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: firstname.lastname@example.org.
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
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