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Available online http://breast-cancer-research.com/content/10/1/R1
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Vol 10 No 1
Research article
Silencing of GM3 synthase suppresses lung metastasis of murine
breast cancer cells
Yuchao Gu*, Junhua Zhang*, Wenyi Mi, Jing Yang, Feng Han, Xinzhi Lu and Wengong Yu
Department of Molecular Biology, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao, China
* Contributed equally
Corresponding author: Wengong Yu, yuwg66@ouc.edu.cn
Received: 5 Aug 2007 Revisions requested: 18 Sep 2007 Revisions received: 24 Oct 2007 Accepted: 3 Jan 2008 Published: 3 Jan 2008
Breast Cancer Research 2008, 10:R1 (doi:10.1186/bcr1841)
This article is online at: http://breast-cancer-research.com/content/10/1/R1
© 2008 Gu et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background Gangliosides are sialic acid containing
glycosphingolipids that are ubiquitously distributed on
vertebrate plasma membranes. GM3, a precursor for most of the
more complex ganglioside species, is synthesized by GM3
synthase. Although total ganglioside levels are significantly
higher in breast tumor tissue than in normal mammary tissue, the
roles played by gangliosides in breast cancer formation and
metastasis are not clear.
Methods To investigate the roles of gangliosides in breast
tumor development, GM3 synthase was silenced in the highly
metastatic 4T1 cells and over-expressed in the non-metastatic
67NR cells. The behavior of breast cancer cells was examined
in vitro using migration assay, invasion assay, and soft agar
assay. Tumor formation and metastasis in vivo were examined
using a well established mouse mammary tumor model.
Results GM3 synthase silencing in 4T1 cells significantly
inhibited cell migration, invasion and anchorage-independent
growth in vitro, and lung metastasis in vivo. In addition, over-
expression of GM3 synthase in nonmetastatic 67NR cells
significantly induced cell migration and anchorage-independent
growth. Further studies indicated that activation of the
phosphoinositide-3 kinase/Akt pathway, and consequently
inhibition of nuclear factor of activated T cell (NFAT)1
expression, could be the mechanism underlying the suppression
of breast cancer migration/invasion induced by GM3 synthase
silencing.
Conclusion Our findings indicate that GM3 synthase silencing
suppressed lung metastasis in murine breast cancer cells. The
molecular mechanism that underlies GM3 synthase mediated
migration and invasion was inhibition of the phosphoinositide-3
kinase/Akt pathway. The findings suggest that GM3 synthase
may be of value as a therapeutic target in breast cancer.
Introduction
Gangliosides are sialic acid containing glycosphingolipids that
are ubiquitously distributed on vertebrate plasma membranes
[1]. They participate in the regulation of various cellular func-
tions, including cell proliferation, apoptosis, migration, and
invasion [2-4]. Numerous studies have demonstrated that
abnormal ganglioside expression is strongly associated with
the malignancy of cancer cells [5]. The ganglioside content is
upregulated in some metastasizing tumor cells, such as lym-
phoma cells, fibrosarcoma cells, and melanoma cells [6-8].
However, there is an inverse relation between metastasis
properties and ganglioside content in some types of tumors,
for instance bladder tumor and liver carcinoma [4,9,10]. Some
observations indicate that the effects of gangliosides on tumor
metastasis are dependent on the ganglioside species and/or
tumor-type specificity. For example, the effects of a given gan-
glioside, such as GM3, on metastatic potential differ between
tumor cell types [10-12]. On the other hand, the metastatic
potential of a given tumor cell type, such as melanoma cells,
may be enhanced or inhibited by different gangliosides
[3,11,13]. Hence, there is a need to define the involvement
and molecular mechanisms of gangliosides in metastasis of
each cancer type.
Among the various gangliosides, GM3 contains the simplest
ganglioside oligosaccharide and it serves as a precursor for
CMP = cytidine 5'-monophosphate; DME-10 = Dulbecco's modified Eagles medium supplemented with 10% fetal calf serum; EGFP = enhanced
green fluorescent protein; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GM3S = CMP-N-acetylneuraminic acid:lactosylceramide 2,3-sia-
lyltransferase; NFAT = nuclear factor of activated T cell; PI3K = phosphoinositide-3 kinase; PTEN = phosphatase and tensin homolog; RT-PCR =
reverse transcription polymerase chain reaction; siRNA = small interfering RNA.
Breast Cancer Research Vol 10 No 1 Gu et al.
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most of the more complex ganglioside species (for example,
GD3, GM2, GD2, and so on) [14]. GM3 is synthesized by
transfer of sialic acid from cytidine 5'-monophosphate (CMP)-
sialic acid to nonreduced terminal galactose residue of lacto-
sylceramide through the α-2,3-glycosyl bond, and the reaction
is catalyzed by GM3 synthase (CMP-N-acetylneuraminic
acid:lactosylceramide 2,3-sialyltransferase [EC 2.4.99.9]; also
known as ST3Gal V, GM3S [the term used in the present
report], and Siat9) [15]. Overexpression and suppression of
GM3S expression are approaches used to determine the
effects of endogenous gangliosides on the metastatic process
[2,16].
Total gangliosides in breast tumor tissues and sera from
breast cancer patients were significantly higher than in those
from healthy individuals [17,18]. GM3 and some other gan-
gliosides (such as GD3 and GM2) have been reported to be
potential targets for breast cancer immunotherapy [19-22],
chemotherapy [23], and radiotherapy [24]. Although ganglio-
side levels have been demonstrated to be elevated in breast
cancer, there are no reports in the literature regarding the roles
played by gangliosides in breast cancer metastasis.
In this study, in order to investigate the roles played by endog-
enous gangliosides in breast cancer formation and metastasis,
GM3S was silenced or over-expressed in different breast
tumor cell lines. The effects of GM3S expression on the malig-
nant properties of breast tumor cells and the molecular mech-
anisms of ganglioside-mediated migration and invasion were
evaluated.
Materials and methods
Cells and cell cultures
67NR and 4T1 (kindly provided by Dr Fred R Miller at Kar-
manos Cancer Institute) are mammary tumor cell lines charac-
terized by enhanced lung metastatic potential that were
derived from a single, spontaneously arising mouse mammary
tumor [25]. The nonmetastatic 67NR cells fail to leave the pri-
mary site, whereas the 4T1 cells can metastasize to lung, liver,
bone, and brain via the hematogenous route. They were cul-
tured in Dulbecco's modified Eagles medium supplemented
with 10% fetal calf serum (DME-10), 1 mmol/l mixed nones-
sential amino acids, and 2 mmol/l L-glutamine.
Plasmid construction
The dual promoter small interfering RNA (siRNA) expression
vector pMEHMpuro was constructed based on a self-inacti-
vating murine stem cell virus plasmid, namely pMSCVpuro.
First, the HindIII restriction site of pMSCVpuro was deleted by
digesting the plasmid with HindIII, blunting the 3' recessed
ends and re-ligating. The enhanced green fluorescent protein
(EGFP) gene was then inserted into the BglII/XhoI sites of
HindIII restriction site deleted pMSCVpuro vector; the result-
ing construct was named pMSCVpuro-EGFP. Then, the dual
promoter siRNA expression cassette was constructed as
described previously [26] and inserted into the XhoI/EcoRI-
digested pMSCVpuro-EGFP plasmid to form pMEHMpuro. In
order to generate siRNA vectors, three pairs of oligonucle-
otides, corresponding to different regions of murine GM3S
encoding gene, were annealed and inserted into BglII/HindIII
double digested pMEHMpuro: siGM3S1, 5'-AGC TTA AAA
AGT AAG GTT GAA CAG TGC GCC TTT TTA-3' and 5'-GAT
CTA AAA AGG CGC ACT GTT CAA CCT TAC TTT TTA-3';
siGM3S2, 5'-AGC TTA AAA AGA CTG CCT TCG ACA TCC
TTC TTT TTA-3' and 5'-GAT CTA AAA AGA AGG ATG TCG
AAG GCA GTC TTT TTA-3'; and siGM3S3, 5'-AGC TTA AAA
AGT GTG ACC ACA GAG ACC AAG TTT TTA-3' and 5'-
GAT CTA AAA ACT TGG TCT CTG TGG TCA CAC TTT
TTA-3'. This process yielded the vectors pMEHM-siGM3S1
(siGM3S1), pMEHM-siGM3S2 (siGM3S2), and pMEHM-
siGM3S3 (siGM3S3), respectively. In addition, annealed oli-
gonucleotides (5'-AGC TTA AAA AGT TCC GTA TGT TGC
ATC ACC TTT TTA-3' and 5'-GAT CTA AAA AGG TGA TGC
AAC ATA CGG AAC TTT TTA-3') that did not match any
known mouse gene was inserted into BglII/HindIII double
digested pMEHMpuro, yielding pMEHM-siMock. The full-
length GM3S encoding gene was amplified from the total
RNA of 4T1 cells using RT-PCR and inserted into XhoI/EcoRI
double digested pMSCVpuro, resulting in the GM3S expres-
sion vector pMSCV-GM3S. All of the resulting constructs
were confirmed by DNA sequencing.
DNA transfection and selection
4T1 cells were transfected with various GM3S-targeting RNA
interference and mock plasmids. 67NR cells were transfected
with GM3S and pMSCVpuro vectors. Transfection was car-
ried out using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA,
USA), and the transfected cells were cultured in the medium
supplied with 4 μg/ml puromycin for 2 weeks. The obtained
cells were named 4T1-siMock, 4T1-siGM3S1, 4T1-siGM3S2,
4T1-siGM3S3, 67NR-Control and 67NR-GM3S.
RNA isolation and real-time RT-PCR
Total RNA was isolated from cells using Trizol (Invitrogen) and
reverse transcription was carried out using the High Capacity
cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA).
Real-Time PCR was carried out using Power SYBR Green
Master PCR mix (Applied Biosystems, Warrington, UK) in trip-
licate, and the reactions were conducted on a 7500 real-time
PCR system (Applied Biosystems, Singapore). The relative
quantitative analysis was normalized to endogenous control
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The
mouse GM3S forward primer was 5'-GCT TCA AGC AAT
GGT AAA AAA TGA-3', and its reverse primer was 5'-TTC
TGC CAC TTG CTT CCA AA-3'. The mouse phosphatase
and tensin homolog (PTEN) forward primer was 5'-TGA AGA
CCA TAA CCC ACC ACA-3', and its reverse primer was 5'-
TCA TTA CAC CAG TCC GTC CCT-3'. Mouse GAPDH for-
ward primer was 5'-AAT TCA ACG GCA CAG TCA AGG-3',
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and its reverse primer was 5'-TGT TAG TGG GGT CTC GCT
CC-3'.
Immunoblotting (IB) analysis
Cells were lysed in lysis buffer (50 mmol/l Tris-HCl [pH 7.4],
150 mmol/l NaCl, 1% NP40, 1 mmol/l EDTA, 1 mmol/l
Na3VO4, 10 mmol/l NaF) containing a protease inhibitor cock-
tail (Roche, Nutley, NJ, USA). Protein samples (50 μg) were
separated by 12% SDS-PAGE and transferred to Immobilon-
P membranes (Millipore, Bedford, MA, USA). Antibodies to
phosphorylated and total Akt (Cell Signaling, Beverly, MA,
USA), phosphorylated (Ser380/Thr382/383) and total PTEN
(Cell Signaling), NFAT1 (Santa Cruz, CA, USA), and GAPDH
(Santa Cruz) were used, with detection by ECL-detecting rea-
gent (Amersham Biosciences, Buckinghamshire, UK). Quanti-
fication of the blots was conducted using Image-Pro Plus
software (version 6.0; Media Cybernetics, Bethesda, MD,
USA).
Flow cytometry analysis
The flow cytometry procedure was as described previously
[27]. Briefly, cells were trypsinized and incubated with anti-
GM3 or anti-GD3 monoclonal antibodies (Seikagaku Corpora-
tion, Tokyo, Japan) and then labeled with rat anti-mouse IgM-
P-RE (Southern Biotechnology Associates, Birmingham, AL,
USA). A total of 1 × 104 labeled cells were analyzed using a
cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Cell proliferation assay
Cells were seeded 1 × 104 per well in a 96-well plate. Cells
were allowed to grow for 24 hours. Then, 20 μl of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5
mg/ml) was added to each well. After 4 hours of incubation at
37°C, cells were lysed by addition of 200 μl dimethylsulfoxide.
Absorbance was measured at 570 nm using a Rainbow micro-
plate reader (Tecan, Groding/Salzburg, Austria).
Anchorage-independent cell growth
Cells (5 × 103 to 1 × 104) were suspended in 1 ml top agar
medium (DME-10 supplied with 0.4% agar), in the presence
or absence of phosphoinositide-3 kinase (PI3K)-specific inhib-
itor LY294002 (Merck, Nottingham, UK). The cell suspension
was then overlaid onto 1.5 ml bottom agar medium (DME-10
supplied with 0.8% agar) in six-well tissue culture plates in
triplicate. DME-10, with or without LY294002, was added to
the plates every 3 days as a feeder layer. On day 12, the
number of colonies was counted in six random fields at 40×
magnification.
Cell migration and invasion assay
Cell migration was assayed using Transwell chambers (6.5
mm; Corning, New York, USA) with 8 μmol/l pore membranes.
The lower chamber was filled with 600 μl NIH-3T3 condi-
tioned medium containing 20 μg/ml fibronectin (BD Bio-
sciences, Bedford, MA, USA) with or without 2 μmol/l
LY294002. Cells (5 × 104) were suspended with 100 μl
upper medium (Dulbecco's modified Eagles medium with 1%
fetal calf serum) and plated into the upper chamber with or
without 2 μmol/l LY294002. After 16 hours, the number of
cells appearing by crystal violet staining on the undersurface
of the polycarbonate membranes was scored visually in five
random fields at 100× magnification using a light microscope.
For invasion assays, the upper face of the membrane was cov-
ered with 70 μl Matrigel (1 mg/ml; BD Biosciences). The inva-
sion assay procedure was the same as for the migration assay,
except that the incubation time of the experiment was pro-
longed to 24 hours.
Primary tumor growth and lung metastases assay
These procedures were performed as described previously
[25,28] with minor modifications. Female BALB/c mice, aged
8 to 10 weeks, were used in the experiment. In brief, mice (six
to eight per group) were anesthetized with sodium pentobar-
bital (50 mg/kg body weight), and tumor cells (5 × 105) in 10
μl DME-10 were injected into the mammary gland. The weight
of the primary tumors and the number of metastatic nodules on
the lung surface were evaluated 30 days after the tumor cells
injection. The animals were housed and cared for in accord-
ance with the guidelines established by the National Science
Council of Republic of China. The lung tissues (six lungs per
group) from in vivo experiments described above were fixed in
10% neutral buffered formalin and embedded in paraffin, and
then thick sections (4.0 μm; three sections per lung tissue)
were cut and stained with hematoxylin and eosin.
Statistic analysis
All of the results were repeated in at least three independent
experiments and consistently yielded similar results. Data were
analyzed by the Student-Newman-Keuls test using the SPSS
11.0 software program (SPSS Inc., Chicago, IL, USA). P <
0.05 was considered statistically significant. Results are
expressed as mean ± standard error of the mean.
Results
GM3S is silenced or overexpressed in breast cancer cell
lines
We found that the expression of GM3S mRNA was greater in
the highly metastatic 4T1 tumor cells than in the nonmetastatic
67NR cells (Figure 1a). These observations led us to regard
GM3S to be a potential candidate for orchestrating metastasis
in breast cancer. To determine whether GM3S played an
important role in breast cancer malignancy, expression of
GM3S in the highly metastatic 4T1 cell line was silenced by
RNA interference. The expression of the GM3S siRNAs
siGM3S2 and siGM3S3, but not that of siGM3S1, drastically
reduced the expression of GM3S mRNA in 4T1 cells (Figure
1b). The results of flow cytometry analysis indicate that GM3S
knockdown significantly suppressed the expression of the
ganglioside GM3 (Figure 2a) and the more complex
Breast Cancer Research Vol 10 No 1 Gu et al.
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ganglioside GD3 (Figure 2b). On the other hand, GM3S was
over-expressed in 67NR cells. The expression of GM3S was
upregulated (Figure 1c) and the levels of expression of GM3
(Figure 2c) and GD3 (Figure 2d) gangliosides were enhanced.
These GM3S-reconstructed cells enabled us to identify the
effects of GM3S expression on the malignant properties of
breast cancer cells, and these cell lines also appeared to be
useful for analyzing the roles played by gangliosides in breast
cancer cells.
GM3S expression enhances the anchorage-independent
growth of breast cancer cells
To investigate whether GM3S expression affected cell prolif-
eration and anchorage-independent growth, MTT and soft
agar colony assays were conducted. Silencing of GM3S in
4T1 cells and over-expression of GM3S in 67NR cells did not
affect the proliferation (Figure 3a,b). The results were also ver-
ified by cell counting experiments (data not shown). Interest-
ingly, GM3S knockdown in 4T1 cells significantly reduced the
number of colonies and slightly decreased the colony size
(Figure 3c), whereas 67NR cells over-expressing GM3S
formed much larger colonies than did control cells, and the
number of colonies was slightly increased (Figure 3d). These
results indicated that endogenous GM3S was important for
maintaining the anchorage-independent growth of breast
tumor cells.
GM3S expression enhances migration and invasion of
breast cancer cells
To investigate the role played by GM3S in breast cancer cell
metastasis, we conducted migration and invasion assays in
vitro. As shown in Figure 4a and 4b, the migration and invasion
abilities were inhibited by silencing of GM3S in 4T1 cells.
Over-expression of GM3S in noninvasive 67NR cells
enhanced their ability to migrate (Figure 4c). However, over-
expression of GM3S was unable to transform noninvasive
67NR cells into invasive ones (data not shown). These find-
ings suggest that GM3S expression is essential but not suffi-
cient for breast cancer cell migration and invasion.
Figure 1
Analysis of GM3S expression of breast tumor cell lines by real-time RT-PCRAnalysis of GM3S expression of breast tumor cell lines by real-time RT-PCR. (a) GM3S mRNA expression was higher in metastatic 4T1 cells than in
noninvasive 67NR cells. (b, c) Relative GM3S mRNA expression in (b) GM3S-silenced 4T1 cells and in (c) GM3S over-expressing 67NR cells.
CMP, cytidine 5'-monophosphate; GM3S, CMP-N-acetylneuraminic acid:lactosylceramide 2,3-sialyltransferase.
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Inhibition of GM3S expression suppresses lung
metastases of breast cancer cells
Based on the roles played by GM3S expression in migration/
invasion and anchorage-independent growth of breast cancer
cells described above, we next examined the effects of GM3S
silencing on tumor formation and metastasis of breast cancer
cells. Consistent with the results in vitro, GM3S silencing in
4T1 cells had no effect on primary tumor weight (Figure 5a)
but it dramatically reduced the number of visible metastatic
nodules on the lung surface of tumor-bearing mice (Figure
5b,c). Histologic analyses confirmed that the number of
micrometastatic lesions was significantly reduced in lungs of
mice carrying 4T1-siGM3S2 and 4T1-siGM3S3 tumors (Fig-
ure 5d).
GM3S promotes breast cancer cells migration and
invasion through the PI3K/Akt pathway
To investigate the molecular mechanism underlying GM3S-
mediated migration and invasion, some metastasis-associated
signaling pathways were assayed by immunoblotting. We
found that suppression of GM3S significantly enhanced the
phosphorylation of Akt at two key residues, namely Thr308
and Ser473 (Figure 6a). Further studies demonstrated that the
PI3K specific inhibitor LY294002 (2 μmol/l) restored Akt
phosphorylation to the level exhibited by 4T1-siMock cells
(Figure 6b) and partially compensated for the inhibition of cell
migration/invasion induced by the GM3S knockdown (Figure
6c).
PTEN is a major negative regulator of the PI3K/Akt signaling
pathway. In the present study, GM3S inhibition suppressed
the expression and phosphorylation of PTEN in 4T1 cells (Fig-
ure 7a,b). These findings suggest that the inhibition of PTEN
expression at least partially contributed to activation of the
PI3K/Akt pathway in 4T1 cells in which GM3S had been
knocked down.
We further investigated the downstream effectors of PI3K/Akt
pathway. GM3S knockdown inhibited NFAT1 expression (Fig-
ure 8a), and inhibition of the PI3K/Akt pathway induced
NFAT1 expression (Figure 8b). In conclusion, as illustrated in
Figure 9, GM3S knockdown suppressed PTEN expression
and resulted in activation of Akt signaling, which reduced the
expression of NFAT1 and inhibited cell migration and invasion.
PI3K/Akt pathway has no effect on anchorage-
independent growth of breast tumor cells
To test whether activation of the PI3K/Akt pathway was
responsible for the anchorage-independent growth affected
by GM3S knockdown in 4T1 cells, we inactivated the PI3K/
Akt pathway with LY294002. The colony forming abilities of
both GM3S-knockdown 4T1 cells and the mock transfected
cells were unaffected by inhibition of the PI3K/AKT pathway
(Figure 10), indicating that the anchorage-independent
growth caused by GM3S was not associated with the PI3K/
Akt pathway. Extra pathway(s) may exist to compensate for the
PI3K/Akt pathway with respect to anchorage-independent
survival of breast tumor cells.
Discussion
Breast cancer is the most common malignant disease of
women. In women with breast cancer, it is not the primary
tumor but its metastasis to distant sites that is the ultimate
cause of death. Improving our understanding of the molecular
mechanisms that underlie the metastatic process might
improve clinical management of the disease [29].
It has been demonstrated that levels of total gangliosides and
GM3 (N-glycolylneuraminic acid) in breast tumor tissue were
significantly higher than in normal tissues [18,24], and ganglio-
side content was fourfold higher in the invasive human breast
cancer cells MDA-MB-231 than in noninvasive MCF-7 cells
[29]. In the present study, the results of flow cytometry analy-
sis indicated that GM3 and GD3 gangliosides were
significantly higher in the highly metastatic 4T1 cells than in
the nonmetastatic 67NR cells. These studies suggested that
gangliosides might play roles in breast cancer formation and
metastasis.
Figure 2
Analysis of ganglioside content on cell surface by flow cytometryAnalysis of ganglioside content on cell surface by flow cytometry.
Expression levels of (a) GM3 and (b) GD3 gangliosides on the surface
of GM3S-silenced 4T1 cells using anti-GM3 and anti-GD3 monoclonal
antibody (shaded peaks: 4T1-siGM3S2; unshaded peaks: 4T1-
siMock). Expression levels of (c) GM3 and (d) GD3 gangliosides on
the surface of GM3S over-expressing 67NR cells using anti-GM3 and
anti-GD3 monoclonal antibody (shaded peaks: 67NR-Control;
unshaded peaks: 67NR-GM3S). CMP, cytidine 5'-monophosphate;
GM3S, CMP-N-acetylneuraminic acid:lactosylceramide 2,3-sialyltrans-
ferase.
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Metastasis is a rather complex process that occurs through a
series of steps that include invasion, intravasation, transport
through the circulatory system, arrest at a secondary site, and
the extravasation and growth in a secondary organ [30]. The
findings presented here suggest that GM3S expression is a
multistep modulator of breast cancer cell metastasis. First,
gain in motility and invasiveness is essential for most steps in
metastasis (the initial step, intravasation, and extravasation).
Our findings indicate that GM3S expression played important
roles in these steps by affecting ability to migrate/invade. In
addition, anchorage-independent growth is thought to be
among the fundamental properties of malignant cells [31,32].
In carcinomas, invasion, intravasation, and extravasation are
either deprived of matrix or exposed to foreign matrix
components. All of these events normally trigger apoptotic
processes [30]. In the present study we demonstrated that
GM3S significantly enhanced anchorage-independent growth
of breast tumor cells. Based on these findings, we hypothe-
sized that GM3S expression could affect almost all steps in
breast cancer metastasis.
Some other studies of the roles played by gangliosides in can-
cer progression support our hypothesis. GD3 and GD2 gan-
gliosides are upregulated in small cell lung cancer cells, and
these gangliosides were able to enhance proliferation and
invasion of small cell lung cancer [27]. In melanoma cells, GD3
has also been demonstrated to promote proliferation and inva-
sion [3]. In many types of tumor cells (for example, erythro-
leukemia, lymphoma, neuroblastoma, melanoma, glioblastoma,
and renal cell carcinoma cells), gangliosides may be shed into
the tumor cell microenvironment and there inhibit antitumor
immune responses [33-36]. It has also been demonstrated
that gangliosides shed from tumor cells can induce cell
migration and in vivo angiogenesis [37-40]. Ganglioside lev-
els in sera from breast cancer patients were also significantly
higher than in sera from healthy individuals [17]; this implies
Figure 3
Analysis of cell proliferation and anchorage-independent growthAnalysis of cell proliferation and anchorage-independent growth. Proliferation of (a) GM3S-silenced 4T1 cells and (b) GM3S over-expressing 67NR
cells was analyzed by MTT assay. Anchorage-independent growth of (c) GM3S-silenced 4T1 cells (5 × 103 cells/well) and (d) GM3S over-express-
ing 67NR cells were analyzed by soft agar assays (1 × 104 cells per well). *P < 0.05, **P < 0.01. CMP, cytidine 5'-monophosphate; GM3S, CMP-
N-acetylneuraminic acid:lactosylceramide 2,3-sialyltransferase.
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that shed breast cancer gangliosides play a role in accelerat-
ing tumor progression.
Although gangliosides affect cell migration and invasion in
some types of tumor cells, only a few investigations have been
conducted that focused on the molecular mechanisms
involved. Hamamura and coworkers [3] demonstrated that
GD3 promoted melanoma cell invasion through activating
p130Cas and paxillin; however, invasion of the human kerati-
nocyte-derived SCC12 cell line was suppressed by GM3
ganglioside via inhibition of matrix metalloproteinase-9 activa-
tion, disrupting its association with integrin [41]. Some gan-
Figure 4
Migration and invasion assaysMigration and invasion assays. The relative (a) migration and (b) invasion assays for GM3S-silenced 4T1 cells. (c) The relative migration assays for
GM3S over-expressing 67NR cells. **P < 0.01. CMP, cytidine 5'-monophosphate; GM3S, CMP-N-acetylneuraminic acid:lactosylceramide 2,3-sia-
lyltransferase.
Figure 5
Tumor formation and lung metastases 30 days after tumor implantationTumor formation and lung metastases 30 days after tumor implantation. (a) Primary tumor weights. (b) The average numbers of lung metastatic nod-
ules. (c) Representative photos of the lungs. The arrows point to the metastatic nodules in lung. (d) Representative hematoxylin and eosin staining
sections of the lungs were photographed at 40× magnification. *P < 0.05. M, metastatic nodule; N, normal.
Breast Cancer Research Vol 10 No 1 Gu et al.
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Figure 6
Involvement of the PI3K/Akt in suppression of breast cancer migration/invasionInvolvement of the PI3K/Akt in suppression of breast cancer migration/invasion. Activation of the PI3K/Akt pathway is the causative mechanism for
the suppression of breast cancer migration/invasion induced by GM3S silencing. (a) Upper panels: the expression and activation of Akt detected by
immunoblotting with anti-Akt, anti-p-Akt Ser (473), and anti-p-Akt Thr (308) antibodies. Lower panels: quantitation of these blots after normalization
with the GAPDH blot. (b) Upper panels: PI3K inhibitor LY294002 restored the phosphorylation of Akt in 4T1-siGM3S2 cells. Lower panels: the
quantitation of the blots after normalization with the GAPDH blot. (c) The migration and invasion abilities of GM3S-silenced 4T1 cells were
enhanced by LY294002. *P < 0.05, **P < 0.01. CMP, cytidine 5'-monophosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM3S,
CMP-N-acetylneuraminic acid:lactosylceramide 2,3-sialyltransferase; PI3K, phosphoinositide-3 kinase.
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gliosides, such as GM3 and GM2, inhibited cell motility
facilitated by the tetraspanins CD9 and CD82 in some tumor
cell lines (for example, colorectal, haptotactic, and bladder
cancer cells) [42-45].
In this study we found that a novel mechanism (silencing of
GM3S) inhibited migration and invasion of breast cancer cells
through activation of the PI3K/Akt pathway. It is generally
accepted that the PI3K/Akt axis promotes tumorigenesis by
enhancing the survival capacity of cancer cells [46]. However,
some recently published evidence indicates that Akt can
inhibit breast cancer cell migration and invasion [47]. Previous
reports have well demonstrated that GM3 gangliosides can
inhibit Akt signaling, and this inhibition occurs mainly through
three processes. First, many studies have demonstrated that
GM3 gangliosides inhibit phosphorylation of the epidermal
growth factor receptor and result in inhibition of PI3K/Akt sig-
naling in varied cell types [48-51]. Second, GM3 and some
other gangliosides interact with integrin and consequently
inhibit the integrin/integrin-linked kinase/Akt signaling pathway
[52-54]. Finally, GM3 treatment markedly increases PTEN
expression, which results in inhibition of Akt signaling in colon
cancer cells [55]. Here, we demonstrated that GM3S silenc-
ing markedly suppressed PTEN expression and subsequently
activated the PI3K/Akt pathway in 4T1 cells. Furthermore, the
PI3K specific inhibitor LY294002 partially restored the cell
migration/invasion ability inhibited by GM3S knockdown in
4T1 cells. This finding clearly indicates that GM3S-associated
breast cancer migration and invasion occurred, at least partly,
through the PI3K/Akt pathway.
Previous studies revealed that the NFAT transcription factor
promoted invasion of breast cancer cells [56,57]. In this report
we demonstrated that GM3S silencing suppressed NFAT1
expression and that inhibition of the PI3K/Akt pathway signifi-
cantly enhanced expression of NFAT1. These findings sug-
gest that inhibition of NFAT1 expression via the activation of
PI3K/Akt signaling plays a causal role in the suppression of
migration and invasion that is induced by GM3S knockdown
in murine breast cancer cells.
Conclusion
This study demonstrates that GM3S silencing can suppress
cell migration, invasion, anchorage-independent growth, and
lung metastasis in murine breast cancer cells. The molecular
mechanism underlying the GM3S-mediated migration and
invasion was found to be inhibition of the PI3K/Akt pathway.
To our knowledge, this report is the first to elucidate the
involvement of GM3S and mechanisms by which it influences
the malignant properties of breast cancer cells.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YG and JZ participated in designing the study, conducted cell
line transfection, immunoblotting analysis and animal experi-
ments, and drafted the manuscript. WM conducted the flow
cytometry analysis and immunoblotting analysis. JY partici-
pated in vector construction. FH and XL participated in the
Figure 7
Expression of PTEN was inhibited by GM3S silencingExpression of PTEN was inhibited by GM3S silencing. (a) Relative
PTEN expression in GM3S-knockdown 4T1 cells analyzed by real-time
RT-PCR. (b) Upper panels: expression and activation of PTEN
detected by immunoblotting. Lower panels: quantitation of these blots
after normalization with the GAPDH blot. *P < 0.05, **P < 0.01. CMP,
cytidine 5'-monophosphate; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; GM3S, CMP-N-acetylneuraminic acid:lactosylcera-
mide 2,3-sialyltransferase; p-PTEN, phosphor-PTEN Ser380/Thr382/
383; PTEN, phosphatase and tensin homolog.
Breast Cancer Research Vol 10 No 1 Gu et al.
Page 10 of 12
(page number not for citation purposes)
Figure 8
Expression of NFAT1 was inhibited through the PI3K/Akt pathway in GM3S-knockdown 4T1 cellsExpression of NFAT1 was inhibited through the PI3K/Akt pathway in GM3S-knockdown 4T1 cells. (a) Upper panels: expression of NFAT1 was
detected by immunoblotting. Lower panels: quantitation of these blots after normalization with the GAPDH blot. (b) Upper panels: expression of
NFAT1 was enhanced by the PI3K inhibitor LY294002. Lower panels: quantitation of these blots after normalization with the GAPDH blot. *P <
0.05, **P < 0.01. CMP, cytidine 5'-monophosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM3S, CMP-N-acetylneuraminic
acid:lactosylceramide 2,3-sialyltransferase; NFAT, nuclear factor of activated T cell; PI3K, phosphoinositide-3 kinase.
Figure 9
Effects of GM3S on Akt-associated cell migration/invasion in breast cancer cellsEffects of GM3S on Akt-associated cell migration/invasion in breast
cancer cells. This schematic diagram shows that GM3S silencing
reduced expression of gangliosides and activated Akt signaling via inhi-
bition of PTEN expression (the major negative regulator of the PI3K/Akt
signaling). It also shows that activation of Akt signaling inhibits cell
migration and invasion, probably via downregulation of NFAT1. CMP,
cytidine 5'-monophosphate; GM3S, CMP-N-acetylneuraminic acid:lac-
tosylceramide 2,3-sialyltransferase; PI3K, phosphoinositide-3 kinase;
PTEN, phosphatase and tensin homolog.
Figure 10
Effects of PI3K/Akt signal on the anchorage-independent growth of breast cancer cellsEffects of PI3K/Akt signal on the anchorage-independent growth of
breast cancer cells. The anchorage-independent growth of both
GM3S-knockdown 4T1 cells and the mock transfected 4T1 cells (5 ×
103 cells/well) were not affected by the inhibition of the PI3K/AKT path-
way. CMP, cytidine 5'-monophosphate; GM3S, CMP-N-acetyl-
neuraminic acid:lactosylceramide 2,3-sialyltransferase; PI3K,
phosphoinositide-3 kinase.
Available online http://breast-cancer-research.com/content/10/1/R1
Page 11 of 12
(page number not for citation purposes)
design of the study and conducted the statistical analysis. WY
conceived of the study, participated in its design and coordi-
nation, and helped to draft the manuscript. All authors read
and approved the final manuscript.
Acknowledgements
We thank Fred R Miller for providing the murine breast tumor cell lines,
Meiyu Geng and Jing Li for technical assistance, and Guanpin Yang for
critical review of the manuscript. This work was supported by the
National Basic Research Program of China (973 Program;
2003CB716402).
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