ligand expression in colon cancer cells undergoing
EGF/bFGF-induced epithelial–mesenchymal transition
Keiichiro Sakumaa, Masahiro Aokia, and Reiji Kannagia,b,c,1
aDivision of Molecular Pathology, Aichi Cancer Center, Chikusa-ku, Nagoya, Aichi 464-8681, Japan;bResearch Complex for Medical Frontiers, Aichi Medical
University, Yazako, Nagakute, Aichi 480-1195, Japan; andcInstitute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan
Edited* by Sen-itiroh Hakomori, Pacific Northwest Research Institute, Seattle, WA, and approved April 5, 2012 (received for review July 11, 2011)
Sialyl Lewis x (sLex) and sialyl Lewis a (sLea) glycans are expressed
on highly metastatic colon cancer cells. They promote extravasa-
tion of cancer cells and tumor angiogenesis via interacting with
E-selectin on endothelial cells. Recently, epithelial–mesenchymal
transition (EMT) has been noted as a critical phenotypic alteration
in metastatic cancer cells. To address the association between sLex/a
expression and EMT, we assessed whether sLex/aare highly ex-
pressed on colon cancer cells undergoing EMT. Treatment of
HT29 and DLD-1 cells with EGF and/or basic FGF (bFGF) induced
EMT and significantly increased sLex/aexpression resulting in en-
hanced E-selectin binding activity. The transcript levels of the gly-
cosyltransferase genes ST3GAL1/3/4 and FUT3 were significantly
elevated and that of FUT2 was significantly suppressed by the
treatment. We provide evidence that ST3GAL1/3/4 and FUT3 are
transcriptionally up-regulated by c-Myc with probable involve-
ment of Ser62 phosphorylation, and that FUT2 is transcriptionally
down-regulated through the attenuation of CDX2. The contribu-
tion of c-Myc and CDX2 to the sLex/ainduction was proved to be
significant by knockdown or forced expression experiments. Inter-
estingly, the cells undergoing EMT exhibited significantly in-
creased VEGF secretion, which can promote tumor angiogenesis
in cooperation with sLex/a. Finally, immunohistological study indi-
cated high E-selectin ligand expression on cancer cells undergoing
EMT in vivo, supporting their coexistence observed in vitro. These
results suggest a significant link between sLex/aexpression and
EMT in colon cancer cells and a pivotal role of c-Myc and CDX2
in regulating sLex/aexpression during EMT.
with more than 1,200,000 new cases and over 600,000 deaths
estimated to have occurred in 2008 (1). Although early detection,
increased awareness, and developments in treatment have in-
creased complete cure rates especially in some advanced coun-
tries, distant metastasis is still a critical event that makes colon
cancer a lethal disease. Therefore, novel therapeutic approaches
to inhibit metastasis are required.
Sialyl Lewis x (sLex) and sialyl Lewis a (sLea) are E-selectin
ligand glycans expressed on the surface of many types of cancer
cells, including colorectal, pancreatic, gastric, breast, prostate,
and lung cancer (2, 3). These glycans play crucial roles in hema-
togenous metastasis through interaction with endothelial cells.
The most established role is promoting extravasation of cancer
cells: circulating cancer cells in blood flow arrest at distant sites
by adhering to endothelial cells, which enables their movement
out of the vasculature (2, 3). Importantly, the interaction between
sLex/aand E-selectin exclusively mediates the adhesion of most
epithelial cancer cells to endothelial cells, whereas sLex/a-in-
dependent interaction with endothelial ICAM-1 and VCAM-1
mediates the adhesion of nonepithelial malignant cells, such as
leukemia and some sarcoma cells, to endothelial cells (4). An-
other important role of sLex/ain hematogenous metastasis is tu-
mor angiogenesis (3, 5), which can facilitate intravasation and
postextravasational proliferation ofcancercells (6–8). Inline with
these observations, high sLex/aexpression levels in colon cancer
patients are correlated with poor prognosis (2). Therefore, these
olon cancer is one of the most prevalent cancers worldwide,
glycans are frequently evaluated as tumor markers. Whereas the
diagnostic utility of sLex/ahas been well established, therapeutic
approaches targeting these glycans are not well developed, partly
because molecular mechanisms of their expression have been
only partially elucidated (9–11).
Recently, epithelial–mesenchymal transition (EMT) has been
noted as a critical event in the early step of cancer metastasis
(12, 13). It is also notable that EMT is known to be associated
with cancer stem cells (14, 15). EMT is defined as a transitional
process from epithelial to mesenchymal phenotype, including
fibroblast-like morphology, down-regulation of E-cadherin by
transcriptional repressors such as SNAIL1, ZEB1, and TWIST,
mesenchymal marker expression such as Vimentin, Fibronectin,
and N-cadherin, and enhanced cell motility. A variety of EMT
inducers have been reported, including TGF-β and receptor ty-
rosine kinase (RTK) growth factors such as hepatocyte growth
factor (HGF), EGF, and basic FGF (bFGF). Although many
studies have focused on TGF-β (16), the TGF-β signaling path-
way is frequently inactivated in colon cancer due to loss-of-
function mutations in TGFBR2 and SMAD genes (17). There-
fore, RTK growth factors are likely to figure more heavily than
TGF-β in EMT of colon cancer cells. Several clinical studies have
suggested the correlation between RTK signaling and metastasis.
EGFR was expressed in ∼85% of patients with metastatic colon
cancer (18) and its expression level and function in colon cancer
cells were correlated with metastatic potential (19, 20). Plasma
bFGF levels were significantly higher in patients with metastatic
colon cancer than in normal controls, whereas those levels were
comparable between patients with nonmetastatic colon cancer
and normal controls (21). Sato et al. demonstrated by quantita-
tive RT-PCR that the transcript levels of FGFR1 in colon cancer
tissues were significantly higher in patients with liver metastasis
than in those without liver metastasis (22).
Despite the significant roles of sLex/aand EMT in cancer
metastasis, their association remains unknown. To address this
issue, we assessed whether sLex/ais highly expressed on cancer
cells undergoing EMT.
Induction of EMT in Colon Cancer Cells by EGF or bFGF. To prepare
colon cancer cells undergoing EMT, we treated HT29 and DLD-
1 cells with EGF (20 ng/mL) and/or bFGF (10 ng/mL) in serum-
deprived medium. Treatment with either EGF or bFGF alone
transiently induced a fibroblast-like appearance (Fig. 1A); however,
it was very difficult to maintain the cells for further experiments.
Treatment with both EGF and bFGF (hereafter EGF/bFGF
treatment) permitted better cell survival and induced a fibroblast-
Author contributions: K.S., M.A., and R.K. designed research; K.S. performed research;
K.S., M.A., and R.K. analyzed data; and K.S., M.A., and R.K. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| May 15, 2012
| vol. 109
| no. 20 www.pnas.org/cgi/doi/10.1073/pnas.1111135109
like appearance (Fig. 1A). The EGF/bFGF treatment increased
the levels of the mesenchymal marker genes SNAIL1, ZEB1, and
Vimentin, whereas it reduced the level of E-cadherin and the
colon-epithelial differentiation marker genes MUC2 (mucin 2)
and ALPI (intestinal alkaline phosphatase) (Fig. 1B). Function-
ally, the treated cells showed significantly enhanced migration
activity (P < 0.05; Fig. 1C). These results indicated that the EGF/
bFGF treatment induced EMT in HT29 and DLD-1 cells.
Induction of sLex/aExpression and E-Selectin Binding Activity in Colon
Cancer Cells by EGF or bFGF. We then evaluated sLex/aexpression
levels on the cells undergoing EMT by flow cytometry. The results
indicated significantly increased sLex/aexpression on the EGF/
bFGF-treated cells as well as on the cells treated with either factor
whether the increased sLex/aexpression could contribute to the
interaction with E-selectin, we examined binding activity of the
treated cells to recombinant E-selectin. The cells exhibited signifi-
nosignificant binding activity was detected for P-selectin (P > 0.05;
Fig. 1E), which selectively binds to sLexdeterminants carried on P-
selectin glycoprotein ligand 1 (PSGL-1), generally expressed on
leukocytes (23). Furthermore, the E-selectin binding activity was
significantly inhibited by anti-sLexantibody, anti-sLeaantibody, or
EDTA (P < 0.000005; Fig. S1), indicating that recombinant E-
selectin did bind to the cells through the interaction with sLex/a.
We previously demonstrated that sLex/aexpressed on cancer
cells promote tumor angiogenesis through interacting with
E-selectin on endothelial cells (5). Because E-selectin is known to
be induced by VEGF (24), we examined whether VEGF was se-
creted into the supernatant of the cells treated with EGF and/or
bFGF. Results of ELISA indicated that the VEGF level was sig-
nificantly increased by the treatment (P < 0.0001; Fig. 1F). These
results suggest that EGF and/or bFGF can strongly promote an-
giogenesis synergistically by inducing sLex/aand E-selectin ex-
pression on colon cancer cells and endothelial cells, respectively.
Altered Expression of ST3GAL1/3/4, FUT3, and FUT2 Induced by EGF or
bFGF. To address the molecular mechanism underlying the EMT-
associated sLex/aexpression, we focused on the glycosyltrans-
FBS or in serum-free medium supplemented with EGF (20 ng/mL) and/or bFGF (10 ng/mL). After culture for 7 d, cells were observed under a phase-contrast
microscope. (Arrows) Cells exhibiting fibroblastic morphology. (B) Expression of marker genes for EMT and colon-epithelial differentiation was examined
by conventional RT-PCR. (C) Cell migration activity was determined with Biocoat Matrigel invasion chambers. (D) Expression levels of sLex/awere determined
by flow cytometry. Bold lines, staining control. (E) Selectin-binding activity was determined by flow cytometry. Bold lines, staining control. (F) Culture
supernatant VEGF levels were measured by ELISA. (C–F) Statistic analysis was performed in three independent experiments by t test. Error bars, SD; asterisks,
P < 0.05 (C), P < 0.01 (D), P < 0.000005 (E), and P < 0.0001 (F) compared with the untreated cells; NS, not significant (P > 0.05).
Induction of EMT and sLex/aexpression in colon cancer cells by EGF or bFGF. (A) HT29 and DLD-1 cells were maintained in culture medium with 10%
Sakuma et al.PNAS
| May 15, 2012
| vol. 109
| no. 20
ferase genes. Sialyltransferases and fucosyltransferases are es-
sential enzymes for the synthesis of sialic acid and fucose resi-
dues of sLex/a, respectively. Screening of the genes involved in
the sLex/asynthesis by conventional RT-PCR using primers listed
in Table S1 revealed that the levels of ST3GAL1/3/4 and FUT3/6
were increased, whereas that of FUT2 was decreased by the
EGF/bFGF treatment of HT29 and DLD-1 cells (Fig. 2A).
Quantitation of the expression levels of these genes by real-time
RT-PCR using the assays listed in Table S2 indicated that the
EGF/bFGF treatment induced significant increases in the
ST3GAL1/3/4 and FUT3 levels (P < 0.005; Fig. 2B) and a sig-
nificant decrease in the FUT2 level (P < 0.00005; Fig. 2C). In
addition, these alterations were also induced by treatment with
EGF or bFGF alone (Fig. 2 B and C). However, the FUT6 level
was not significantly changed by any of the treatment (P > 0.05;
Fig. 2B). Therefore, we focused on ST3GAL1/3/4, FUT3, and
FUT2 for further experiments. ST3GAL1/3/4 catalyze the addi-
tion of N-acetylneuraminic acid (NeuAc) to the nonreducing
terminal galactose (Gal) residue of glycans, and FUT3 catalyzes
addition of fucose (Fuc) to the N-acetylglucosamine (GlcNAc)
residue (Fig. 2D). Therefore, up-regulation of ST3GAL1/3/4 and
FUT3 results in increased sLex/aexpression. In contrast, FUT2
catalyzes addition of Fuc to the nonreducing terminal Gal, com-
peting with NeuAc addition by sialyltransferases (Fig. 2D). Down-
regulation of FUT2 thus contributes to increased sLex/aexpression.
As expected, quantitative analysis by flow cytometry indicated that
EGF/bFGF treatment induced significantly higher increase in
the levels of sLexand sLeacompared with those of Leyand Leb,
respectively (P < 0.01; Fig. S2).
Involvement of c-Myc in the Induction of ST3GAL1/3/4 and FUT3
Expression by EGF/bFGF. To explore the mechanism of the EGF/
bFGF-induced alteration in the glycogene transcription, we
next searched for potential transcription factor binding sites in
the 5′-regulatory regions of the glycogenes identified above and
noticed potential c-Myc binding sites in the promoters of
ST3GAL1/3/4 and FUT3 (Fig. S3 A–D). ChIP assays using pri-
mers listed in Table S3 indicated increased recruitment of c-Myc
to their promoters in the EGF/bFGF-treated cells (Fig. 3A). To
determine the role of c-Myc in the sLex/ainduction, we per-
formed c-Myc knockdown experiments. Namely, we introduced
a c-Myc shRNA-expressing vector into HT29 and DLD-1 cells
(Fig. 3B) and treated the cells with EGF/bFGF. Knockdown
of c-Myc significantly inhibited the maximal induction of
ST3GAL1/3/4 and FUT3 expression (P < 0.05; Fig. 3C). Con-
sequently, the maximal sLex/ainduction was also inhibited (P <
0.0005; Fig. 3D). These results suggested a pivotal role of c-Myc
in the sLex/ainduction through the transcriptional regulation
of ST3GAL1/3/4 and FUT3 under the EGF/bFGF treatment.
To gain insights into the molecular mechanism by which the
EGF/bFGF treatment induced the glycogenes through c-Myc,
we performed Western blot analysis. Unexpectedly, the level of
total c-Myc was reduced by the treatment in HT29 cells (Fig. 3E)
likely caused by a decrease in the transcript level (Fig. 3F).
However, the level of phospho–c-MycSer62/Thr58was strongly en-
hanced by the treatment both in HT29 and DLD-1 cells (Fig.
3E). It is known that a priming phosphorylation of c-Myc at
Ser62 can be followed by phosphorylation at Thr58 by GSK3β
(25). Because Western blotting revealed a decrease in the level of
total GSK3β and an increase in that of phospho-GSK3βSer9, the
inactivated form of GSK3β (Fig. S4), the increase in the phos-
pho–c-MycSer62/Thr58level most likely reflects the hyperphos-
phorylation of the Ser62 site, which has been implicated in the
enhanced recruitment of c-Myc to the promoter of its target
thesis was screened by conventional RT-PCR. (B and C) Expression levels of ST3GAL1/3/4, FUT3/6 (B), and FUT2 (C) were determined by quantitative RT-PCR.
The mean 2
(C) compared with the untreated cells; NS, not significant (P > 0.05). (D) Scheme of the sLex, Ley, sLea, and Lebsynthetic pathways showing the roles of
ST3GAL1/3/4, FUT3, and FUT2. Note that FUT2 competes with ST3GAL3/4 for sLexsynthesis and with ST3GAL1 for sLeasynthesis, respectively.
Altered glycogene expression induced by EGF or bFGF. (A) Expression of the sialyltransferase and fucosyltransferase genes involved in the sLex/asyn-
-ΔΔCTvalues ± SD from three independent experiments are shown. Statistic analysis was performed by t test. Asterisks, P < 0.005 (B) and P < 0.00005
| www.pnas.org/cgi/doi/10.1073/pnas.1111135109Sakuma et al.
genes as well as in the enhanced transcriptional activity of c-Myc
Involvement of CDX2 in the EGF/bFGF-Induced Transcriptional
Suppression of FUT2. We next examined the mechanism of the
the 5′-regulatory regions of ST3Gal1/3/4 and FUT3. (B) Effect of c-Myc shRNA (shMYC) on the transcript level of c-Myc was evaluated by quantitative RT-PCR.
(C) Effects of shMYC on the expression levels of ST3Gal1/3/4 and FUT3 were examined by quantitative RT-PCR. (D) Expression levels of sLex/awere examined by
flow cytometry. Dotted lines, staining control; bold lines, nontarget shRNA (shNT)-introduced cells; filled histogram, shMYC-introduced cells. (E) Levels of c-
Myc and phospho–c-MycSer62/Thr58were determined by Western blotting. (F) Expression of c-Myc was examined by conventional RT-PCR. (B–D) Statistic analysis
was performed in three independent experiments by t test. Error bars, SD; asterisks, P < 0.00001 (B), P < 0.05 (C), and P < 0.0005 (D) compared with the shNT-
transfected cells (B) or to the shNT-introduced cells treated with EGF/bFGF (C and D).
Involvement of c-Myc in the transcriptional regulation of ST3Gal1/3/4 and FUT3. (A) ChIP assays were performed to examine the binding of c-Myc to
transcript level of CDX2 was determined by quantitative RT-PCR. (B) ChIP assays were performed to examine the binding of CDX2, SNAIL1, and ZEB1 to the 5′-
regulatory region of FUT2. (C and D) Effect of CDX2 forced expression (C) or CDX2 shRNA (shCDX2; D) on the expression levels of CDX2 and FUT2 were
determined by quantitative RT-PCR. (E) Expression levels of Ley/band sLex/awere examined by flow cytometry. Dotted lines, staining control; bold lines, mock
vector-introduced cells; filled histogram, CDX2 expression vector-introduced cells. (A and C–E) Statistic analysis was performed in three independent
experiments by t test. Error bars, SD; asterisks, P < 0.000001 (A), P < 0.0005 (C and D), and P < 0.05 (E) compared with the untreated cells (A), to the mock
vector-transfected cells (C and E) or to the nontarget shRNA (shNT)-transfected cells (D); NS, not significant (P > 0.05).
Involvement of CDX2 in the transcriptional regulation of FUT2. (A) Expression of CDX1 and CDX2 was examined by conventional RT-PCR. The
Sakuma et al. PNAS
| May 15, 2012
| vol. 109
| no. 20
EGF/bFGF-induced transcriptional suppression of FUT2. We
noticed potential binding sites for CDX1 and CDX2, transcrip-
tion factors known to regulate several colon-specific genes (28–
31), in the 5′-regulatory region of FUT2 (Fig. S3E). Both HT29
and DLD-1 cells showed good levels of CDX2 expression, which
were significantly reduced by EGF/bFGF treatment (P <
0.000001; Fig. 4A). ChIP assays revealed that binding of CDX2
to the FUT2 promoter was abolished by the treatment (Fig. 4B).
On the other hand, SNAIL1 and ZEB1, EMT-related tran-
scriptional repressors, were not recruited to the promoter (Fig.
4B). To determine the role of CDX2 in the transcriptional reg-
ulation of FUT2, we introduced a CDX2 expression vector into
HT29 and DLD-1 cells. The cells exhibited significantly elevated
FUT2 expression compared with the mock vector-transfected
cells (P < 0.0005; Fig. 4C). In contrast, HT29 and DLD-1 cells
introduced with CDX2 shRNA showed significantly reduced level
of FUT2 (P < 0.0005; Fig. 4D). Furthermore, forced expression
of CDX2 elevated Ley/bexpression and suppressed sLex/aexpres-
sion in HT29 and DLD-1 cells (Fig. 4E). These results suggest
that the EGF/bFGF-induced down-regulation of CDX2 contrib-
utes to the sLex/ainduction via suppression of FUT2 transcription.
E-Selectin Ligand Glycan Expression on Colon Cancer Cells Undergoing
EMT in Vivo. Finally, we examined association between E-selectin
ligand glycan expression and EMT in clinial samples by immu-
nohistochemical analysis. We focused on sLeain this experiment,
because this glycan is preferentially expressed on cancer cells,
whereas sLexis broadly expressed on various normal cells in-
cluding leukocytes and might complicate the results. We perfor-
med double staining with antibodies against sLeaand E-cadherin
on sections from five colorectal cancer patients. In one section
from a 70-y-old male patient with colon cancer, we identified
a small area of cancer cells that lacked cell-surface E-cadherin
expression at the invasion front (Fig. 5A). Most interestingly,
these cancer cells exhibited high sLeaexpression, whereas cancer
cells with cell-surface E-cadherin exhibited no sLeaexpression
(Fig. 5A). Furthermore, double staining with antibodies against
SNAIL1 and sLeaidentified a subset of cancer cells that coex-
results were obtained by double staining with antibodies against
of E-selectin ligand expression and EMT observed in vitro.
The major findings of this study are as follows: (i) sLex/aex-
pression is strongly induced during EMT of colon cancer cells
triggered by EGF or bFGF, and (ii) c-Myc and CDX2 play key
roles in the sLex/ainduction by EGF or bFGF.
Our present results demonstrate that c-Myc contributes to
sLex/aexpression by transcriptional induction of ST3GAL1/3/4
and FUT3 in the EGF/bFGF-treated cells. Although the detailed
mechanism of this glycogene induction by c-Myc remains unclear,
we speculate the possible involvement of Ser62 phosphorylation
of c-Myc as described above (Fig. 3E). Ser62 of c-Myc is known
to be phosphorylated by ERK or cyclin-dependent kinase (CDK)
2 (25–27). The kinase that contributed to this phosphorylation
in the EGF/bFGF-treated cells remains to be identified.
In this study, we demonstrated that the transcription of CDX2
was down-regulated by the EGF/bFGF treatment, which resulted
in a decrease in the transcript level of FUT2. Although the
mechanism underlying the down-regulation of CDX2 by the
treatment remains unknown, SNAIL1 may be involved because
transcription of CDX2 is known to be repressed by SNAIL1 (32),
and SNAIL1 expression was increased by EGF/bFGF (Fig. 1B).
Although several lines of evidence indicate that CDX2 is a tumor
suppressor (33), the association between CDX2 and metastasis
EMT in vivo. (A) Expression of E-cadherin (green) and sLea(red) was exam-
ined on human colon cancer sections by immunohistochemistry. (Left) Re-
gion with cancer cells exhibiting high cell-surface E-cadherin expression.
(Center) Another region in the same section containing cancer cells without
cell-surface E-cadherin expression, a part of which are surrounded by
a square (k) and magnified in the Right column. Arrowheads indicate cells
with decreased E-cadherin and increased sLeaexpression on the cell surface.
(Blue) Hoechst 33342. (B) Expression of SNAIL1 (a, green), ZEB1 (b, green), and
sLea(c and d, red) was examined on sections from the same patient. (e and f)
E-selectin ligand glycan expression on colon cancer cells undergoing
Hoechst 33342. Cells with arrowheads (g and h) are magnified (i and j), with
arrows showing nuclear SNAIL1 and ZEB1, respectively. (Scale bars, 50 μm.)
| www.pnas.org/cgi/doi/10.1073/pnas.1111135109Sakuma et al.
has been unclear. Clinically, Baba et al. reported that the loss of
CDX2 expression in colon cancer tissues was significantly cor-
related with stage IV disease (34). Our present findings may
explain at least a part of the mechanisms by which the loss of
CDX2 contributes to metastasis.
We previously reported that hypoxia induced sLex/aexpression
in colon cancer cells (9). In that report, we documented that the
transcription of ST3GAL1, FUT7, and UGT1 (UDP-galactose
transporter 1), which are all involved in the E-selectin ligand
glycan synthesis, was elevated under a hypoxic condition. Hyp-
oxia-inducible factor-1α (HIF-1α) was involved in the induction of
these glycogenes. The present study provides additional in-
formation on the transcriptional regulation of the sLex/asynthesis-
Recently, Guan et al. reported a significant association be-
tween glycans and EMT, demonstrating that the expression
levels of GM2 and Gg4 glycosphingolipids were significantly
decreased during TGF-β–induced EMT and that the gluco-
sylceramide synthase inhibitor EtDO-P4 induced EMT (35).
From their subsequent observations demonstrating that exoge-
nous addition of Gg4 abrogated the EMT process and that Gg4
was closely associated with E-cadherin and β-catenin, they pro-
posed that Gg4 may be important in maintaining epithelial cell
membrane organization (36). Together with these reports, our
present study demonstrates a drastic alteration in the glycan ex-
pression during the EMT process. It remains an interesting issue
whether the alteration in sLex/aexpression further promotes the
EMT process as the alteration in the Gg4 expression did.
We demonstrated that sLeawas preferentially expressed on
the cancer cells with low expression of membranous E-cadherin,
nuclear SNAIL1, and nuclear ZEB1 in a clinical sample of colon
cancer. These results are consistent with the coincidence of
sLex/aexpression and EMT observed in vitro and suggest that
these glycans may serve as a good marker of EMT in cancer
patients. Our results indicate that RTK signaling activation
confers both EMT and sLex/aexpression on cancer cells. As RTK
signaling pathways provide effective therapeutic targets, these
glycans may serve as surrogate markers for evaluating thera-
peutic effects of such modalities.
Materials and Methods
Additional information can be found in SI Materials and Methods.
Human colon cancer cell lines,HT29 and DLD-1, were maintained in DMEM
and RPMI1640 medium (Invitrogen), respectively, supplemented with 10%
(vol/vol) FBS. For treatment with EGF and/or bFGF, recombinant human EGF
(Sigma; 20 ng/mL) and/or recombinant human bFGF (Sigma; 10 ng/mL) were
added to the serum-free medium with recombinant human insulin (Sigma;
25 μg/mL), human holo-transferrin (Sigma; 100 μg/mL), putrescine dihydro-
chloride (Sigma; 10 μg/mL), and sodium selenite (Sigma; 5 ng/mL).
ACKNOWLEDGMENTS. This work was supported in part by Grants-in-Aid for
Young Scientists (B) 20790583 and 22790774 from the Japan Society for the
Promotion of Science, Grants-in-Aid 24590364 and (on priority areas)
23112520 from the Ministry of Education, Culture, Sports, Science and
Technology, Grants-in-Aid for the Third-Term Comprehensive Ten-Year
Strategy for Cancer Control from the Ministry of Health and Welfare, and
a grant from Uehara Memorial Foundation, Japan.
1. Jemal A, et al. (2011) Global cancer statistics. CA Cancer J Clin 61:69–90.
2. Kannagi R (1997) Carbohydrate-mediated cell adhesion involved in hematogenous
metastasis of cancer. Glycoconj J 14:577–584.
3. Kannagi R, Izawa M, Koike T, Miyazaki K, Kimura N (2004) Carbohydrate-mediated
cell adhesion in cancer metastasis and angiogenesis. Cancer Sci 95:377–384.
4. Takada A, et al. (1993) Contribution of carbohydrate antigens sialyl Lewis A and sialyl
Lewis X to adhesion of human cancer cells to vascular endothelium. Cancer Res 53:
5. Tei K, et al. (2002) Roles of cell adhesion molecules in tumor angiogenesis induced by
cotransplantation of cancer and endothelial cells to nude rats. Cancer Res 62:6289–
6. Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other diseases. Nature 407:
7. Tien YW, et al. (2001) Tumor angiogenesis and its possible role in intravasation of
colorectal epithelial cells. Clin Cancer Res 7:1627–1632.
8. Zetter BR (1998) Angiogenesis and tumor metastasis. Annu Rev Med 49:407–424.
9. Koike T, et al. (2004) Hypoxia induces adhesion molecules on cancer cells: A missing
link between Warburg effect and induction of selectin-ligand carbohydrates. Proc
Natl Acad Sci USA 101:8132–8137.
10. Miyazaki K, et al. (2004) Loss of disialyl Lewis(a), the ligand for lymphocyte inhibitory
receptor sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) associated with
increased sialyl Lewis(a) expression on human colon cancers. Cancer Res 64:
11. Yusa A, Miyazaki K, Kimura N, Izawa M, Kannagi R (2010) Epigenetic silencing of the
sulfate transporter gene DTDST induces sialyl Lewisx expression and accelerates
proliferation of colon cancer cells. Cancer Res 70:4064–4073.
12. Kalluri R, Weinberg RA (2009) The basics of epithelial-mesenchymal transition. J Clin
13. Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial-mesenchymal transitions
in development and disease. Cell 139:871–890.
14. Mani SA, et al. (2008) The epithelial-mesenchymal transition generates cells with
properties of stem cells. Cell 133:704–715.
15. Radisky DC, LaBarge MA (2008) Epithelial-mesenchymal transition and the stem cell
phenotype. Cell Stem Cell 2:511–512.
16. Ikushima H, Miyazono K (2010) TGFbeta signalling: A complex web in cancer pro-
gression. Nat Rev Cancer 10:415–424.
17. Xu Y, Pasche B (2007) TGF-beta signaling alterations and susceptibility to colorectal
cancer. Hum Mol Genet 16(Spec No 1):R14–R20.
18. Normanno N, et al. (2009) Implications for KRAS status and EGFR-targeted therapies
in metastatic CRC. Nat Rev Clin Oncol 6:519–527.
19. Radinsky R, et al. (1995) Level and function of epidermal growth factor receptor
predict the metastatic potential of human colon carcinoma cells. Clin Cancer Res 1:
20. Goldstein NS, Armin M (2001) Epidermal growth factor receptor immunohistochem-
ical reactivity in patients with American Joint Committee on Cancer Stage IV colon
adenocarcinoma: Implications for a standardized scoring system. Cancer 92:
21. George ML, Tutton MG, Abulafi AM, Eccles SA, Swift RI (2002) Plasma basic fibroblast
growth factor levels in colorectal cancer: A clinically useful assay? Clin Exp Metastasis
22. Sato T, et al. (2009) Overexpression of the fibroblast growth factor receptor-1 gene
correlates with liver metastasis in colorectal cancer. Oncol Rep 21:211–216.
23. McEver RP, Cummings RD (1997) Perspectives series: Cell adhesion in vascular biology.
Role of PSGL-1 binding to selectins in leukocyte recruitment. J Clin Invest 100:
24. Kim I, et al. (2001) Vascular endothelial growth factor expression of intercellular
adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and
E-selectin through nuclear factor-kappa B activation in endothelial cells. J Biol Chem
25. Sears R, et al. (2000) Multiple Ras-dependent phosphorylation pathways regulate Myc
protein stability. Genes Dev 14:2501–2514.
26. Benassi B, et al. (2006) c-Myc phosphorylation is required for cellular response to
oxidative stress. Mol Cell 21:509–519.
27. Hydbring P, et al. (2010) Phosphorylation by Cdk2 is required for Myc to repress Ras-
induced senescence in cotransformation. Proc Natl Acad Sci USA 107:58–63.
28. Dang DT, Mahatan CS, Dang LH, Agboola IA, Yang VW (2001) Expression of the gut-
enriched Krüppel-like factor (Krüppel-like factor 4) gene in the human colon cancer
cell line RKO is dependent on CDX2. Oncogene 20:4884–4890.
29. Yamamoto H, Bai YQ, Yuasa Y (2003) Homeodomain protein CDX2 regulates
goblet-specific MUC2 gene expression. Biochem Biophys Res Commun 300:
30. Chan CW, et al. (2009) Gastrointestinal differentiation marker Cytokeratin 20 is
regulated by homeobox gene CDX1. Proc Natl Acad Sci USA 106:1936–1941.
31. Kakizaki F, et al. (2010) CDX transcription factors positively regulate expression of
solute carrier family 5, member 8 in the colonic epithelium. Gastroenterology 138:
32. Gross I, et al. (2008) The intestine-specific homeobox gene Cdx2 decreases mobility
and antagonizes dissemination of colon cancer cells. Oncogene 27:107–115.
33. Guo RJ, Suh ER, Lynch JP (2004) The role of Cdx proteins in intestinal development
and cancer. Cancer Biol Ther 3:593–601.
34. Baba Y, et al. (2009) Relationship of CDX2 loss with molecular features and prognosis
in colorectal cancer. Clin Cancer Res 15:4665–4673.
35. Guan F, Handa K, Hakomori SI (2009) Specific glycosphingolipids mediate epithelial-
to-mesenchymal transition of human and mouse epithelial cell lines. Proc Natl Acad
Sci USA 106:7461–7466.
36. Guan F, Schaffer L, Handa K, Hakomori SI (2010) Functional role of gangliote-
traosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia
and by TGF-β. FASEB J 24(12):4889–4903.
Sakuma et al.PNAS
| May 15, 2012
| vol. 109
| no. 20