α2,6-hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells
Acta Pharmacologica Sinica
?2,6-Hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells
Jin QIAN 0
Cai-hua ZHU 0
Shuai TANG 1
Ai-jun SHEN 0
Jing AI 1
Jing LI 1
Mei-yu GENG 0 1
Jian DING 0
0 Division of Anti-tumor Pharmacology, Shanghai Institute of Materia Medica , Shanghai 201203 , China
1 Department of Pharmacology, Marine Drug and Food Institute, Ocean University of China , Qingdao 266003 , China
Aim: We aimed to investigate the potential modification of previously unrecognized surface glycoprotein(s) by ?2,6-sialylation other than by integrins. Methods: The expression of ?-galactoside ?2,6-sialyltransferase (ST6Gal-I) in the colon cancer cell line HCT116 was reduced by siRNA. The adhesion and Boyden chamber assay were used to detect the variation in cell motility. ?2,6-Sialylation proteins were detected with lectin affinity assay. The mRNA expression, protein expression and downstream signaling modulation with siRNA were detected using reverse transcription-polymerase chain reaction, flow cytometry analysis, and Western blot. Results: In HCT116 cells, the knockdown of ST6Gal-I inhibited cell motility, but did not affect cell adhesion. This selectively altered cell migration was caused by the loss of ?2,6-sialic acid structures on c-Met. Moreover, STAT3 was dephosphorylated at tyrosine 705 in ST6Gal-I-knockdown (ST6Gal-I-KD) HCT116 cells. Conclusion: c-Met is the substrate of ST6Gal-I. The hyposialylation of c-Met can abolish cell motility in ST6Gal-I-KD HCT116 cells.
cell motility; c-Met; hyposialylation; ST6Gal-I
Cell surface proteins in mammals are typically elaborated
with a complex array of asparagine-linked (N-linked) glycans.
Sialic acids are usually found at the non-reducing terminal
position of these N-glycans. This terminal sialylation imparts
a negative charge at physiological pH values and mediates
many biological functions. Altered expression of certain sialic
acid types or their linkages is closely associated with cellular
adhesion, migration and metastasis in tumor cells[1, 2].
Overexpression of ?-galactoside ?2,6-sialyltransferase
(ST6Gal-I) has been observed in numerous types of human
tumors, and it results in the increase of N-glycans in the
Sia6LacNAc stucture in various cancer cells . Emerging
evidences showed that hypersialylation of ?1-integrin stimulated
both cell attachment and migration on collagen I and induced
variation in cell motility[4, 5]. However, hypersialylation of
?1-integrin could not explain all phenotypic events. We
therefore hypothesized that ST6Gal-I could modify other surface
glycoprotein(s), which might directly or in synergy with
integrins be responsible for the biological behavior.
In the present study, we aimed to investigate the
potential modification of previously unrecognized surface
glycoprotein(s) by ST6Gal-I. The expression of ST6Gal-I in
the human HCT116 colon carcinoma cell line was transiently
knocked down with siRNA. Finally, we demonstrated a
functional role for ST6Gal-I-driven terminal sialylation of c-Met
glycoprotein in colon cancer progression, which might be
distinct from integrin-dependent cancer progression.
Materials and methods
Cell lines and culture conditions
The colon cancer adenocarcinoma cell line, HCT116, was
obtained from the American Type Culture Collection
(Manassas, VA, USA). Cells were propagated and maintained in
McCoy?s 5A Medium (Sigma, St Louis, MO, USA) containing
100 U/mL penicillin and supplemented with 10% fetal bovine
serum (FBS, Gibco, Grand Island, NY, USA). Cells were
maintained at 37 ?C under a humidified 95% and 5% (v/v) mixture
of air and CO2.
Short interfering (si)RNA transfection
siRNA duplexes (D3) 5?-AACTCTCAGTTGGTTACCACA-3?
specific for the human ST6Gal-I gene sequences, as well as
control nontargeting siRNA (NC), were purchased from
Shanghai Genepharma Co Ltd (Shanghai, China). Briefly,
HCT116 cells were grown to 75% confluence, exposed to
siRNA (100 nmol/L) for 6 h in the presence of oligofectamine
(Invitrogen, Carlsbad, CA, USA), and then incubated at 37?C
for 24 h before use in assays.
RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from transfected cells using TRIzol
(Invitrogen) after cells had reached about 90% confluence.
After RT, cDNA was amplified by PCR using Taq DNA
polymerase, deoxynucleoside triphosphates and the indicated
primers, as described previously, and 30 cycles of
amplification were performed. The PCR products (10 ?L) were
analyzed by electrophoresis on 2% agarose gels. After being
stained with ethidium bromide, gel images were obtained
using Genesnap Version 6.0026 software (Syngene,
ST6Gal-I activity detection
The activity of ST6Gal-I was determined by an
enzyme-linkedimmunosorbent assay (ELISA) using asialofetuin (Sigma)
precoated plates. Equivalent amounts of cell lysates were loaded
and cytidine-5?-monophospho-N-acetylneuraminic acid
sodium salt (CMP-NeuAC, Sigma) was then added to initiate
the reaction. After being washed and blocked, the sialylated
fetuin was bound with SNA-biotin (Vector Lab, Burlingame,
CA, USA), and probed with streptavidin-horseradish
peroxidase. Finally, 100 ?L of solution (0.03% H2O2, 2 mg/mL
o-phenylenediamine in citrate buffer 0.1 mol/L, pH 5.5) was added
and the reaction was terminated with H2SO4. The absorbance
at 492 nm was measured using a multiwell spectrophotometer
(VERSAmax, Molecular Devices, Union City, CA, USA).
Fluorescence activated cell sorting (FACS) analysis
For analysis of the cell surface ?2,6-sialic acid structure and
c-Met protein, adherent HCT116 cells were trypsinized and
placed in FACS blocking buffer at a concentration of 5?105
cells/mL. The cells were then incubated with 2 ?g SNA-biotin
or 0.5 ?g c-Met antibody (R&D, Minneapolis, MN, USA) for 30
min on ice, washed twice in blocking buffer, and then exposed
to 0.25 ?g R-phycoerythrin (RPE)-conjugated streptavidin for
an additional 30 min. Labeled samples were analyzed by flow
Cell adhesion assay
Adhesion assays were performed in 96-well plates pre-coated
with collagen (10 ?g/mL), fibronectin (10 ?g/mL) or laminin
(20 ?g/mL) (BD, San Jose, CA, USA). After being blocked
with 1% bovine serum albumin, 0.4?105 cells were seeded into
each well and allowed to adhere at 37 ?C for 1 h.
Non-adherent cells were rinsed with phosphate-buffered saline (PBS);
the remaining cells were fixed with 4% paraformaldehyde and
then stained with 0.1% crystal violet for 15 min. After being
rinsed with water, the remaining cells were solubilized by
acetic acid. Absorbance spectroscopy (595 nm) was used to
quantify the number of substrate-adherent cells.
Transwell Boyden chambers (Costar, Bethesda, MD, USA)
were coated with gelatin. After being soaked with serum-free
5A medium, 2?105 transfected cells suspended in 5A
containing 2% FBS were loaded into the upper well. The lower
compartment was filled with culture medium alone or medium
containing 100 ng hepatocyte growth factor (HGF, R&D) and
then incubated at 37 ?C for 30 or 48 h. After the non-migratory
cells were removed, the migrated cells were fixed and stained
as previously described and counted.
Preparation of total cell extracts and immunoblot analysis
After transfection with siRNA for 24 h, cells were harvested
and resuspended in RIPA lysis buffer (50 mmol/L Tris-HCl,
150 mmol/L NaCl, 1 mmol/L EDTA, 0.1% SDS, 0.5%
deoxycholic acid, 0.02% sodium azide, 1% NP-40, 2.0 ?g/mL
aprotinin, 1 mmol/L phenylmethylsulfonylfluoride). The effects of
HGF stimulation were detected by serum starving transfected
cells for 24 h and then stimulating with 50 ng HGF for 20 min.
Equivalent amounts of proteins were used for Tyr1349-MET,
Erk1/2, phosphorylated-Erk1/2, AKT, phosphorylated-AKT,
STAT3, Tyr705-STAT3, Ser727-STAT3 (Cell Signaling
Technology, Beverly, MA, USA) and/or ?-subunit-specific antibody
for MET (Santa Cruz Biotech, Santa Cruz, CA, USA)
Lectin affinity assays
Lysates were centrifuged at 10 000?g for 15 min at 4 ?C. One
milligram of supernatant was incubated for 4 h at 4 ?C with
6 ?g SNA. Streptavidin-agarose beads (Sigma) were then
added and incubated for an additional 4 h at 4 ?C with
rotation. After being briefly centrifuged and washed, precipitated
proteins were released from the bead complexes by boiling in
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) sample buffer and analyzed directly by
SDSPAGE and immunoblotting.
For analysis of the cell surface ?2,3-sialic acid structure,
adherent HCT116 cells were trypsinized with trypsin-EDTA
using standard methods and placed in FACS blocking buffer
(0.15 mol/L PBS with 2% BSA) at a concentration of 5?105
cells/mL. The cells were then incubated with 2 ?g
MAAbiotin (Vector Lab, Burlingame, CA) for 30 min on ice, washed
twice in blocking buffer, and then exposed to 0.25 ?g
RPEconjugated streptavidin for an additional 30 min. Labeled
samples were analyzed by flow cytometry.
5-bromo-2?-deoxyuridine (BrdU) assay
DNA synthesis was monitored by measuring incorporation
of the artificial thymidine nucleotide analog BrdU (Sigma, St
Louis, MO, USA) into newly synthesized DNA. Cells (1.5?105
per well) were cultured in 6-well plates and transfected
with siRNAs. Forty-eight hours after transfection, cells
were refreshed with complete medium containing 100
?mol/L BrdU and incubated for an additional 30 min for
BrdU incorporation. After BrdU incubation, cells were washed
three times with PBS and fixed in 4%
paraformaldehydePBS for 20 min. Next, the fixed cells were permeabilized
with 0.5% Triton X-100-PBS. After partial denaturation of the
DNA with 2 mol/L HCl, cells were incubated with anti-BrdU
mouse monoclonal antibody (Santa Cruz Biotechnology, CA),
at a 1:50 dilution for 1 h. Cells were then washed with PBS
three times and incubated with Alexa Fluor 633 anti-mouse
antibody (Invitrogen, Carlsbad, CA, USA) at a 1:200 dilution
for 1 h. After three PBS washes, cells were counterstained with
4?,6?-diamidino-2-phenylindole for 5 min and analyzed using
an Olympus DP70 digital microscope camera. Quantitative
analysis of immunofluorescence data was carried out with
Image-Pro Plus software.
Knockdown of ST6Gal-I reduced endogenous ST6Gal-I expression in HCT116 cells
Cell surface sialylation of the metastasizing HCT116 cell line
correlates with in vivo tumorigenicity, and overexpression
of ST6Gal-I has been implicated in increased cell motility.
Here, we sought to determine the effect of in vitro knockdown
of ST6Gal-I expression on the motility of HCT116 cells. We
transiently transfected ST6Gal-I-targeting siRNA into HCT116
cells (D3) and verified that ST6Gal-I mRNA expression was
reduced by RT-PCR detection in a time-dependent manner
The activity of ST6Gal-I in siRNA transfected HCT116 cells
(D3) was compared with that in parental HCT116 cells (P)
and HCT116 cells transfected with nonspecific siRNA (NC)
by ELISA assay. After interference with ST6Gal-I-targeting
siRNA, the ?2,6-sialic acid decoration was significantly
decreased in D3 cells (Figure 1B).
The ?2,6-sialic acid structures on the cell surface were
evaluated by FACS analysis. As expected, the ?2,6-sialic acid
structures on the D3 cell surface were remarkably reduced
compared with those in P and NC cells (Figure 1C), whereas the
?2,3-sialic acid structures were not affected (Figure S1). These
results indicated that siRNA effectively reduced the
expression of ST6Gal-I.
Decreased expression of ST6Gal-I reduces cell migration but not cell adhesion
To determine the effect of cell surface ?2,6-sialylation on cell
motility, we evaluated cell migration using a modified Boyden c-Met is hyposialylated in ST6Gal-I-KD HCT116 cells
chamber assay with FBS as a chemoattractant. The migration It has been postulated that changes in sialic acid structure
of D3 cells was significantly less than that of P or NC cells (Fig- on membrane glycoconjugates can modulate the adhesion
ure 1D). The growth kinetics study showed that the reduc- of cancer cells to ECM components in an integrin-dependent
tion in ST6Gal-I had no effect on cell proliferation (Figure S2). manner[7?10]. Because we did not find any change in
ST6GalThese results indicate that the ST6Gal-I regulated cell motility I-KD HCT116 cell adhesion, we suggest that integrins alone
but not cell growth. cannot fully explain this observation. Sialylation of membrane
We next examined the effects of ST6Gal-I siRNA on cell glycoproteins other than integrins might be responsible for the
adhesion, specifically to three main extracellular matrix (ECM) ST6Gal-I-dependent motility phenotype in HCT116 cells. To
components: laminin, fibronectin, and collagen. The ability of identify the related membrane glycoproteins in the regulation
D3 cells to attach to these three components was similar to that
of P and NC cells (Figure 1E), suggesting that downregulation
of ST6Gal-I expression had no effect on cell adhesion.
of cell motility, we precipitated ?2,6-sialylated glycoproteins
using SNA and then examined the glycoproteins of interest,
c-Met and CXCR4, by immunoblotting. Remarkably, c-Met
was clearly present in SNA precipitates prepared from cells
with normal ST6Gal-I expression (P and NC), whereas little
c-Met was present in ST6Gal-I-KD HCT116 cells (D3) (Figure
2A). In contrast, CXCR4 was not co-precipitated by SNA (data
To further examine the possibility that the decreased cell
motility of ST6Gal-I-KD HCT116 cells was caused by the
abolition of normal c-Met function, we evaluated HGF-stimulated
migration using Boyden chamber assays. In D3 cells, HGF
stimulation induced less migration and also eliminated the
ability to trigger the development of multicellular-branched
structures compared with P and NC cells (Figure 2B). These
results indicate that down-regulation of ST6Gal-I expression
resulted in c-Met hyposialylation and inhibition of
HGFinduced motility of HCT116 cells.
Insufficient terminal ?2,6-sialylation abolishes c-Met maturation
To elucidate how insufficient terminal ?2,6-sialylation
determines the fate of the c-Met molecule, we examined c-Met
mRNA and protein expression in ST6Gal-I-KD HCT116 cells.
Reduced terminal ?2,6-sialylation was associated with a
decrease in c-Met expression on the surface of D3 cells relative
to P or NC cells (Figure 3A). However, there were no
differences in mRNA expression among the three tested cell lines
(Figure 3B). These findings indicate that ST6Gal-I affected
c-Met function at the post-translational level, rather than at the
transcriptional or translational level.
The endoplasmic reticulum (ER)-resident precursor form of
c-Met is glycosylated and cleaved into two Golgi-processed
mature subunits (? and ?) that are further linked via disulfide
bonds into a functional heterodimer on the cell surface. We
therefore investigated whether the terminal deficiency in
?2,6linkage disrupted c-Met maturation in ST6Gal-I-KD HCT116
cells. Using ?-subunit-specific antibodies, we found that
mature ?-subunit was significantly decreased in D3 cells
compared with P or NC cells. Consistently, a reduction in
phosphorylated c-Met was noted, mimicking the protein
expression profile (Figure 3C).
Tyr705, but not Ser727 phosphorylation of STAT3 responds to hyposialylation in HCT116 cells
We investigated three key downstream targets of c-Met in
these transfected HCT116 cells under normal culture
conditions. Phosphorylation of STAT3 was down-regulated at
Tyr705, rather than Ser727, in ST6Gal-I-null cells, whereas
the total level of STAT3 remained unchanged. The other two
downstream effectors, Erk1/2 and AKT, were not affected
(Figure 4A). Moreover, there was no significant modulation
on any of the downstream signaling pathways when cells were
starved and stimulated with HGF alone (Figure 4B). These
results indicate that insufficient terminal ?2,6-sialylation of
c-Met or other unidentified glycoproteins directly or indirectly
regulate STAT3 Tyr705 dephosphorylation.
Increased levels of ST6Gal-I and ?2,6-sialic acid have been
observed in various cancer cells and are associated with a poor
prognosis in cancer progression. In vitro cell culture studies
suggest that ST6Gal-I up-regulation may contribute to
metastasis by regulating invasiveness and cell motility. The role of
the integrin family members has been emphasized in this
context by modifying adhesion to ECM components. However,
some studies have reported conflicting results. For example,
terminal sialylation reduces attachment to ECM glycoproteins
in melanoma and in myeloid cells, but enhances adhesion
in erythroleukemia K562 cells and colon epithelial SW948
cells. These observations support the possibility that
integrins are not the only relevant targets of ST6Gal-I-mediated
sialylation in cancer cell adhesion and suggest that other surface
glycoproteins may also be involved.
Using the siRNA technique in combination with an SNA
lectin-blotting approach, we found, for the first time, that
ST6Gal-I knock-down specifically abolished HCT116 cell
motility via terminal sialylation of c-Met. Despite numerous
reports on the glycoprotein nature of c-Met, the potential
glycosylation pattern of N-linked carbohydrates on c-Met has not
been extensively studied. In our study, ST6Gal-I deficiency
caused a reduction in ?2,6-sialylation of c-Met, supporting the substrate of ST6Gal-I and that its function is regulated by
idea that c-Met is the substrate of ST6Gal-I. modification of its ?2,6-sialic terminal structure. We also
Under physiological conditions, c-Met activation is a rela- found that elimination of a single terminal sialic acid structure
tively transient event, whereas in tumor cells, c-Met is often could affect signaling events in HCT116 cells. These findings
constitutively activated. c-Met activation in human tumor suggest that therapies targeting toward the ST6Gal-I
glycosylcells can be initiated through various mechanisms, including transferase gene may be beneficial, either directly or through
overexpression, structural alterations, and receptor deregula- synergistic interactions, in treating malignant tumors
harbortion. The results of the current study suggest that the c-Met ing constitutively activated c-Met.
hyposialylation is likely to result in long-lasting phenotypic
changes. In contrast to the rapid and transient regulatory Acknowledgments
dynamic characteristics of mature forms of c-Met, the altered This work was supported by the National Basic Research
Proglycosylation patterns typical of immature c-Met prevent its gram Grant (No 2003CB716400) of China, the Natural Science
efficient trafficking and thus limit its localization to the cell Foundation of China for Distinguished Young Scholars (No
surface. In this context, ST6Gal-I-driven c-Met activation 30725046) and the Natural Science Foundation of China for
could account for the long-term changes in c-Met-mediated Innovation Research Group (No 30721005).
cell motility that are characteristic of neoplastic cells.
Similar to many other growth factor receptor tyrosine Author contribution
kinases, c-Met exerts its oncogenic potential through deregu- Prof Mei-yu GENG, Prof Jian DING and Prof Jing LI designed
lating the activation of a number of protein phosphorylation- the research. Jin QIAN performed the research, analyzed
dependent signaling cascades. These include the Erk/ data and wrote the paper. Cai-hua ZHU, Shuai TANG, Ai-jun
MAPK and PI3K pathways, which are important for cell SHEN, and Jing AI contributed to the research.
adhesion, proliferation, and survival. In the present study,
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