Snail Contributes to the Maintenance of Stem Cell-Like Phenotype Cells in Human Pancreatic Cancer
et al. (2014) Snail Contributes to the Maintenance of Stem Cell-Like Phenotype Cells in Human Pancreatic Cancer. PLoS
ONE 9(1): e87409. doi:10.1371/journal.pone.0087409
Snail Contributes to the Maintenance of Stem Cell-Like Phenotype Cells in Human Pancreatic Cancer
Wei Zhou 0
Ran Lv 0
Weilin Qi 0
Di Wu 0
Yunyun Xu 0
Wei Liu 0
Yiping Mou 0
Liewei Wang 0
Michael Klymkowsky, University of Colorado, Boulder, United States of America
0 1 Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University , Hangzhou , P. R. China , 2 Departments of Anesthesiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University , Hangzhou , P. R. China , 3 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic , Rochester, Minnesota , United States of America
Snail, a potent repressor of E-cadherin expression, plays a key role in epithelial-to-mesenchymal transition (EMT) in epithelial cancer. Recently, EMT and stemness programs are found linked together. In the current study, the expression of Snail and its contribution to cancer stem cell (CSC) marker expression, invasiveness, self-renewal, clonogenicity, and tumorigenicity of pancreatic cancer cells were studied. Our results showed that Snail was highly expressed in CSChigh cell line Panc-1. Stable, short hairpin RNA (shRNA)-mediated Snail knockdown decreased invasion in Panc-1 cells, in line with increased E-cadherin expression and its translocation from the nucleus to the membrane. Snail silencing in Panc-1 also inhibited CSC marker ALDH expression, together with decreased sphere and colony forming capacity, which was highly consistent with the expression of stem cell associated transcription factors like Sox2 and Oct4. In mouse xenograft models, knockdown of Snail led to a reduced number of tumor-bearing mice and a reduced average size of tumors, which had a stronger membrane staining of E-cadherin and lighter staining of Oct4. Collectively, these findings implicate Snail is required for the maintenance of stem cell-like phenotype in pancreatic cancer, and inhibition of Snail could be an efficient strategy to treat pancreatic cancer by targeting CSCs.
Funding: This work was supported by the National Natural Science Foundation of China . The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Pancreatic ductal adenocarcinoma is a highly aggressive
epithelial cancer with a reported 5-year survival rate of
approximately 5%. Only 20% of pancreatic cancer patients
are eligible for surgical resection, and metastatic disease frequently
develops even after surgery, while current chemo- and
radiotherapies are largely ineffective. Therefore, Understanding the
molecular events underlying the development and progression of
pancreatic cancer is urgently needed, which may hold the key to
the development of more efficacious and novel therapeutic
An increasing amount of scientific evidence indicates that
tumors contain a small subpopulation of cells, i.e., cancer stem-like
cells (CSCs) or cancer-initiating cells (CICs), which exhibit a
selfrenewing capacity, resistant to conventional chemotherapy and
are responsible for therapy failure, cancer relapse and metastasis
. Although the CSCs hypothesis suggests that tumors can arise
from stem or progenitor cells, studies from some laboratories
indicate that epithelial-mesenchymal transition (EMT), a
developmental process in which cells lose epithelial characteristics and
acquire mesenchymal properties such as increased motility and
invasion, can endow cells with stem-cell like characteristics.
EMT is induced by repression of E-cadherin expression by
EMT regulators such as Snail, Slug, and Twist. The Snail family
of zinc-finger transcriptional repressors directly represses
Ecadherin in vitro and in vivo via an interaction between their
COOH-terminal region and the 59-CACCTG-39 sequence in the
E-cadherin promoter . In human colorectal cancer cells,
overexpression of Snail was reported to induce not only EMT
but also a CSC-like phenotype, which enhanced cell migration
and invasion in vitro and an increase in metastasis formation in
vivo. Studies have also shown that Snail plays an essential role
in the progression and metastatic process of human pancreatic
cancer[9,10]. In clinical setting, Snail overexpression has
previously been associated with poorer prognosis and a more invasive
phenotype in many malignancies. However, few reports
exist regarding the link between Snail expression and the gain of
pancreatic cancer stem cell properties. We therefore evaluated the
Snails function on stem cell marker expression, self-renewal
capacity in pancreatic cancer cell line in vitro and xenograft
tumors formation in vivo. Our work reveals that gene regulation
mediated by Snail may support human pancreatic cancer growth
by maintaining the pancreatic cancer stem cell compartment.
Materials and Methods
The human pancreatic cancer cell lines Panc-1 and BxPC-3
were obtained from the American Type Culture Collection
(Manassas, VA). Cells were cultured and maintained in DMEM
medium supplemented with 10% fetal bovine serum (Gibco/
Invitrogen, CA), penicillin-streptomycin (Flow Laboratories,
Rockville, MD). Both cell lines were maintained in a humidified
atmosphere at 37uC with 5% CO2. Gross cell morphology for the
presence or absence of morphologic characteristics consistent with
EMT was assessed by two observers blinded to the treatment
conditions. Images of cell lines were taken using a Nikon Eclipse
TS100 inverted microscope and Pro-MicroScan camera (Oplenic).
Evaluation of aldehyde dehydrogenase activity
Aldefluor substrate (2.5 ml, Aldagen, Inc., Durham, NC) was
added to 16106 tumor cells in 500 ml assay buffer and incubated
for 60 min at 37uC. Cells were analyzed on a FACSCalibur flow
cytometer (Becton Dickinson) according to the instructions of the
manufacturer. Treatment of cells with 5 ml of the ALDH inhibitor
diethylamino-benzaldehyde (DEAB) served as negative control.
Lentiviral vectors without fluorescence were used for cell
transfection during FACS Analysis.
Sphere formation assay
Sphere formation assay was performed as described
elsewhere. In brief, cells were plated in six-well ultralow
attachment plates (Corning Inc., Corning, NY) at a density of
1,000 cells/ml in DMEM supplemented with 1% N2 supplement
(Gibco, Grand land, NY), 2% B27 supplement (Gibco, Grand
island, NY), 20 ng/ml human platelet growth factor
(SigmaAldrich), 100 ng/ml epidermal growth factor (PeproTech, Rocky
Hill, NJ) and 1% antibiotic-antimycotic (Invitrogen) at 37uC in a
humidified atmosphere of 95% air and 5% CO2. Sphere cultures
were passaged after 7,10 days. To passage spheres, media was
removed and spheres were collected and incubated at room
temperature for 5 min in 0.05% trypsin (Solarbio, Beijing, China)
and observed under the microscope to verify dissociation. The
cells obtained from dissociation were sieved through a 40-mm filter
and counted by counter using trypan blue dye before replating.
Soft agar assay
To assess clonogenic potential, the colony formation assay was
performed as follows. Each well of a six-well culture dish was
coated with 2 ml bottom agar mixture (DMEM, 10% (v/v) FCS,
0.6% (w/v) agar). After the bottom layer solidified, 2 ml top agar
medium mixture (DMEM, 10% (v/v) FCS, 0.3% (w/v) agar)
containing 16104 cells was added, and the dishes were incubated
at 37uC for 3 weeks. Plates were stained with 0.5 ml of 0.005%
crystal violet for 1 h and then a dissecting microscope was used to
count the number of colonies .50 cells .
Transwell invasion assay
For invasion assay, the 24-well plate Transwell system with an
8-mm pore size polycarbonate filter membrane (Corning Costar,
Corning, NY) was used. 16105 cells in 100 ml serum-free medium
were added to the top chamber coated with Matrigel (BD
Bioscience, Bedford, MA). The lower chamber contained 10%
FBS containing medium. The cells were incubated for 48 h and
cells that had invaded through the Matrigel-coated membrane
were fixed and stained with crystal violet, then counted under a
light microscope in four random fields in a blinded fashion.
Real-time RT-PCR analysis for gene expression
For real-time RT-PCR analysis, the total RNA of cells was
extracted by using the Trizol kit (Invitrogen, Carlsbad, CA).
cDNA was synthesized using equivalent amounts of total RNA
(1 mg) with random primers in a 20 ml reverse transcriptase
reaction mixture (Promega, Madison, WI). Real-time PCR
primers were designed and purchased from Ruisai Inc (Shanghai,
China) as follows:
Snail, forward (59-GCTGCAGGACTCTAATCCAGA-39)
and reverse (59-ATCTCCGGAGGTGGGATG-39);
Slug, forward (59-AGCGAACTGGACACACATAC-39)
and reverse (59-TCTAGACTGGGCATCGCAG-39);
Twist 1, forward (59-CACTGAAAGGAAAGGCATCA-39)
and reverse (59-GGCCAGTTTGATCCCAGTAT-39);
and reverse (59-ACCCAGACTGCGTCACATGTCTT-39);
ZEB2, forward (59-GAGTTGATGCCTCGGCTATTGC-39)
and reverse (59- CTGGACATTGAGCTGCTTCGATC-39);
(59-GGTGTGAACCATGAGAAGTATGand reverse (59- GATGGCATGGACTGTGGTCAT-39).
The amplification was carried out in a total volume of 20 ul
containing 1 ul of each primer, 10 ul LightCycler FastStart DNA
Master SYBR green I (Roche Diagnostics, Pleasanton, CA) and
1 ul of 1:10 diluted cDNA. PCR reactions were prepared in
duplicate and heated to 95uC for 2 min followed by 40 cycles of
denaturation at 95uC for 15 sec, annealing at 55uC for 20 sec, and
extension at 72uC for 20 sec. All assays were performed in
triplicate and were calculated on the basis of DDCt method. The
nfold change in mRNAs expression was determined according to
the method of 2DDCT.
Lentiviral-mediated RNAi of Snail
The pcDNA6.2-GW/EmGFP-miR vector was purchased from
Invitrogen (Carlsbad, CA). Double-stranded shRNAs targeting
human Snail were designed by BLOCK-i TTM RNAi Designer.
The targeted sequence 1 was
59-GCCTAACTACAGCGAGCTG-39; targeted sequence 2 was
59-GGATCTCCAGGCTCGAAAG-39. Both targeted sequences were verified as specific for
Snail by Blast searching against the human genome. Universal
non-targeting control shRNA (sequences:
59-CCTGAAATGTACTGCGTGGAGACGTCAGTCAGTGGCCAAAACGTCTCCACGCGCAGTACATTTC-39) was used as negative control.
shRNAs were synthesized and cloned into pcDNA6.2-GW/
EmGFP-miR, then transferred into the lentiviral expression
plasmid pLenti6/V5-DEST with the Gateway recombination
technology. Lentiviral production was done by transfection of
293 T cells with shRNA or negative control plasmid and
packaging Mix (Invitrogen) in the presence of POLOdelivererTM
3000 Transfection Reagent (Ruisai Inc, shanghai, China).
Supernatants were collected 48 h after transfection and then were
filtered; the viral titers were then determined by real-time PCR.
Subconfluent cells were infected with lentivirus at a multiplicity of
infection of 50 in the presence of 8 mg/ml polybrene
(SigmaAldrich). Panc-1 cells with stable silencing of Snail gene were
selected with 2 ug/ml of Blasticidin for 4 weeks. Then cells were
cultured with 1 ug/ml Blasticidin. Another negative control
shRNA plasmid (sc-108060) from Santa Cruz Biotechnology,
which encodes a scrambled shRNA sequence, was used for
transient transfection according to the protocol.
Western blot analysis
For whole cell protein extraction, cells were lysed with ice-cold
RIPA buffer containing 1 mM PMSF and centrifuged at 14,000 g
for 5 min. Supernatant containing the isolated protein was
quantified by a commercially available modified Bradford assay
(Bio-Rad Laboratories, Hercules, CA). Western blot protein
samples were prepared by boiling isolated proteins with
denaturing sample buffer. Equal amounts of protein were resolved by
SDS-PAGE and transferred to nitrocellulose membranes. The
membranes were then blocked with 5% nonfat dry milk in TBS
and 0.1% Tween 20 for 1 h and probed with the appropriate
primary antibody overnight at 4uC. The membranes were then
washed and incubated with the appropriate horseradish
peroxidaseconjugated secondary antibody (Sigma Aldrich, St Louis,
MO) for 1 h at room temperature. Membranes were then washed
and protein bands were visualized by using a commercially
available enhanced chemiluminescence (ECL) kit (Thermo
Scientific). To verify the accuracy of loading of protein isolated from
whole-cell lysate, the blots were stripped, washed and reprobed
with GAPDH antibody (Bioworld) as a loading control. Images
were visualized using the ECL Detection System (Amersham,
Arlington Heights, IL). Antibodies used for Western blot analyses
were as follows: rabbit mAb anti-Snail, rabbit mAb
anti-ECadherin, rabbit mAb anti-vimentin, rabbit mAb anti-Bmi1,
rabbit mAb anti-Nanog, rabbit mAb anti-Oct4, rabbit mAb
antiSox2 (Cell Signaling Technology, Inc.). Densitometry of Western
blots was analyzed by using Image J software.
Cells were grown on glass coverslips, fixed in 4% formaldehyde
in PBS for 10 min, permeabilized with 0.2% Triton X-100 for
10 min, and blocked in 10% goat serum in PBS-0.2% Tween for
1 h. Coverslips were incubated with E-cadherin antibody (BD
Biosciences, 1:200 dilution) in blocking solution for 1 h at room
temperature. After washing the cells with PBS-0.2% Tween, we
incubated the cells for 1 h with goat anti-rabbit IgG Alexa 594
(1:1000 dilution; Invitrogen). Nuclei were counterstained with
DAPI. The slides were washed extensively with PBS and mounted
with Fluoromount-G. Samples were photographed using
In vivo analysis of tumor growth
All procedures involving animals were approved by the Animal
Care and Use Committee of Zhejiang University School of
Medicine (ZYXK2010-0149). Single cells suspensions (26105 in a
total volume of 100 mL of 1/1 (v/v) PBS/Matrigel) were injected
subcutaneously into the right and left midabdominal area of male
nude mice (BALB/c strain) aged 8 weeks. The tumor size
monitored daily with calipers and the tumor volume was
calculated according to the formula (Length6Width2)/2. Animals
underwent autopsy at 28 days after cell implantation and tumor
growth was assessed.
Immunohistochemistry analysis of xenograft tissue
The subcutaneous tumors formed in mice were fixed in 10%
phosphate-buffered formalin and embedded in paraffin. 4-mm
thick sections were deparaffinized using xylene, and hydrated by a
graded series of ethanol washes. Endogenous peroxidase activity
was quenched by 10-min incubation in 3% H2O2. After
incubation with blocking solution for 30 min, sections were
incubated with primary antibody from BD Biosciences (anti-Snail,
1:200 dilution; anti-E-cadherin, 1:200 dilution, and anti-Oct4,
1:300 dilution)for 1 h, a biotinylated secondary antibody for
20 min and then with streptavidin horseradish peroxidase (HRP)
for 10 min. The antibody was visualized with diaminobenzidine
(DAB) chromogen, and sections were counterstained with H&E.
The experiments were repeated at least two times. Results are
expressed as mean6SD. Statistically significant differences were
determined by Students t test for independent samples when
appropriate using SPSS statistical analysis software (version 13.0)
(SPSS, Inc., Chicago, IL). Significant differences among groups
were calculated at P,0.05.
Snail expression and stem cell marker ALDH in pancreatic
Under the microscope, BxPC-3 cells were morphologically
epithelial in nature. In contrast, Panc-1 cells were mixed
populations of epithelial and spindle-shaped mesenchymal type
cells (Figure 1 A). Using flow cytometry, poorly-differentiated cell
line Panc-1 was characterized as CSChigh cell with more
ALDHhigh population, while well-differentiated cell line BxPC-3
as CSClow cell with less ALDHhigh population (Figure 1 B). Sphere
formation assay also revealed that Panc-1 formed more and larger
spheres than BxPC-3 did (Figure 1 A). To gain insight into the
crucial role of the EMT regulators, we examined the basal
expression of Snail, Slug, Twist1, ZEB1 and ZEB2 in Panc-1 and
BxPC-3. Notably, real-time RT-PCR analyses showed that Panc-1
cells had a significantly higher expression of Snail and ZEB1
mRNA (approximately 6-fold and 4-fold respectively, P,0.01) in
comparison to BxPC-3 cells (Figure 1 C). There was a correlation
between poor differentiation, Snail and ZEB1 expression levels
and sphere-forming capacity in these two pancreatic cancer cell
lines. We chose the Panc-1 cell line for later Snail silencing
experiments due to its relatively high expression level of Snail and
excellent lentiviral transfection efficiency.
Snail induces EMT-like changes in pancreatic cancer cell
To examine the role of Snail in EMT induction and CSC
generation, we produced a stably Snail knockdown Panc-1 cell line
(Panc-1/shSnail) by lentiviral transfection.
Two independent stable shSnail-expressing clones (S1, S2) and
a stable negative control shRNA clone (NC) were analyzed for
Snail mRNA expression. S1 and S2 showed up to 80%
downregulation of Snail mRNA levels, whereas control clone did
not exhibit any significant reduction in Snail mRNA (Figure 2A).
We chose S1 clone as Panc-1/shSnail for following experiment
because of its higher extent of Snail silencing. Knockdown of Snail
in Panc-1 cells was confirmed by Western blot analysis (Figure 2B
and C). Rabbit mAb anti-Snail was validated against cell lysates
from Panc-1, MIA-PaCa2, BxPC-3, and Capan-1 cell lines. The
staining pattern was similar to that of previously published data
(Figure S1) [9,16]. To rule out heterogeneity caused by stable
transfection and selection protocol, we used another negative
control shRNA plasmid (sc-108060) for transient transfection.
Real-time RT-PCR and Western blot revealed no significant
difference of Snail expression between stably and transiently
transfected control clones (Figure S2).
After lentivirus infection, blinded investigators observed
differences in the gross appearance of cells. Stable infection of Panc-1
cells by shSnail significantly increased intercellular adhesion and
cobble-stone-like shape, as compared with control cells (Figure 3A).
Next, we evaluated the expression and localization of E-cadherin
using immunofluorescence staining. Compared with Panc-1/NC
cells, Panc-1/shSnail cells had increased levels of expression of
Ecadherin and relocation of E-cadherin from the nuclear
compartment to cell plasma membrane (Figure 3B). These changes were
typical of cells with an epithelial phenotype, indicating that the
cells were undergoing mesenchymal-to-epithelial transition (MET)
after Snail silencing. To further confirm the observation, we
assessed the expression of epithelial adhesion molecule E-cadherin,
mesenchymal cell marker vimentin and other EMT transcription
Figure 1. Differences of epithelial-mesenchymal features and CSC properties in Panc-1 and BxPC-3 cells. A. Morphology of Panc-1 and
BxPC-3 cells and their spheres. Note that Panc-1 cells have more spindle-shaped mesenchymal populations and can form more and larger spheres. **
P,0.01 compared with BxPC-3. B. ALDH activity in Panc-1 and BxPC-3 cells. Dot plots of cells analyzed by flow cytometry for ALDH activity. Cells were
treated with Aldefluor substrate in the presence or absence of ALDH inhibitor DEAB. After treatment, the samples were analyzed by flow cytometry
for the presence of ALDHhigh cells. The values presented are the averages of three independent experiments. C. Real-time RT-PCR quantifing Snail,
Slug, Twist1, ZEB1, and ZEB2 mRNA expression in Panc-1 and BxPC-3 cells. Bar graphs show the ratio of the expression level in Panc-1 cells to that in
BxPC-3 cells. ** P,0.01.
Figure 2. Changes of epithelial-mesenchymal makers after Snail silencing in Panc-1 cells. A. Snail mRNA expression of stable
shSnailexpressing (S1, S2) and negative control shRNA-expressing (NC) Panc-1 clones. Values are the averages and standard deviations of triplicate
measurements. **P,0.01 compared with NC. B. Western blot showing epithelial-mesenchymal markers Snail, Slug, Twist1, ZEB1, ZEB2, E-cadherin and
vimentin in Panc-1 cells transfected with lentivirus-mediated negative control shRNA (Panc-1/NC) or shSnail (Panc-1/shSnail). GAPDH was used as
loading control. C. Quantification of protein levels of Snail, Slug, Twist1, ZEB1, ZEB2, E-cadherin and vimentin in Panc-1/NC and Panc-1/shSnail cells. *
factors Slug, Twist1, ZEB1 and ZEB2 by Western blotting on cell
lysates. As expected, after silencing of Snail in Panc-1, expression
of the E-cadherin was observed to increase. Conversely, a decrease
in the expression of vimentin and ZEB1 was observed after Snail
knockdown. No significant differences were observed in protein
levels of Slug, Twist1 and ZEB2 (Figure 2 B, C). These results
show a clear relationship between Snail and E-cadherin, and
also suggest a direct or indirect interaction between Snail and
ZEB1, both of which hold the potential to repress E-cadherin
Snail increases cell invasion ability in pancreatic cancer
Since the processes of EMT have been linked with cell invasion,
we next asked if Snail expression has some effect on cell invasion
capacity in pancreatic cancer cells. Using the Matrigel in vitro
invasion assay, we found Panc-1 cells with Snail silencing had
significantly decreased capacity for invasion when compared to
control cells (Figure 3 C). These data confirm that Snail expression
enhances the invasive capacity of pancreatic cancer cells, and the
inactivation of Snail leads to MET with less invasive
Snail enhances stem-cell like properties in pancreatic
As Snail is highly expressed in CSChigh compared to CSClow
cells, we therefore examined whether Snail could affect the
expression of stem cell marker ALDH. Snail silenced Panc-1 cells
showed a significant decrease in the ALDHhigh population (1.60%)
in comparison to their control counterparts (6.01%) (Figure 4 A).
Next, we used in vitro sphere formation assay to examine whether
Snail participates in CSC renewal upon serial passaging. We
showed that Snail knockdown not only affected the initial
formation of spheres, but also led to an ongoing reduction of
sphere numbers in subsequent generations. Colony formation test
also showed that silencing of Snail significantly blocked colony
growth of Panc-1 cells (Figure 4 B). These experiments implicate
that Snail has an important role in the regulation of pancreatic
CSC content and is necessary for the self-renewing capacity.
Snail increases the expression of stem cell associated
As Snail expression has some relationship with CSC content, we
used Western blotting to determine if Snail expression has roles in
changing expression of Bmi1, Nanog, Sox2, and Oct4, which are
required for maintaining pluripotency in stem cells. As shown in
Figure 4 C and D, lentiviral mediated transfection of Snail shRNA
induced a dramatic reduction in the expression of Sox2 and Oct4
(P,0.05 and P,0.01, respectively) in Panc-1 cells, which was
consistent with the loss of CSC phenotype, suggesting that the
expression of these factors may be important to Snail-induced
CSC formation in pancreatic cancer cells.
Snail enhances pancreatic cancer cell tumorigenicity in
Since our in vitro studies suggested that Snail plays a regulatory
role in pancreatic cancer cell invasion and CSC formation, we
implanted subcutaneously equal numbers of Panc-1/NC and
Panc-1/shSnail in the nude mice and measured the resultant
tumor growth. When injected 26105 cells, Panc-1/NC had 100%
(8/8) tumor formation while only 2/8 mice injected with Panc-1/
shSnail showed pancreatic tumor engraftment. Moreover, a trend
to delayed tumor formation and a decrease in tumor sizes were
observed in tumors derived from Snail silencing cells compared to
those from control cells (Figure 5A). Immunohistochemistry of
tumors showed a lighter staining of Snail and Oct4, while a
stronger membrane staining of E-cadherin in Panc-1/shSnail
tumors, as compared with those of Panc-1/NC tumors (Figure 5B).
These data are in accord with our in vitro observations and
support the role of Snail in the maintenance of the pancreatic CSC
Our study demonstrates that Snail which is related to EMT of
human pancreatic cancer cells can also regulate expression of stem
cell markers and pluripotency maintaining transcription factors,
modulating self-renewal capacity and clonogenicity. Together with
the tumor implantation study, our results indicate that the
activation of Snail is required for the maintenance of cancer stem
cell-like traits, which directly impacts tumor initiation, growth in
vivo. We have convincingly demonstrated that inhibition of Snail
could be considered as a novel strategy to enhance the biological
effects of anticancer and chemopreventive agents.
Researchers usually identified CSCs from pancreatic cancer
based on the expression of the cell surface antigens like CD44,
CD24, epithelial-specific antigen (ESA), and CD133 .
There are different conclusions in separate studies as to which
marker best enriches for pancreatic CSCs. However, recent studies
have found that cell populations enriched for high ALDH activity
alone are sufficient for efficient tumor-initiation with enhanced
tumorigenic potential. In our experiment, knockdown of the
key EMT inducer Snail significantly decreased the ALDHhigh cell
population. These data indicate that EMT endows tumor cells
with stem cell-like properties. Our results are in accordance with
the research from Chen et al, which found ALDHhigh cells in head
and neck squamous cancer having EMT shifting and
endogenously co-expressed Snail. Furthermore, the knockdown of Snail
expression significantly decreased the expression of ALDH,
inhibited cancer stem-like properties. These observations were
further confirmed by sphere formation in vitro and tumor
implantation in vivo, where Snail knockdown led to less and
smaller engraftment. These findings are consistent with those of
Mani and colleagues who have showed that forced expression
of EMT-associated molecules such as Snail and Twist results in
cells with a cancer stem cell phenotype. EMT offers an alternative
way to generate cancer stem cell properties from differentiated
epithelial tumor cells. This hypothesis of dedifferentiation is
supported by the recently described generation of pluripotent stem
cells from seemingly terminally differentiated somatic cells.
It is generally considered that Sox2 and Oct4 are key
transcription regulators of embryonic stem cell (ESC) self-renewal
and pluripotency. Accumulating evidence indicates that
these transcription factors of ESCs are expressed by CSC-like cells
in different types of cancer. Knockdown of these genes could lead
to diminished CSC phenotype, reduce the clonogenic and
tumorigenic capabilities of cancer cells. . They are also
sufficient to reprogram human somatic cells to pluripotent stem
cells that exhibit the essential characteristics of ESCs. In the
present study, we found that the expression levels of Sox2 and
Oct4 were decreased in Snail-silencing Panc-1 cells compared with
the control cells. This suggests that Snail functions as a master
switch during regulating the pluripotent potentials of the stem cell.
Similar results are found in other researches, in which high
expression of Oct4 and Nanog promotes EMT, and is associated
with drug resistance, tumor metastasis and poor prognosis in
various human malignances.[30,31]. Our findings, together with
Figure 3. Changes of epithelial-mesenchymal features after Snail silencing in Panc-1 cells. A. Morphological alterations of Panc-1 cells
after Snail silencing indicate a change in the cellular growth pattern from a mesenchymal towards an epithelial phenotype. B. Immunofluorescence
staining for the expression and cellular localization of E-cadherin in stable clones of Panc-1/NC and Panc-1/shSnail. Nuclear DNA was detected by
DAPI staining. Stable Snail knockdown leads to an increased expression of E-cadherin and its translocation from the nucleus to the membrane. C.
Snail silencing inhibits Panc-1 invasiveness in in vitro Matrigel invasion assays. **P,0.01 compared with Panc-1/NC. Data shown here are the mean 6
SD of three experiments.
the previous studies strongly indicates a molecular link or
crosstalk between pluripotency factors and EMT regulatory factors,
and thus the activation of these signaling pathways appears to be
mechanistically associated with the acquisition of EMT and CSC
phenotype of pancreatic cancer cells.
EMT is an embryonic program in which epithelial cells lose
their characteristics and gain mesenchymal features. Accumulating
evidence suggests that EMT plays an important role during
malignant tumor metastasis. It is believed that sustained metastatic
growth requires the dissemination of a CSC from the primary
tumor followed by its reestablishment in a secondary site. Rhim
silencing significantly inhibits the ability of sphere formation with serial passaging and the capability of the clonogenicity in Panc-1 cells.
Representative pictures of colony are shown above the column diagram. Similar experiments were repeated three times. ** P,0.01, compared with
Panc-1/NC. C. Western blot analysis of cell extracts from Panc-1/NC and Panc-1/shSnail cells for Bmi1, Nanog, Sox2, and Oct4 expression. GAPDH was
used as a loading control. D. Quantification of protein levels of Bmi1, Nanog, Sox2, and Oct4 in Panc-1/NC and Panc-1/shSnail cells. * P,0.05, **P,
0.01 compared with Panc-1/NC.
Figure 5. Validation of the role of Snail in tumorigenicity in mouse. A. Graphical representation of growth rates of subcutaneous xenograft
tumors using cells of Panc-1/NC and Panc-1/shSnail. 26105 of each cell was transplanted in eight mice per group. All mice in the Panc-1/NC group,
while only 2 mice in the Panc-1/shSnail group had tumor formation. B. Expression of Snail, E-cadherin, and Oct4 in xenograft tumors. Representative
examples of Snail, E-cadherin, and Oct4 expression determined by immunohistochemistry. Strongly positive Snail expression is present in the nucleus
and cytoplasm of Panc-1/NC tumors (a) but not in Panc-1/shSnail tumors (b). Expression of E-cadherin is weak and heterogeneous in the Panc-1/NC
tumors (c), but more intensive in the membrane of Panc-1/shSnail tumors (d). Lower level of Oct4 expression is present in the Panc-1/shSnail tumors
(f), as compared to that of Panc-1/NC tumors (e). Scale bars: 10 mm.
and colleagues have found that EMT causes epithelially-derived
cells to migrate into bloodstream, and seed in liver even before
pancreatic tumor formation at the primary site. It is now
accepted that aberrant activation of an EMT-stemness program,
which are characteristics of cancer cells at the invasive edge of
tumor, separates a few of the tumor cells from the primary lesion
and exhibits stem cell properties, enables migrating cancer stem
cells (MCSCs) to enter blood vessels. The MCSCs are not easily
eliminated by conventional therapies due to their drug resistance
and quiescence state. Therefore they can seed into distant organs,
form micrometastases and finally colonize to macrometastases[33
35]. This process is associated with the activation of genes like
Snail, which is triggered by environmental factors such as
inflammation and hypoxia. Interestingly, Zhang et al
demonstrated that hypoxia-stabilized HIF1a promoted EMT through
increasing Snail transcription in hepatocellular carcinoma
cells. TNFa, a key regulator of the inflammatory response,
can also increase the expression of transcription factor Snail and
induce EMT in colorectal cancer cells.
The highly conserved EMT is frequently associated with the
downregulation of E-cadherin and upregulation of vimentin and
several transcription factors including Snail, ZEB1 and Slug.
Recent researches have identified a link between p53, microRNA
miR-34, and Snail in the regulation of cancer cell EMT programs.
In the absence of wild-type p53 function, Snail-dependent EMT is
activated in cancer cells as a consequence of a decrease in miR-34
levels. Conversely, the transcription factors Snail bound to
Eboxes in the miR-34 promoters, thereby repressing miR-34
expression. So, miR-34 and Snail form a double-negative
feedback loop to regulate epithelial-mesenchymal transitions.
Further studies are needed to clarify whether this feedback loop
functions in the EMT-stemness program. Expression of
pri-miR200c is also repressed by Snail. The inhibition of ZEB1 by shSnail
in our study may be due to the increased expression of miR-200c,
since ZEB1 is a known target of miR-200 c .
Based on the current study, Snail is thought to be a critical
component of the machinery that maintains CSC compartment.
Considering that Snail is just one of EMT regulators, the different
contributions of molecular machinery, including the Slug, ZEB1
signaling pathways, might also influence the CSC phenotype.
Further analyses would be necessary to clarify the mechanisms
underlying the regulation of CSC in pancreatic cancer. Thus, the
discovery of molecular knowledge related to CSC characteristics
and EMT in pancreatic cancer is becoming an important area of
research, and such knowledge is likely to be helpful in the
discovery of novel molecular targets and strategies for the
prevention of pancreatic cancer by targeting CSCs.
Figure S2 Snail mRNA and protein expression in Panc-1
cells after stable and transient negative control shRNA
transfection. A. Panc-1 cells were transfected by
lentivirusmediated negative control shRNA (NC) or plasmid-mediated
negative control shRNA (NC-p). Snail mRNA expression was
evaluated by Real-time RT-PCR. B. Protein levels of Snail in
stable and transient negative control shRNA-expressing Panc-1
Conceived and designed the experiments: WZ RL YM. Performed the
experiments: WZ WQ DW YX WL. Analyzed the data: WZ RL.
Contributed reagents/materials/analysis tools: WQ DW LW. Wrote the
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