally, a(1,3/1,4) fucosyltransferase
(FucT-III) catalyzes the addition of fucose to the chain in an a1,3
linkage, resulting in the formation of C2-O-sLeX. Functional
activity of both C2GnT1 and FucT-III enzymes is necessary for
We targeted the C2GnT1 gene to disrupt C2-O-sLeX
biosynthesis in carcinoma cells. We chose this gene because high
C2GnT1 expression in carcinomas has previously been correlated
with vessel invasion, depth of tumor invasion, and metastasis
[31,32,33]. Using the CHO-131 monoclonal antibody (mAb)
which specifically detects C2-O-sLeX, we directly examined
the ability of tumor cells transduced with C2GnT1 shRNA and
expressing low levels C2-O-sLeXto bind to selectins. We have
previously shown that C2-O-sLeXis a tumor-associated ligand that
is abundant on the invasive front of human colon carcinoma
tissues, and mediates E-selectin binding when expressed on a colon
carcinoma cell line [25,26]. In this report, we directly demon-
strated for the first time that expression and activity of the
C2GnT1 gene responsible for C2-O-sLeXsynthesis in colon and
hepatic carcinoma cell lines regulated invasion of tumor cells, a
key property that facilitates metastasis. We found that the sLeX
epitope was predominantly distributed on O-glycans compared to
N-glycans and was an important E-selectin ligand in these cell
Human carcinoma cell lines highly express genes
involved in C2-O-sLeXsynthesis and bind to E-selectin
We examined mRNA expression of C2GnT1 and FucT-III
genes in colon (LS174T) and hepatic (HepG2) cell lines by RT-
PCR. Both cell lines expressed C2GnT1 and FucT-III mRNA
necessary for C2-O-sLeXsynthesis (Fig. 2A). To investigate
whether mRNA expression of the C2GnT1 and FucT-III genes
resulted in cell surface expression of C2-O-sLeX, we tested the
carcinoma cell lines for reactivity with CHO-131 mAb by flow
cytometry. Approximately 75% of un-manipulated LS174T
colorectal adenocarcinoma cells (MFI, 5866) and 55% of HepG2
hepatic carcinoma cells (MFI, 1659) reacted positively with CHO-
131 mAb but did not react with an isotype control mAb (Fig. 2B).
The binding of the sLeXepitope on colon carcinoma cells to E-
selectin has been associated with a greatly increased metastatic
potential and a poor prognosis . We therefore examined the
participation of C2-O- sLeXin E-selectin binding of LS174T and
HepG2 cells by detecting the ability of these carcinoma cell lines
expressing C2-O-sLeXto bind to a mouse E-selectin Fc chimera by
flowcytometry. Selectin bindinginteractions arecalcium dependent
and are dissociated in the presence of EDTA, a chelator of calcium
ions. LS174T and HepG2 cells with high endogenous C2-O-sLeX
expression bound avidly to E-selection (98% and 95% cellular
Figure 1. Pathway of C2-O-sLeXbiosynthesis. A simplified version of C2-O-sLeXbiosynthesis is shown. Core 1 O-glycans are synthesized by
addition of b1,3 galactose to N-acetylgalactosamine. The core 2 b1,6-N-acetylglucosaminyltransferase (C2GnT1) enzyme catalyzes the addition of a
b1,6 branch to a core 1 O-glycan to form a core 2 b1,6 branched O-glycan. The core 2 O-glycan structure is further modified by a series of enzymatic
reactions (omitted for clarity) including modification by a2,3 sialyltransferase and a1,3 fucosyltransferase (FucT-III) to form a sLeXterminus (dotted
box). These modifications result in the synthesis of the sLeX-modified core 2 b1,6 O-glycan (C2-O-sLeX) structure. GalNAc, N-acetylgalactosamine; Gal,
galactose; GlcNAc, N-acetylglucosamine; NeuAc, sialic acid; Fuc, fucose.
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Figure 2. C2GnT1 and FucT-III genes are endogenously expressed in human carcinoma cell lines. (A) Endogenous C2GnT1 and FucT-III
mRNA transcripts in LS174T colorectal adenocarcinoma (lane 1) and HepG2 hepatic carcinoma (lane 2) cell lines were detected by RT-PCR. (B) Flow
cytometric analysis of positive staining of LS174T and HepG2 cells labeled with CHO-131 mAb (anti-C2-O-sLeX). A representative example of 3
experiments is shown for (A-B). (C) LS174T (*p=0.008) and HepG2 cells (#p=0.001) substantially bind to E-selectin in the presence of calcium ions
(Ca2+). Binding is impeded in the presence of EDTA, a calcium ion chelator. The average of two experiments is shown.
C2-O-sLeXon Human Carcinomas Regulate Invasion
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binding for LS174T and HepG2 cells respectively compared to
binding in the presence of 10 mM EDTA) (Fig. 2C).
Knock-down of the C2GnT1 gene results in decreased
Currently, a specific inhibitor of C2GnT1 expression and
activity is not available. To directly evaluate the influence of
C2GnT1 activity on the generation of C2-O-sLeXepitopes on
tumor cells and the binding to E-selectin, we instead used short
hairpin RNA (shRNA) gene silencing techniques to target the
C2GnT1 gene in HepG2 and LS174T cells. The four lentiviral
pGIPZ-C2GnT1-shRNA vectors, the PLKO.1-C2GnT-shRNA
vector, the scrambled pGIPZ-shRNA vector, and the empty
PLKO.1 control vector were tested for silencing of the C2GnT1
gene in LS174T colon carcinoma and HepG2 hepatic carcinoma
cells. All shRNA sequences achieved consistent gene knockdown in
the cell lines tested indicating that our observed effects were
specifically due to loss of the C2GnT1 gene. The clones that were
most efficient at gene knockdown were selected for subsequent
experiments. Thus, LS174T cells were stably transduced with one
of the lentiviral pGIPZ-C2GnT1-shRNA vectors or with a
scrambled pGIPZ-shRNA vector. HepG2 cells were transiently
transfected with the lentiviral PLKO.1-C2GnT-shRNA vector or
with an empty PLKO.1 control vector. The C2GnT1 shRNA
sequences were homologous to only the human C2GnT1 gene
when compared to the human genome using NCBI Nucleotide
BLAST. The scrambled shRNA vectors were not complementary
to any gene in the human genome.
As shown in Fig. 3A, mRNA levels of the C2GnT1 gene were
considerably lower in LS174T cells transduced with C2GnT1
shRNA compared to cells transduced with the scrambled shRNA
vector by densitometry. Similarly, mRNA levels of C2GnT1 were
lower in HepG2 cells transfected with C2GnT1 shRNA compared
to cells transfected with the control vector (Fig. 3B). In contrast, in
both cell lines, FucT-III mRNA levels were not affected by
silencing of the C2GnT1 gene, indicating that the shRNA clones
did not have off-target effects. The C2GnT1 protein was detected
in LS174T cells transduced with the scrambled shRNA vector but
not in cells transduced with C2GnT1 shRNA after immunopre-
cipitation and Western blotting with an anti-C2GnT1 antibody
(Fig. 3C and D). The percentage of cells that was reactive with
CHO-131 mAb, indicating expression of cell surface C2-O-sLeX
before and after C2GnT1 gene knock-down, was assessed by flow
cytometry. In multiple experiments using LS174T and HepG2
cells, we consistently achieved approximately 30–40% reduction of
C2-O-sLeXexpression after C2GnT1 gene knock-down and a
representative experiment is shown in Fig. 3D. We verified
silencing of the C2GnT1 gene by examining C2GnT1 enzyme
activity in LS174T cells and observed a significant ,40% decrease
in enzyme activity of LS174T cells transduced with C2GnT1
shRNA compared to those cells transduced with scrambled
shRNA (p=0.02, Fig. 3E). For all groups of cells, viability was
assessed by trypan blue exclusion and was greater than 95%.
For LS174T and HepG2 cells in which the C2GnT1 gene was
silenced and for control cells, reactivity with a mouse E-selectin/Fc
chimera was assessed by flow cytometry. We did not observe a
significant decrease in E-selectin binding of LS174T cells
containing C2GnT1 shRNA compared to un-manipulated cells
and cells transduced with scrambled shRNA (Fig. 4A). However,
we observed a significant 40% decrease in E-selectin binding for
C2GnT1 shRNA-transfected HepG2 cells compared to those cells
transfected with the control vector, (p=0.02, Fig. 4B).
To further assess the influence of C2GnT1 expression on E-
selectin binding of LS174T cells, we performed more stringent
hydrodynamic shear flow assays that closely simulate forces
occurring in the microvasculature. The adhesive behavior of cells
was examined at a range of shear stresses between 0.5 and 1.5
dynes/cm2and chimera concentrations between 0.1 and 5 mg/ml
in the presence of 10 mg/ml of an anti-E-selectin function-blocking
mAb or an isotype-matched control mAb. We observed optimal E-
selectin binding of isotype control mAb treated un-manipulated
LS174T cells and LS174T cells transduced with C2GnT1 shRNA
at a concentration of 1 mg/ml of the E-selectin/Fc chimera and a
shear stress of 0.5 dynes/cm2(Fig. 4C). Under these conditions,
accumulation of LS174T cells transduced with C2GnT1 shRNA
was reduced by 36% compared to accumulation of un-manipu-
lated LS174T cells, p=0.04. For both groups of cells, fewer cells
accumulated on the E-selectin chimera as the shear stress was
increased to 1.5 dynes/cm2. In order to assess the specificity of the
interaction of cells with E-selectin, both groups of cells were
exposed to an anti-E-selectin function-blocking mAb (10 mg/ml) or
an isotype-matched control mAb at the same concentration.
Accumulated cells at a shear stress of 0.5 dynes/cm2are shown in
Fig. 4D and binding of both groups of cells exposed to the isotype
control mAb is shown for comparison. The presence of an E-
selectin blocking mAb greatly diminished the numbers of un-
manipulated LS1704T (p=0.004) and C2GnT1 transduced cells
bound to E-selectin (p=0.0001). The addition of 20 mM EDTA
to perfusates prevented the binding of cells to E-selectin (data not
LS174T and HepG2 cells predominantly express sLeXon
core 2 O-glycans
In order to determine the nature of the carbohydrate structure
to which the sLeXepitope is attached in un-manipulated LS174T
and HepG2 cells, we treated cells with agents to inhibit O-
glycosylation (BGN) and N-glycosylation (SWN) because these
glycans are the main carriers of sLeX. Treated and untreated cells
were labeled with CSLEX1 mAb that detects sLeXon any
structure to assess the percentage of sLeXpositive cells on O-
glycans versus N-glycans by flow cytometry. For both LS174T and
HepG2 cells in the presence of BGN but not SWN, a significant
decrease in the percentage of cells reactive with CSLEX1 mAb
was observed compared to untreated cells (Fig. 5A and B,
p=0.0004 for LS174T cells and p,0.0001 for HepG2 cells)
indicating that sLeXwas mainly expressed on O-glycans in these
cell types. Treatment of LS174T and HepG2 cells with both BGN
and SWN inhibitors did not cause a further reduction in cells
reactive with CSLEX1 mAb but the decrease was significant when
compared to untreated cells, (p=0.002 and p,0.0001 respective-
ly), and was likely due to the effects of BGN.
LS174T and HepG2 cells, either untreated or treated with
BGN, were subsequently labeled with CHO-131 mAb that
specifically detects C2-O-sLeXpositive cells to determine the
percentage of the subset of core 2 O-glycans that carried sLeX. We
observed that reactivity with CHO-131 mAb was significantly
reduced by 40% in BGN-treated LS174T and by 60% in HepG2
cells compared to untreated cells (Fig. 5C, p,0.0001). Binding to
E-selectin was also evaluated in untreated cells and in cells treated
with the inhibitors. As shown in Fig. 5D and E, BGN treatment
significantly decreased E-selectin binding of LS174T cells by 38%
(p=0.003) and of HepG2 cells by 67% (p=0.0005) compared to
untreated cells. A combination of BGN and SWN treatment did
not result in further reductions in E-selectin binding of LS174T
and HepG2 cells but significant differences were observed when
compared to untreated cells, p=0.0005 and p,0.0001 respec-
tively, indicating the effects of BGN treatment.
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C2-O-sLeXmediates the invasion of carcinoma cells
We examined the influence of silencing of the C2GnT1 gene on
a Matrigel invasion assay that mimics active invasion of tumor cells
across a basement membrane, we found that after 48 hours, the
number of C2GnT1 shRNA-transduced LS174T cells that invaded
across the Matrigel membrane was significantly decreased compared
to LS174T cellstransduced with scrambled shRNA(meannumberof
cells/field =27 versus 162, p,0.0001) (Fig. 6A, C, and D). Similarly,
we observed a significant decrease in invasion of HepG2 cells
transfected with C2GnT1 shRNA compared to cells transfected with
a control vector, (mean number of cells/field =14 versus 99,
p,0.0005) (Fig. 6B, E, and F). Our results indicated that C2GnT1
gene expression mediated the invasive properties of LS174T and
HepG2 cells by an unknown mechanism.
In the metastatic process, circulating cancer cells in the
bloodstream arrest and adhere to endothelium, extravasate and
invade into organs distant from the primary tumor, survive,
proliferate, and promote angiogenesis. These multistep events
Figure 3. C2GnT1 gene knockdown decreases C2-O-sLeXglycoproteins and C2GnT1 activity. (A) C2GnT1 suppression by shRNA resulted
in decreased mRNA levels of C2GnT1 but not FucT-III in LS174T cells, (scrambled shRNA vector, lane 1, and C2GnT1 shRNA vector, lane 2). (B) Similar
effects on C2GnT1 mRNA but not FucT-III mRNA were observed in HepG2 cells after transient transfection with C2GnT1 shRNA (empty control vector,
lane 1, and targeting shRNA vector, lane 2). Densitometry values are normalized to b-actin. (C) The C2GnT1 glycoprotein (50 kDa) was present in
scrambled shRNA transduced LS174T cells (lane 1) but absent in C2GnT1 shRNA transduced cells (lane 2) as detected by immunoprecipitation and
Western blotting using a polyclonal anti-C2GnT1 antibody. (D) The procedures were repeated using the C2GnT1 antibody for immunoprecipitation
and a negative control IgG antibody for Western blotting. Note the absence of a detected C2GnT1 protein 50 kDa band. (D) We observed a 30–40%
decrease in reactivity with CHO-131 mAb by flow cytometry for LS174T and HepG2 cells that contained C2GnT1 shRNA. Representative data from
multiple experiments are shown. (E) C2GnT1 enzyme activity, measured as nmols of N-acetylglucosamine (GlcNAc) transferred per mg protein per
hour in a glycosyltransferase assay, was also significantly decreased in C2GnT1 shRNA transduced LS174T cells, *p=0.02. Mean values 6 standard
deviations of two independent experiments are presented.
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require complex adhesive interactions between ligands on cancer
cells and cellular adhesion molecules and other substrates present
in the tumor microenvironment [21,34]. Many types of cancers
over-express altered carbohydrates that profoundly impact tumor
growth, adhesion, invasion, and metastasis. Alterations in
glycosylation, including the production of sLeXepitopes, are
generated by glycosyltransferases, yet surprisingly the role of
glycosyltransferases in cancer malignancy is still not well defined.
In this report, we have directly shown that the glycosyltrans-
ferase C2GnT1 and its product cell membrane expressed C2-O-
sLeXregulate E-selectin binding and invasion of colon and hepatic
carcinoma cells. After gene silencing of C2GnT1 and treatment
with O- and N-glycosylation inhibitors, we observed significant
reductions in the high levels of C2-O-sLeXpresent on cells,
binding to E-selectin, and cellular invasiveness into Matrigel.
Binding of carcinoma cells to E-selectin was specific as evidenced
Figure 4. C2GnT1 gene knock-down results in decreased binding of cells to E-selectin. (A) Significant differences in E-selectin binding
were not observed among LS174T un-manipulated cells, cells transduced with scrambled shRNA, or with C2GnT1 shRNA by flow cytometry. (B) E-
selectin binding was significantly decreased for HepG2 cells transfected with C2GnT1 shRNA compared to cells transfected with the control vector,
*p=0.02. (C) Shear flow assays were performed at an E-selectin/Fc chimera concentration of 1 mg/ml and shear stresses ranging from 0.5 – 1.5 dynes/
cm2in the presence of 10 mg/ml of an IgG isotype control mAb. At a shear stress of 0.5 dynes/cm2, significantly fewer C2GnT1 shRNA transduced cells
accumulated on E-selectin than un-manipulated LS174T cells, *p=0.04. (D) In the same run of experiments, at a shear stress of 0.5 dynes/cm2,
significantly fewer un-manipulated LS174T cells treated with a 10 mg/ml of a blocking anti-E-selectin mAb accumulated on E-selectin than those cells
treated with an isotype control mAb,#p=0.004. Significantly fewer LS174T cells transduced with C2GnT1 shRNA and treated with a blocking anti-E-
selectin mAb accumulated on E-selectin than those cells treated with an isotype control mAb,&p=0.01. The same assay as shown in (C) at 0.5 dynes/
cm2is included for comparison. For both groups of cells treated with an isotype control mAb, significantly fewer un-manipulated LS174T cells
accumulated on E-selectin than LS174T cells transduced with C2GnT1 shRNA, *p=0.04. Each continuous shear flow assay was performed in duplicate
for each shear stress and the bars represent the mean 6 standard deviations. Representative data from three independent experiments are shown.
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Figure 5. LS174T and HepG2 cells predominantly express sLeXon core 2 O-glycans. (A) The percentage of sLeXpositive LS174T cells was
significantly decreased after treatment with BGN (an O-glycosylation inhibitor) *p=0.0004, or with BGN and SWN (an N-glycosylation inhibitor,
**p=0.002) compared to untreated cells (None). (B) For HepG2 cells, a reduction in the percentage of sLeXpositive cells was observed after treatment
with BGN *p , 0.0001, or with BGN and SWN **p , 0.0001, compared to untreated cells. (C) The cells used in (A) and (B) before and after treatment
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by the lack of appreciable binding in the presence of a function
blocking anti-E-selectin mAb. Our findings are novel because they
indicate that targeting of the C2GnT1 gene and subsequent down-
regulation of C2-O-sLeXexpression, a selectin ligand on colon and
hepatic carcinoma cells, altered key properties of metastasis that
are likely occur in vivo: decreased attachment of circulating tumor
cells to endothelium and invasion into tissues. The possibility of
the cellular toxicity of the gene silencing clones introduced into
cells was excluded because the viability of all groups of cells was
Figure 6. C2GnT1 mediates invasion. (A) LS174T cells transduced with C2GnT1 shRNA were significantly less invasive than cells transduced with
scrambled shRNA, *p,0.0001, in Matrigel transwell invasion assays. (B) Similarly, decreased invasion was observed for HepG2 cells that were
transfected with C2GnT1 shRNA compared to transfection with a control vector, *p=0.0005. (C) Representative photomicrographs from two separate
experiments of invasion of LS174T cells transduced with the scrambled shRNA vector compared to (D) cells transduced with C2GnT1 shRNA and (E)
HepG2 cells transfected with the control vector compared to (F) HepG2 cells transfected with C2GnT1 shRNA. The invading cells were stained and
counted in five separate fields of view at 100X magnification. Arrows indicate invasive cells.
with BGN were also labeled with CHO-131 mAb to specifically detect C2-O-sLeX. Note the significant reduction in the percentage of C2-O-sLeX
positive LS174T and HepG2 cells after treatment with BGN compared to untreated cells, * and **p , 0.0001. (D) A significant decrease in E-selectin
binding was observed for LS174T cells after treatment with BGN *p=0.003, and after BGN and SWN **p=0.0005, compared to untreated cells.
(E) Similar results were observed for HepG2 cells after BGN,#p=0.0005, and after BGN and SWN,##p,0.0001, compared to untreated cells.
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The observations reported here agree with our previous findings
that C2GnT1 gene expression results in synthesis of C2-O-sLeX
that binds to E-selectin [25,26]. We have found that C2-O-sLeXis
predominantly expressed at the advancing edge of invasive
colorectal adenocarcinomas and high mRNA levels of C2GnT1
are present in these carcinomas compared to normal colonic
tissues . Our results support previous studies that suggest that
C2GnT1 plays an important role in cancer. Earlier reports found
that C2GnT1 activity is increased in human leukemia cells and
metastatic murine tumor cell lines [35,36,37]. C2GnT1 gene
expression is more highly associated with progression of colon and
lung cancers than expression of sialyl Lewis structures [31,32] and
also promotes prostate cancer progression . The cell surface
sialyl Lewis ligands on cancer cells generated by C2GnT1
expression increase the binding efficiency of clonal cancer cell
lines to E-selectin on endothelial cells which is directly propor-
tional to their metastatic potential .
In this study, we did not further examine the possible
mechanisms by which down-regulation of C2GnT1 and C2-O-
sLeXexpression led to reduced invasiveness of cancer cells but we
are currently investigating potential processes. We speculate that
changes in C2GnT1 and C2-O-sLeXexpression may alter the
expression and function of other adhesion molecules on cancer
cells or regulate components of the extracellular matrix known to
participate in invasive events. C2GnT1 may regulate the
expression of tumor-associated epitopes such as cell surface
associated mucins which may favor tumor progression . For
instance, mucins such as MUC1 and MUC4 present on cancer
cells participate in signal transduction pathways that contribute to
the invasive and metastatic activities of adenocarcinomas .
Over-expression of MUC1 is associated with invasive and
metastatic colon cancer [42,43], and MUC1 is also expressed on
liver cancers . Oligosaccharide and protein structures on
MUC1 can mediate adhesion events by binding to E-selectin .
Conceivably, C2-O-sLeXmay be one of the oligosaccharides
attached to MUC1 or another mucin present on cancer cells that
may modulate their invasive properties.
Matrix metalloproteinases (MMPs) are a large group of proteins
that degrade the extracellular matrix and facilitate the invasion
and metastasis of malignant cells into distant tissue sites. MMPs
promote motility, migration, and invasion of both colon and
hepatocellular carcinomas [46,47]. The membrane type MMPs,
and in particular MT1-MMP bound to tumor cells promote
cancer cell motility and invasion. MT1-MMP has also been
reported to cleave MUC1 , and the cell adhesion molecule
CD44: an E-selectin ligand that participates in adhesion and
invasion . MT1-MMP also participates in processing the av
subunit of integrins, which are heterodimeric transmembrane
glycoprotein receptors that function as cell anchoring and
signaling molecules and are also known to be involved in invasion
and tumor progression [50,51]. It is possible that C2GnT1 and/or
C2-O-sLeXexpression in tumors may modulate the activities of
MMPs and consequently their targets such as MUC1, CD44, or
integrins and thereby indirectly regulate invasion.
Other enzymes can compete with C2GnT1 for the core 1
substrate and prevent the formation of core 2 O-glycans, including
C2-O-sLeX, and therefore it is important to determine the
structure to which C2-O-sLeXis attached. Differences in this
structure may considerably influence the adhesion, invasion, and
metastatic potential of cancer cells. Several scaffold molecules
containing complex core 2 O-glycans, other O-glycan structures,
N-glycans, or glycolipids are also terminally decorated with sLeX
[53,54,55]. We have shown here that in a human colon carcinoma
and hepatic carcinoma cell line, sLeXwas primarily distributed on
core 2 O-glycans forming C2-O-sLeX. Several reports indicate
that high expression of C2GnT1 mRNA transcripts in carcinoma
cells positively correlates with metastasis [31,32,33]. This report
suggests that C2GnT1 activity, resulting in the generation of C2-
O-sLeXcarbohydrates on tumor cells, in addition participates in
cancer invasion. Thus, C2-O-sLeXmay have distinct functions
depending on its site of expression: on circulating tumor cells it
could enhance attachment of tumor cells to endothelium and C2-
O-sLeXcould facilitate invasion on malignant cells that have
extravasated and invaded into distant metastatic sites from the
Abnormal glycosylation is a prominent feature of malignant
transformation and cancer progression . Although our studies
indicate that abundant C2-O-sLeXstructures present on colon and
hepatic carcinoma cells mediate invasion, this moiety is not the
only participant in E-selectin binding and invasion because knock-
down of the C2GnT1 gene and treatment with O- and N-glycan
inhibitors did not abrogate selectin binding and invasion,
nevertheless, C2-O-sLeXmay act in concert with other molecules
to promote these processes.
C2GnT1, also known as C2GnT-L (leukocyte type), is a key
branching enzyme that controls mammalian core 2 O-glycan
synthesis [57,58]. Huang et al. recently reported that gene
expression of another C2GnT isotype, C2GnT-M (mucin type),
is downregulated in colorectal carcinomas and that C2GnT-M
expression suppressed cell growth, adhesion, motility, invasion,
and colony formation ability . C2GnT1 and C2GnT-M may
have alternate functions in regulating the progression of
carcinomas at various stages of malignancy of the tumor.
C2GnT-M may act predominantly as an inhibitor of early stages
of colorectal carcinoma growth. As the tumor progresses to more
advanced stages, C2GnT1 activity may dramatically increase
resulting in a corresponding increase in C2-O-sLeXexpression,
and increased invasion of cancer cells.
C2-O-sLeXis specifically recognized by the CHO-131 mAb
that has been extensively characterized in cancer and immune
cells [25,26,28,60,61]. Other antibodies such as CSLEX1 mAb
recognize the sLeXepitope alone that can be present on several
types of glycan structures including core 2 O-glycans and thus,
these antibodies have broader recognition of glycan structures
. The reactivity of CHO-131 mAb is unique in that it requires
the functional activity of the glycosyltransferases C2GnT1, a2,3-
sialyltransferase, and a1,3-fucosyltransferase [28,29,30]. CHO-
131 mAb is not a function blocking antibody, however, and at high
concentrations does not inhibit interactions of C2-O-sLeX
terminally displayed on leukocyte PSGL1 with P-selectin .
In summary, our results indicate that the C2GnT1 enzyme, that
regulates C2-O-sLeXsynthesis of cancer cells, participates in
invasion of colon and hepatic carcinomas. Our findings are novel
because we have identified a previously un-described function of
the carbohydrate enzyme C2GnT1 and its associated product C2-
O-sLeXin mediating the invasive properties of colon and hepatic
carcinoma cells. It is well established that inflammatory responses
contribute to tumor development including the invasive behavior
of tumor cells. Invasion requires extensive proteolysis of the
extracellular matrix at the invasive front. Our study highlights the
complex interactive roles that molecules in the tumor microenvi-
ronment play in neoplastic development. C2GnT1 and C2-O-
sLeXappear to contribute to tumor invasion but are not likely to
be the only candidates responsible for invasive events. On
carcinoma cells, C2-O-sLeXmay act in concert with other
adhesion molecules or proteases to promote invasion and
ultimately metastasis. Our results inspire additional questions
about the specific mechanisms involving C2GnT1 and C2-O-sLeX
C2-O-sLeXon Human Carcinomas Regulate Invasion
PLoS ONE | www.plosone.org9 January 2011 | Volume 6 | Issue 1 | e16281
that mediate invasion and we are currently designing studies to
directly investigate these events. We expect that our findings will
impact and support the design of therapies aimed at preventing the
formation of specific carbohydrates or interrupting their tumor-
promoting effects in colon and hepatic cancer.
Materials and Methods
The human cell lines LS174T (colorectal adenocarcinoma) and
HepG2 (hepatocellular liver carcinoma) were obtained from
American Type Culture Collection (Manassas, VA) and authen-
tication testing, including confirmation of a negative status for
mycoplasma, bacteria, fungi contamination, confirmation of
species identity, and cytogenetic analysis, was performed prior to
purchase. Cell lines were passaged for less than 6 months after
thaw for use in these studies. LS174T and HepG2 cells were
grown in Minimal Essential Medium (MEM) supplemented with
1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES,
1.0 mM sodium pyruvate, 1mM non-essential amino acids, 10%
fetal bovine serum, and a combination of 100 U/ml penicillin/
100 mg/ml streptomycin.
Flow cytometric analysis
A FACSCanto instrument and BD FACSDiva software (Becton
Dickinson Biosciences, San Jose, CA) were used for flow
cytometric analyses as previously described [25,63]. Labeling of
cells (56106cells per sample) was performed with CHO-131
mouse anti-human IgM monoclonal antibody (mAb) that detects
C2-O-sLeX(10 mg/ml) and was generously provided by Dr. Bruce
Walcheck, University of Minnesota, St. Paul, MN or with mouse
anti-human CSLEX1 IgM mAb (BD Biosciences (San Jose, CA).
For the flow cytometric adhesion assays, cells were tested for
binding to 10 mg/ml of a mouse E-selectin/human Fc chimera (R
& D Systems, Minneapolis, MN) in buffer containing 2mM CaCl2
because binding is calcium dependent, and with and without
2 mM EDTA (a calcium ion chelator), or with 10 mM EDTA
alone as controls. Bound antibodies were detected with a
phycoerythrin (PE) conjugated F(ab’)2 goat anti-mouse IgM
secondary antibody and bound chimera was detected with a PE-
conjugated F(ab’)2 goat anti-human-IgG secondary antibody
(Jackson Immunoresearch Laboratories, West Grove, PA). To
evaluate the levels of background staining, cells were labeled with
only the secondary antibody.
Hydrodynamic shear flow assays
Cancer cell adhesion mechanisms were examined under
hydrodynamic shear stress with a parallel plate flow chamber
system (Glycotech, Gaitherburg, MD) as previously described .
A functional site density was defined as the minimum concentra-
tion of chimeric molecules in the correct orientation for binding
required for attachment of ,90% cancer cells at a hydrodynamic
shear stress of 0.5 dynes/cm2and was determined to be 0.5 mg/ml
for an E-selectin/Fc chimera. LS174T colon carcinoma cells were
perfused over 1 mg/ml of the E-selectin/Fc chimera under shear
stresses ranging from 0.5 to 1.5 dynes/cm2that were within the
range previously described to occur in post-capillary venules .
Each assay was performed in triplicate in the presence of 10 mg/ml
of an E-selectin function blocking mAb (R & D Systems,
Minneapolis, MN) or the same concentration of an isotype-
matched control mAb (Invitrogen, Carlsbad, CA, USA). Attached
cells were enumerated at 100X magnification after 3 minutes of
Quantitative Reverse-Transcriptase-PCR (RT-PCR) analysis
Total RNA was extracted from 26106cells of each cell line
using Trizol reagent (Invitrogen) and DNA was removed using
DNase I treatment. Reverse transcription was performed to
synthesize cDNA using a First-Strand Synthesis System (Invitro-
gen) and 20 pmols each of primers specific for human C2GnT1,
human FucT-III, and human b-actin. Quantitative real-time PCR
was performed on an icycler thermocycler (Bio-Rad Laboratories,
Hercules, CA) using Taq DNA polymerase (Continental Lab
Products, San Diego, CA, USA) according to the manufacturer’s
instructions. Reactions were performed for 30 cycles with the
following parameters: 94uC, 50 sec; 58uC, 50 sec; 72uC, 50 sec.
Gene silencing of C2GnT1
A pGIPZ-C2GnT1-short hairpin (sh)RNAmir vector engi-
neered to silence human C2GnT1 gene expression, a control
pGIPZ vector expressing scrambled shRNA, four lentiviral
C2GnT1-shRNA vectors in a pLK01 vector system, and an
empty pLK01 vector were purchased from Open Biosystems
(Huntsville, AL). All vectors were tested for silencing of the
C2GnT1 gene in human hepatic carcinoma cells (HepG2) and
human colon carcinoma cells (LS174T) and the vector that was
most efficient at gene silencing was used in subsequent assays with
each cell line. Thus, HepG2 cells were transiently transfected with
a C2GnT1 shRNA-pLKO1 vector or with the empty pLK01
vector. LS174T cells were stably transduced with the pGIPZ-
C2GnT1-shRNAmir vector or with the scrambled pGIPZ-shRNA
vector. C2GnT1 gene silencing was confirmed in transfected or
transduced cells by RT-PCR, Western blot, and flow cytometry to
detect cell surface expression of C2-O-sLeX. The specificity of
gene silencing was assessed by the use of more than one shRNA
vector, by comparing the C2GnT1 and scrambled shRNA
sequences to the complete human genome using NCBI Nucleotide
BLAST, and by investigation of the effects of the C2GnT1 shRNA
vectors on the human FucT-III gene.
Immunoprecipitations and Western blot analyses
Whole cell lysates of LS174T cells were prepared with lysis
buffer (SolObuffer-200, FabGennix International Inc, Frisco, TX)
according the manufacturer’s instructions. Protein concentration
was quantified by the Bradford assay (Bio-Rad, Hercules, CA).
Approximately 500 mg of protein was immunoprecipitated with
4 ml of a rabbit polyclonal anti-C2GnT1 primary antibody in
immunoprecipitation buffer (50mM Tris-HCl, pH 8.0, 150mM
NaCl, 1% Igepal, and protease inhibitor). Protein complexes were
precipitated with 40 ml of Protein G beads. Products were
subjected to 10% SDS-PAGE and transferred to an Immobilon-
P transfer membrane (Millipore Corporation, Medford, MA),
blocked with 5% blotting grade blocker non-fat dry milk, and
probed with the C2GnT1 antibody or with a rabbit IgG negative
control antibody and subsequently incubated with a HRP-
conjugated goat anti-rabbit-IgG antibody. Protein bands were
visualized with an ECL system (Pierce, Rockford, IL).
C2GnT1 glycosyltransferase assay
The activity of C2GnT1 glycosyltransferase in LS174T cells was
evaluated as previously described by Prorok-Hamon et al . Cells
were lysed in buffer containing 120mM NaCL, 40mM Tris, 1%
Triton X-100 and 20% glycerol (Fisher, Pittsburgh, PA), and 1%
Protease inhibitor (Roche Diagnostics Corporation, Indianapolis,
IN). The substrate was 10 mM Gal b1,3 GalNAca-O-paranitrophe-
nyl (Toronto Research Chemicals, Downsview ON, Canada) and
10 mg/ml of proteins were used as the source of transferases.
C2-O-sLeXon Human Carcinomas Regulate Invasion
PLoS ONE | www.plosone.org 10January 2011 | Volume 6 | Issue 1 | e16281
Treatment of cells with O-glycan and N-glycan inhibitors
HepG2 and LS174T cells (36105cells/well) were treated with
either 2mM benzyl 2-acetamido-2-deoxy-alpha-D-galactopyrano-
side (BGN, an O-glycosylation inhibitor), 10 ug/ml of swainosine
(SWN, an N-glycosylation inhibitor), or a combination of both
agents for 3–5 days. Using untreated and treated cells, the
presence of membrane-associated sLeX
CSLEX1 mAb and membrane-associated C2-O-sLeXwas detect-
ed with CHO-131 mAb. For both cell types, binding to an E-
selectin/Fc chimera was assessed by flow cytometry.
was detected with
Matrigel invasion assays
Twenty-four well BioCoatTMMatrigelTMInvasion Chambers
(Becton-Dickinson, Bedford, MA, USA) were used for the tumor
cell invasion assays according to the manufacturer’s instructions.
Cell suspensions (2.56104cells/chamber) in 0.5 ml serum-free
medium were added to the upper chamber of each well and 0.5 ml
complete medium containing 10% fetal bovine serum was added
to each bottom chamber. Chambers were incubated for 24 hours
in a humidified tissue culture incubator at 37uC and 5% CO2. The
invading cells were stained and counted in five separate fields of
view at 100X magnification. Digital images were captured using a
Zeiss Axiovert 200 inverted microscope and an AxioCam MRc
camera using AxioVision 4.1 software (Carl Zeiss Inc., Germany).
Differences between groups were analyzed using a conventional
two-tailed distribution Student’s t test, with two-sample unequal
variance where appropriate. Reported p-values were considered
significant at p#0.05.
We thank Drs. Bruce Walcheck and Kaylee Schwertfeger for review of the
manuscript and Katherine Krieser for technical assistance and review of
Conceived and designed the experiments: CAS. Performed the experi-
ments: MF DBH. Analyzed the data: CAS DBH MF. Contributed
reagents/materials/analysis tools: CAS. Wrote the paper: CAS MF.
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PLoS ONE | www.plosone.org12 January 2011 | Volume 6 | Issue 1 | e16281