EGF-induced EMT and invasiveness in serous borderline ovarian tumor cells: a possible step in the transition to low-grade serous carcinoma cells?
ABSTRACT In high-grade ovarian cancer cultures, it has been shown that epidermal growth factor (EGF) induces cell invasion by activating an epithelial-mesenchymal transition (EMT). However, the effect of EGF on serous borderline ovarian tumors (SBOT) and low-grade serous carcinomas (LGC) cell invasion remains unknown. Here, we show that EGF receptor (EGFR) was expressed, that EGF treatment increased cell migration and invasion in two cultured SBOT cell lines, SBOT3.1 and SV40 large T antigen-infected SBOT cells (SBOT4-LT), and in two cultured LGC cell lines, MPSC1 and SV40 LT/ST-immortalized LGC cells (ILGC). However, EGF induced down-regulation of E-cadherin and concurrent up-regulation of N-cadherin in SBOT cells but not in LGC cells. In SBOT cells, the expression of the transcriptional repressors of E-cadherin, Snail, Slug and ZEB1 were increased by EGF treatment. Treatment with EGF led to the activation of the downstream ERK1/2 and PI3K/Akt. The MEK1 inhibitor PD98059 diminished the EGF-induced cadherin switch and the up-regulation of Snail, Slug and ZEB1 and the EGF-mediated increase in SBOT cell migration and invasion. The PI3K inhibitor LY294002 had similar effects, but it could not block the EGF-induced up-regulation of N-cadherin and ZEB1. This study demonstrates that EGF induces SBOT cell migration and invasion by activating EMT, which involves the activation of the ERK1/2 and PI3K/Akt pathways and, subsequently, Snail, Slug and ZEB1 expression. Moreover, our results suggest that there are EMT-independent mechanisms that mediate the EGF-induced LGC cell migration and invasion.
- SourceAvailable from: Umut A Gurkan[show abstract] [hide abstract]
ABSTRACT: Seventy-five percent of patients with epithelial ovarian cancer present with advanced-stage disease that is extensively disseminated intraperitoneally and prognosticates the poorest outcomes. Primarily metastatic within the abdominal cavity, ovarian carcinomas initially spread to adjacent organs by direct extension and then disseminate via the transcoelomic route to distant sites. Natural fluidic streams of malignant ascites triggered by physiological factors, including gravity and negative subdiaphragmatic pressure, carry metastatic cells throughout the peritoneum. We investigated the role of fluidic forces as modulators of metastatic cancer biology in a customizable microfluidic platform using 3D ovarian cancer nodules. Changes in the morphological, genetic, and protein profiles of biomarkers associated with aggressive disease were evaluated in the 3D cultures grown under controlled and continuous laminar flow. A modulation of biomarker expression and tumor morphology consistent with increased epithelial-mesenchymal transition, a critical step in metastatic progression and an indicator of aggressive disease, is observed because of hydrodynamic forces. The increase in epithelial-mesenchymal transition is driven in part by a posttranslational up-regulation of epidermal growth factor receptor (EGFR) expression and activation, which is associated with the worst prognosis in ovarian cancer. A flow-induced, transcriptionally regulated decrease in E-cadherin protein expression and a simultaneous increase in vimentin is observed, indicating increased metastatic potential. These findings demonstrate that fluidic streams induce a motile and aggressive tumor phenotype. The microfluidic platform developed here potentially provides a flow-informed framework complementary to conventional mechanism-based therapeutic strategies, with broad applicability to other lethal malignancies.Proceedings of the National Academy of Sciences 05/2013; · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Glycogen synthase kinase 3 beta (GSK3β) is highly inactivated in epithelial cancers and is known to inhibit tumor migration and invasion. The zinc-finger-containing transcriptional repressor, Slug, represses E-cadherin transcription and enhances epithelial-mesenchymal transition (EMT). In this study, we find that the GSK3β-pSer9 level is associated with the expression of Slug in non-small cell lung cancer. GSK3β-mediated phosphorylation of Slug facilitates Slug protein turnover. Proteomic analysis reveals that the carboxyl terminus of Hsc70-interacting protein (CHIP) interacts with wild-type Slug (wtSlug). Knockdown of CHIP stabilizes the wtSlug protein and reduces Slug ubiquitylation and degradation. In contrast, nonphosphorylatable Slug-4SA is not degraded by CHIP. The accumulation of nondegradable Slug may further lead to the repression of E-cadherin expression and promote cancer cell migration, invasion and metastasis. Our findings provide evidence of a de novo GSK3β-CHIP-Slug pathway that may be involved in the progression of metastasis in lung cancer.Oncogene advance online publication, 15 July 2013; doi:10.1038/onc.2013.279.Oncogene 07/2013; · 7.36 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Embryo implantation requires a precise synchronism between the receptive uterus and activated blastocyst and is regulated by complicated molecular networks. Although many implantation-related genes have been identified, the crosstalk among them is still unknown. Snail, a transcription repressor, plays a central role during epithelial-mesenchymal transition. Our previous study showed that Snail is highly expressed at implantation site in mouse uterus. This study was to examine how Snail is related with other implantation-related genes in mice. Uterine stromal cells were isolated from mouse uteri on day 4 of pregnancy and treated with HB-EGF. Snail was induced significantly by HB-EGF. By using specific inhibitors and siRNA, we demonstrated that HB-EGF induction on Snail expression is dependent on the EGFR-ERK-Stat3 pathway. Cox-2 was regulated by Snail. The current findings demonstrate that Snail can relate with HB-EGF, Stat3 and Cox-2 and may play a role during mouse embryo implantation and decidualization.Molecular and Cellular Endocrinology 08/2013; · 4.04 Impact Factor
EGF-Induced EMT and Invasiveness in Serous Borderline
Ovarian Tumor Cells: A Possible Step in the Transition to
Low-Grade Serous Carcinoma Cells?
Jung-Chien Cheng, Nelly Auersperg, Peter C. K. Leung*
Department of Obstetrics and Gynecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada
In high-grade ovarian cancer cultures, it has been shown that epidermal growth factor (EGF) induces cell invasion by
activating an epithelial-mesenchymal transition (EMT). However, the effect of EGF on serous borderline ovarian tumors
(SBOT) and low-grade serous carcinomas (LGC) cell invasion remains unknown. Here, we show that EGF receptor (EGFR) was
expressed, that EGF treatment increased cell migration and invasion in two cultured SBOT cell lines, SBOT3.1 and SV40 large
T antigen-infected SBOT cells (SBOT4-LT), and in two cultured LGC cell lines, MPSC1 and SV40 LT/ST-immortalized LGC cells
(ILGC). However, EGF induced down-regulation of E-cadherin and concurrent up-regulation of N-cadherin in SBOT cells but
not in LGC cells. In SBOT cells, the expression of the transcriptional repressors of E-cadherin, Snail, Slug and ZEB1 were
increased by EGF treatment. Treatment with EGF led to the activation of the downstream ERK1/2 and PI3K/Akt. The MEK1
inhibitor PD98059 diminished the EGF-induced cadherin switch and the up-regulation of Snail, Slug and ZEB1 and the EGF-
mediated increase in SBOT cell migration and invasion. The PI3K inhibitor LY294002 had similar effects, but it could not
block the EGF-induced up-regulation of N-cadherin and ZEB1. This study demonstrates that EGF induces SBOT cell
migration and invasion by activating EMT, which involves the activation of the ERK1/2 and PI3K/Akt pathways and,
subsequently, Snail, Slug and ZEB1 expression. Moreover, our results suggest that there are EMT-independent mechanisms
that mediate the EGF-induced LGC cell migration and invasion.
Citation: Cheng J-C, Auersperg N, Leung PCK (2012) EGF-Induced EMT and Invasiveness in Serous Borderline Ovarian Tumor Cells: A Possible Step in the
Transition to Low-Grade Serous Carcinoma Cells? PLoS ONE 7(3): e34071. doi:10.1371/journal.pone.0034071
Editor: Karl X. Chai, University of Central Florida, United States of America
Received December 8, 2011; Accepted February 21, 2012; Published March 30, 2012
Copyright: ? 2012 Cheng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by an operating grant from the Canadian Institutes of Health Research to PCKL. PCKL is the recipient of a Child & Family
Research Institute Senior Investigator Award. JCC is the recipient of BC Foundation for Non-Animal Research-Evelyn Martin Memorial Fellowship. No additional
external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The epithelial-mesenchymal transition (EMT) is a highly
conserved biological process during which there are multiple
biochemical changes. This process results in the conversion of
polarized, immotile epithelial cells into mesenchymal cells with a
motile phenotype. This important process was initially recognized
during critical phases of embryonic development, and recently, it
has been shown that EMT is involved in promoting cancer cell
invasion and metastasis .
A defining feature of EMT is a reduction in E-cadherin levels
and a concomitant induction of N-cadherin . Loss of E-
cadherin expression is mainly due to an up-regulation of Snail,
Slug, Twist, ZEB1 and other transcription factors that repress E-
cadherin . There is increasing evidence indicating that EMT is
stimulated by signals from the tumor microenvironment, including
a variety of growth factors and cytokines. In addition, EMT has
been shown to be regulated by a series of intracellular signaling
networks, including ERK1/2, PI3K/Akt, Smads, RhoB and b-
Epithelial ovarian cancer is the fifth leading cause of cancer-
related deaths among women in developed countries. Most deaths
from ovarian cancer are due to metastases that are resistant to
conventional therapies. The epithelial growth factor receptor
(EGFR) family consists of four members, EGFR (HER1), ErbB2
(HER2), ErbB3 (HER3), and ErbB4 (HER4), and has been shown
to play an important role in metastasis and tumorigenesis in many
types of human cancers [5,6]. Amplifications and overexpression
of the EGFR family have been reported in high-grade ovarian
cancer and are associated with more aggressive clinical behavior
and a poor prognosis [7,8]. It has been shown that EGF can
induce EMT in ovarian surface epithelium (OSE) and ovarian
cancer cells, suggesting that EGF may be involved in ovarian
cancer pathogenesis and metastasis [9,10]. Ovarian cancer cells
with low E-cadherin expression are more invasive, and the
absence of E-cadherin expression in ovarian cancers is predictive
of poor survival [11,12]. Serous borderline ovarian tumors
(SBOT) are non-invasive and are considered to be distinct entities
that give rise to invasive low-grade serous carcinomas (LGC),
which have a relatively poor prognosis when compared to SBOT
and are unrelated to high-grade serous carcinomas . Studies
using clinical samples have shown that EGFR is expressed in
borderline ovarian tumors [7,14]. Although the function of EGFR
signaling in cultured ovarian cancer cells has been studied, its
function in the borderline tumors and in LGC is still unknown due
to the lack of a suitable in vitro model. We recently established an in
PLoS ONE | www.plosone.org1March 2012 | Volume 7 | Issue 3 | e34071
vitro culture system with human SBOT cells. Cultured SBOT cells
grow slowly, are essentially non-invasive and exhibit limited
motility. These characteristics resemble the cells’ behavior in vivo
. Our recent study showed that p53 regulates the transition of
SBOT cells from non-invasive to invasive ovarian carcinomas by
activating the PI3K/Akt pathway and decreasing the expression of
E-cadherin, indicating that EMT is a critical process for the
regulation of SBOT cell invasion [16,17].
In this study, we report for the first time that the EGFR is
expressed in two cultured SBOT cell lines, SBOT3.1 and SBOT4-
LT, and in two LGC-derived cell lines, MPSC1 and ILGC cells,
and that EGF treatment induces cell migration and invasion in all
cell lines. Interestingly, EGF only induces the cadherin switch in
SBOT cells, which leads to SBOT cell migration and invasion. We
also show that the underlying mechanisms involve the activation of
the ERK1/2 and PI3K/Akt pathways. The information derived
from this study provides critical insight into the role of EGFR
activation in the down-regulation of E-cadherin, which plays a key
role in increasing SBOT cell migration and invasion.
Materials and Methods
The SBOT3.1 , SV40 LT-infected SBOT (SBOT4-LT)
 and SV40 LT/ST immortalized LGC (ILGC)  cell lines
were established in our laboratory. SBOT and ILGC cells were
grown in a 1:1 (v/v) mixture of M199/MCDB105 medium
(Sigma, Oakville, ON) supplemented with 10% fetal bovine serum
(FBS, Hyclone Laboratories Inc., Logan, UT). The MPSC1 cell
line, which was established from a LGC (provided by Dr. Ie-Ming
Shih, Department of Pathology, Johns Hopkins Medical Institu-
tions, Baltimore, MD), was maintained in RPMI 1640 (Invitrogen,
Burlington, ON) supplemented with 10% FBS . Cultures were
maintained at 37uC in a humidified atmosphere of 5% CO2in air.
Antibodies and reagents
Polyclonal anti-EGFR and anti-b-actin antibodies were ob-
tained from Santa Cruz Biotechnology (Santa Cruz, CA). The
monoclonal anti-E-cadherin and anti-N-cadherin antibodies were
obtained from BD Biosciences (Mississauga, ON). Monoclonal
anti-phospho-EGFR (Tyr1173), anti-phospho-ERK1/2 (Thr202/
Tyr204) anti-ZEB1 and anti-HER2 antibodies and polyclonal
anti-ERK1/2, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-
p38 MAPK, anti-phospho-Akt (Ser473) and anti-Akt antibodies
were obtained from Cell Signaling Technology (Danvers, MA).
Polyclonal anti-Snail and anti-Slug antibodies were obtained from
Abgent (San Diego, CA). Horseradish peroxidase-conjugated goat
anti-mouse IgG and goat anti-rabbit IgG were obtained from Bio-
Rad Laboratories (Hercules, CA). Horseradish peroxidase-conju-
gated donkey anti-goat IgG was obtained from Santa Cruz
Biotechnology. Human epidermal growth factor (EGF), AG1478,
SB203580 and LY294002 were obtained from Sigma. PD98059
was obtained from Calbiochem (San Diego, CA).
In the migration and invasion assays, cells with 80% confluence
or cells treated with siRNA were treated with EGF for 24
(migration) and 48 (invasion) hr, respectively. After EGF
treatment, cells were trypsinized and seeded into transwell inserts.
For the general EGF treatment experiments, cells were cultured
until 80% confluent and treated with 50 ng/ml EGF. The effect of
EGF on the mRNA levels of E-cadherin, N-cadherin, Snail, Slug,
Twist and ZEB1 were examined after 24 hr EGF treatment. The
effect of EGF on the protein levels of those molecules were
examined after 48 hr EGF treatment. The levels of specific mRNA
and protein were examined by RT-qPCR and western blot,
respectively. To knockdown EGFR, the cells were cultured until
60% confluent and then transfected with ON-TARGETplus
SMARTpool EGFR (50 nM) siRNA (Dharmacon Research, Inc.,
Lafayette, CO) using Lipofectamine RNAiMAX (Invitrogen) for
48 hr. The siCONTROL NON-TARGETINGpool siRNA (Dhar-
macon) was used as the transfection control.
Cells were lysed in lysis buffer (Cell Signaling Technology), and
protein concentrations were determined using a DC protein assay
kit with BSA as the standard (Bio-Rad Laboratories). Equal
amounts of protein were separated by SDS polyacrylamide gel
electrophoresis and transferred to PVDF membranes. Following
blocking with TBS containing 5% non-fat dry milk for 1 hr,
membranes were incubated overnight at 4uC with primary
antibodies, followed by incubation with HRP-conjugated second-
ary antibody. Immunoreactive bands were detected with enhanced
chemiluminescent substrate. Membranes were stripped with
stripping buffer and reprobed with anti-b-actin as a loading
control. Band intensities were quantified using the Scion Image
software and normalized to b-actin.
Reverse transcription quantitative real-time PCR
Total RNA was extracted using TRIzol reagent (Invitrogen)
according to the manufacturer’s instructions. Reverse transcription
was performed with 3 mg of RNA, random primers and M-MLV
reverse transcriptase (Promega, Madison, WI). The primers used
for SYBR Green reverse transcription-qPCR (RT-qPCR) were as
follows: E-cadherin, 59-ACA GCC CCG CCT TAT GAT T-39
(sense) and 59-TCG GAA CCG CTT CCT TCA-39 (antisense);
N-cadherin, 59-GGA CAG TTC CTG AGG GAT CA-39 (sense)
and 59-GGA TTG CCT TCC ATG TCT GT-39 (antisense);
Snail, 59-CCC CAA TCG GAA GCC TAA CT-39 (sense) and 59-
GCT GGA AGG TAA ACT CTG GAT TAG A-39 (antisense);
Slug, 59-TTC GGA CCC ACA CAT TAC CT-39 (sense) and 59-
GCA GTG AGG GCA AGA AAA AG-39 (antisense); Twist, 59-
GGA GTC CGC AGT CTT ACG AG-39 (sense) and 59-TCT
GGA GGA CCT GGT AGA GG-39 (antisense); ZEB1, 59- GCA
CCT GAA GAG GAC CAG AG-39 (sense) and 59-TGC ATC
TGG TGT TCC ATT TT-39 (antisense); and GAPDH, 59-GAG
TCA ACG GAT TTG GTC GT-39 (sense) and 59-GAC AAG
CTT CCC GTT CTC AG-39 (antisense). RT-qPCR was
performed on an Applied Biosystems 7300 Real-Time PCR
System (Perkin-Elmer, Wellesley, MA) equipped with a 96-well
optical reaction plate. All RT-qPCR experiments were run in
triplicate, and a mean value was used for the determination of
mRNA levels. Relative quantification of the mRNA levels was
performed using the comparative Ct method with GAPDH as the
reference gene and with the formula 22DDCt.
Transwell migration and invasion assay
Migration and invasion assays were performed in Boyden
chambers with minor modifications . Cell culture inserts (24-
well, pore size 8 mm; BD Biosciences, Mississauga, ON) were
seeded with 16105cells in 250 mL of medium with 0.1% FBS. Un-
coated inserts were used for migration assays, whereas inserts pre-
coated with growth-factor-reduced Matrigel (40 ml, 1 mg/ml, BD
Biosciences) were used for invasion assays. Medium with 10% FBS
(750 ml) was added to the lower chamber and served as a
chemotactic agent. After 24 hr (migration) or 48 hr (invasion)
incubation, non-migrating/invading cells were wiped from the
upper side of the membrane. Cells that penetrated the membrane
EGF Induces SBOT Cell Invasion by Stimulating EMT
PLoS ONE | www.plosone.org2March 2012 | Volume 7 | Issue 3 | e34071
were fixed with cold methanol, and cell nuclei were stained with
Hoechst 33258 and counted by epifluorescence microscopy with
Northern Eclipse 6.0 software (Empix Imaging, Mississauga, ON).
Triplicate inserts were used for each individual experiment, and
five microscopic fields were counted per insert.
Results are presented as the mean 6 SEM of at least three
independent experiments. Two-sample data were analyzed by
Excel with the two-sample t-test assuming unequal variances.
Multiple comparisons were analyzed by one-way ANOVA
followed by Tukey’s multiple comparison test using PRISM
software. Significant differences were defined as p,0.05.
Expression of E-cadherin, N-cadherin, EGFR and HER2 in
cultured SBOT and LGC cells
Our recent studies showed that EMT is a critical process that
contributes to the progression of non-invasive SBOT to invasive
LGC [16,17]. To confirm this result, we compared the basal
expression levels of E-cadherin and N-cadherin in two SBOT lines,
SBOT3.1 and SBOT4-LT, and two LGC-derived cell lines,
MPSC1 and ILGC. SBOT3.1 cells grew slowly, whereas SBOT4-
LT, MPSC1 and ILGC cells were grew faster. As shown in
Figure 1A, SBOT3.1 and MPSC1 exhibited an epithelial
morphology. With the introduction of SV40 LT or LT/ST,
SBOT4-LT and ILGC exhibited a more atypical and scattered
morphology. To compare the expression levels of E-cadherin and
N-cadherin, cells were grown until they were fully confluent, and
then the total proteins were collected. As shown in Figure 1B, the
expression levels of E-cadherin were high in SBOT3.1 cells and low
in MPSC1 cells, whereas the levels of E-cadherin were almost
absent in SV40 immortalized SBOT4-LT and ILGC cells, which is
consistent with our previous data showing that E-cadherin is down-
regulated by the inhibition of p53 [16,18]. These results indicate
that MPSC1 cells are a more mesenchymal-like cell type compared
to SBOT3.1 cells. To date, whether cultured SBOT and LGC cells
express EGFR or HER2 remains unclear. As shown in Figure 1C,
both SBOT and LGC cells expressed EGFR and HER2. The
expression level of EGFR was lower in SBOT3.1 cells than in
others, whereas all cell lines expressed similar levels of HER2.
EGF treatment increases cell migration and invasion in
SBOT and LGC cells
Transwell migration and invasion assays showed that SBOT3.1
cells were essentially non-motile and non-invasive, whereas
SBOT4-LT, MPSC1 and ILGC cells were highly motile and
invasive (Figure 2A). Interestingly, EGF treatment resulted in a
significant increase in cell migration (Figure 2B) and invasion
(Figure 2C) in a dose-dependent manner in all SBOT and LGC
cell lines. To confirm the involvement of EGFR in EGF-induced
cell invasion, EGFR-specific siRNA was used to knock down the
endogenous EGFR. Western blot analysis showed that EGFR
siRNA significantly knocked down the endogenous expression of
EGFR. Moreover, EGFR siRNA abolished EGF-induced cell
migration and invasion (Figure 2D). These results confirmed that
EGFR is required for EGF-induced cell migration and invasion.
EGF induces a down-regulation of E-cadherin and an up-
regulation of N-cadherin in SBOT cells
A characteristic of EMT is a switch from E-cadherin to N-
cadherin expression. In SBOT3.1 and SBOT4-LT cells, RT-
qPCR analysis showed that EGF treatment down-regulated E-
cadherin mRNA levels. Concurrently, N-cadherin mRNA levels
increased with EGF treatment. Unexpectedly, EGF treatment did
not alter the mRNA levels of E-cadherin or N-cadherin in MPSC1
and ILGC cells (Figure 3A). Similarly, western blot analysis
performed on total cell lysates collected from cells treated with
EGF for 48 hr showed that EGF down-regulated E-cadherin and
up-regulated N-cadherin total protein levels in SBOT3.1 cells, but
not in MPSC1 cells (Figure 3B). In addition, the effects of EGF on
the mRNA and protein levels of E- and N-cadherin in SBOT3.1
cells were eliminated by treatment with the EGFR inhibitor,
AG1478 (Figures 3C and D). Moreover, EGFR siRNA abolished
the EGF-inducedswitch from
(Figure 3E). It has been shown that the binding of EGF to EGFR
rapidly induces clustering and internalization of the ligand-
receptor complexes, ultimately resulting in lysosomal degradation
of both EGF and its receptor . This process was supported by
the data in Figure 3E, which shows that EGFR was down-
regulated in SBOT3.1 cells in response to EGF treatment.
EGF up-regulates Snail, Slug and ZEB1, but not Twist, in
To investigate whether EGF down-regulates E-cadherin
expression by modulating the transcriptional regulation of E-
cadherin, we used RT-qPCR to examine the mRNA levels of the
E-cadherin transcriptional repressors Snail, Slug, Twist and ZEB1.
Treatment with EGF significantly increased Snail, Slug and ZEB1
mRNA levels in SBOT3.1 and SBOT4-LT cells. However, EGF
treatment did not alter the mRNA levels of Twist. In addition, the
effects of EGF on these transcription factors were not detected in
MPSC1 and ILGC cells, confirming that the E-cadherin is not
transcriptionally regulated by EGF in LGC cells (Figure 4A). In
addition, treatment with AG1478 abolished the effects of EGF on
Snail, Slug and ZEB1 mRNA levels in SBOT3.1 cells (Figure 4B).
Moreover, western blot analysis showed that EGFR siRNA
abolished EGF-induced Snail, Slug and ZEB1 expression in
SBOT3.1 cells (Figure 4C).
Activation of ERK1/2 and PI3K/Akt pathways are
mediated by EGF-induced EMT and cell migration and
invasion in SBOT cells
It has been shown that the ERK1/2, p38 MAPK and PI3K/
Akt pathways are involved in EGF-induced EMT [9,22].
However, it is unknown whether these signaling pathways are
also involved in EGF-induced EMT in SBOT cells. As shown in
Figure 5, treatment with EGF induced the activation of ERK1/2
and Akt with the maximal effect observed at 5 min followed by a
decrease after 180 min treatment. Interestingly, treatment with
EGF did not activate p38 MAPK in SBOT3.1 cells. In contrast,
EGF induced ERK1/2, p38 MAPK and Akt activation in MPSC1
cells. In SBOT3.1 cells, the EGF-induced down-regulation of E-
cadherin and the up-regulation of N-cadherin mRNA and protein
levels were diminished by treatment with the MEK1 inhibitor
PD98059. Interestingly, treatment with the PI3K inhibitor
LY294002 only diminished the EGF-induced down-regulation of
E-cadherin but did not affect the EGF-induced up-regulation of
N-cadherin (Figure 6A). In addition, treatment with PD98059 and
LY294002 diminished EGF-induced up-regulation of Snail and
Slug mRNA levels. However, the EGF-induced up-regulation of
ZEB1 mRNA levels was only blocked by treatment with PD98059
and not with LY294002 (Figure 6B). Furthermore, EGF-induced
cell migration and invasion were blocked by PD98059 and
LY294002 treatments, although the inhibitory effect of LY294002
EGF Induces SBOT Cell Invasion by Stimulating EMT
PLoS ONE | www.plosone.org3March 2012 | Volume 7 | Issue 3 | e34071
was less than that of PD98059 (Figure 6C). In MPSC1 cell,
inhibition of ERK1/2, p38 MPAK and PI3K/Akt by PD98059,
SB203580 and LY294002 attenuated EGF-induced cell migration
and invasion (Figure 6D). Taken together, these results indicated
that the ERK1/2 and PI3K/Akt pathways are involved in EGF-
induced EMT and cell migration and invasion in SBOT cells. In
addition, although EGF did not induce EMT in MPSC1 cells, our
results indicate that ERK1/2, p38 MPAK and PI3K/Akt
signaling pathways are involved in EGF-induced MPSC1 cell
migration and invasion.
There is increasing evidence indicating that the activation of
EGFR signaling contributes to cellular invasion in ovarian cancer
by a variety of mechanisms. EGF treatment is known to increase
cultured ovarian cancer cell migration, invasion, and proteolytic
activity [23,24]. Although the contributions of EGF and EGFR
signaling have been described in ovarian cancer, the majority of
studies have been performed only on high-grade ovarian cancer
cells. In borderline tumors, immunohistochemical studies have
shown that EGF and the EGFR are expressed, but there is no
difference in EGFR staining intensity between benign, borderline
and malignant ovarian tumors [25,26]. Despite reports of EGFR
expression in borderline tumors, the EGFR-mediated cell
functions remain largely unknown. In the present study, we show
that, consistent with previous immunohistochemical results, EGFR
is expressed in cultured SBOT and LGC cells. It is well known
that SV40 large T antigen (LT) inactivates p53 and retinoblastoma
protein (Rb), whereas SV40 small T antigen (ST) inhibits the
activity of the protein phosphatase 2A (PP2A) [27,28]. It has been
shown that the cell motility can be regulated by p53 and PP2A
[28,29]. In the present study, we used two SBOT cell lines which
one is infected with SV40 LT (SBOT4-LT) and the other one is
not (SBOT3.1). In addition, ILGC is the SV40 LT/ST
immortalized LGC cell line, whereas MPSC1 is the LGC-derived
cell line which does not carry SV40 LT/ST. Interestingly,
although the four cell lines used in this study have different
genetic backgrounds, our results show that treatment with EGF
induced cell migration and invasion in all SBOT and LGC cell
lines. These results suggest that p53/Rb and PP2A may not affect
the EGF-induced cell migration and invasion in SBOT and LGC
It has been shown that none of the EGF family of peptides can
bind HER2, and this is important because HER2 is the preferred
dimerization partner for all the other EGFR family members .
Overexpression of HER2 has been shown in high-grade ovarian
cancer [30,31]. However, other studies showed no relationship
between HER2 expression and survival among patients with high-
grade ovarian cancer [32,33]. In SBOT and LGC, similar to high-
grade ovarian cancer, HER2 expression and its association with
prognosis are controversial [34,35]. In the present study, we found
that the expression levels of HER2 were similar in two SBOT and
Figure 1. Expression of E-cadherin, N-cadherin, EGFR and HER2 in SBOT3.1, SBOT4-LT, MPSC1 and ILGC cells. A, The morphology of
SBOT3.1, SBOT4-LT, MPSC1 and ILGC cells. The scale bar represents 100 mm. B, Endogenous protein levels of E-cadherin and N-cadherin were
analyzed by western blot. C, Endogenous protein levels of EGFR and HER2 were analyzed by western blot.
EGF Induces SBOT Cell Invasion by Stimulating EMT
PLoS ONE | www.plosone.org4March 2012 | Volume 7 | Issue 3 | e34071
two LGC cell lines. However, whether HER2 is involved in EGF-
induced SBOT and LGC cell motility remains unknown.
In ovarian cancer, based on molecular genetic and morpho-
logical studies, it has been suggested that there are two pathways of
tumorigenesis that correspond to the development of low-grade
and high-grade serous ovarian carcinoma . In type I tumors,
invasive LGC develops from a non-invasive SBOT. Histopatho-
logic and molecular genetic studies suggest that SBOT may arise
from ovarian surface epithelium (OSE) or cystadenomas . In
humans, OSE has either a flat or a cuboidal appearance. Flat OSE
does not express E-cadherin. In the ovary, E-cadherin expression
is limited to rare regions such as cuboidal and columnar OSE,
where cells resemble metaplastic epithelium [38,39]. Immunohis-
tochemical studies showed that membranous E-cadherin expres-
sion is detected in benign and serous borderline ovarian tumors.
Importantly, reduced expression of E-cadherin correlates with the
presence of microinvasion in serous borderline tumors . Our
recent study in cultured SBOT cells also showed that down-
regulation of E-cadherin contributes to the progression of SBOT
to invasive LGC . Taken together, these results suggest that
the expression of E-cadherin occurs intermittently during the
progression from OSE to SBOT to invasive LGC and may be
Figure 2. EGF induces cell migration and invasion in SBOT3.1, SBOT4-LT, MPSC1 and ILGC cells. A, The intrinsic migration and invasion
of cells. B and C, Cells were treated with increasing doses of EGF (20, 50 and 100 ng/ml). D, Cells were transfected with control siRNA (si-Ctrl) or EGFR
siRNA (si-EGFR) for 48 hr and then treated with 50 ng/ml EGF. After treatment cells were seeded into un-coated (migration) and Matrigel-coated
(invasion) transwell inserts. After 24 hr (migration) and 48 hr (invasion) incubation, non-invading cells were wiped from the upper side of the filter
and the nuclei of invading cells were stained with Hoechst 33258. Top panels show representative photos of the migration or invasion assay. Scale
bar represents 200 mm. Bottom panels show summarized quantitative results which are expressed as the mean 6 SEM of at least three independent
experiments. Western blots show the knockdown of EGFR by EGFR siRNA. *p,0.05 compared with Ctrl.#p,0.05 compared with EGF or EGF in si-Ctrl.
EGF Induces SBOT Cell Invasion by Stimulating EMT
PLoS ONE | www.plosone.org5March 2012 | Volume 7 | Issue 3 | e34071
required for the initiation of tumorigenesis in type I tumors.
Therefore, we hypothesize that once normal OSE acquires the
expression of E-cadherin, which may play a role in early events
leading to the malignant phenotype, the subsequent EMT may be
required for the progression of a non-invasive tumor to an invasive
Although the key feature of EMT is the down-regulation of E-
cadherin and up-regulation of N-cadherin, there still are some
other molecular markers that are used for EMT, such as increased
expression of vimentin, fibronectin and nuclear localization of b-
catenin and decreased expression of the tight junction protein,
occluding . However, the transition from epithelial to
mesenchymal cell characteristics encompasses a spectrum of inter-
and intracellular changes, not all of which are always seen during
EMT . In the present study, we show that EGF treatment
induced a switch from E-cadherin to N-cadherin expression in
SBOT cells. However, the effect of EGF on other EMT markers
requires further investigation. Here, we show that EGF treatment
down-regulates E-cadherin expression in SBOT cells. In contrast,
no such changes were observed in LGC cells. The western blot
results show that the EGFR level was higher in SBOT3.1 cells
than in MPSC1 cells, indicating that the effects of EGF on
cadherin switch are not related to the levels of EGFR. A recent
study showed that different binding affinities between EGF and
EGFR activate different signaling pathways. High-affinity EGF
binding is sufficient for activation of most canonical signaling
pathways, whereas low-affinity EGF binding is required for the
activation of the STATs and PLCc1 . Many signaling
pathways have been reported to be involved in the EMT in
ovarian cancer . It will require further investigation to
examine whether the divergent effects of EGF on the cadherin
switch result from the different binding affinities between EGF and
EGFR in SBOT and LGC cells. In high-grade ovarian cancer
cells, we recently showed that H2O2mediates the EGF-induced
down-regulation of E-cadherin expression in SKOV3 ovarian
cancer cells and suggested that the lack of an effect of EGF on E-
cadherin in OVCAR3 cells may reflect an uncoupling of EGFR
activation from H2O2 production . However, because the
EGFR is functional, as shown by detection of activated EGF-
induced EGFR phosphorylation, ERK1/2, p38 MAPK and
PI3K/Akt, it is unclear whether the lack of an effect of EGF on
E-cadherin expression in MPSC1 cells is due to the lack of H2O2
production after EGF treatment.
Reduced expression of E-cadherin in human cancers is
associated with metastasis, whereas in high-grade ovarian cancer,
forced expression of E-cadherin inhibits tumor metastasis . We
have shown that endogenous E-cadherin plays an important
regulatory role in cell invasion and that EGF-induced cell invasion
is mediated by the down-regulation of E-cadherin expression in
high-grade ovarian cancer cells . In SBOT cells, our recent
Figure 3. EGF induces cadherin switch in SBOT3.1 and SBOT4-LT cells, but not in MPSC1 and ILGC cells. A, Cells were treated with
50 ng/ml EGF for 24 hr. E-cadherin and N-cadherin mRNA levels were analyzed by RT-qPCR. B, Cells were treated with 50 ng/ml EGF for 48 hr. E-
cadherin and N-cadherin protein levels were analyzed by western blot. C and D, SBOT3.1 cells were treated with the EGFR inhibitor, AG1478 (10 mM),
in the presence or absence of 50 ng/ml EGF, and the levels of E-cadherin and N-cadherin mRNA (24 hr EGF treatment) and protein (48 hr EGF
treatment) were analyzed. E, SBOT3.1 cells were transfected with control siRNA (si-Ctrl) or EGFR siRNA (si-EGFR) for 48 hr and then treated with 50 ng/
ml EGF for 48 hr. The protein levels of E-cadherin and N-cadherin were analyzed by western blot. The results are expressed as the mean 6 SEM of at
least three independent experiments. *p,0.05 compared with time-matched Ctrl.#p,0.05 compared with EGF or EGF in si-Ctrl.
EGF Induces SBOT Cell Invasion by Stimulating EMT
PLoS ONE | www.plosone.org6 March 2012 | Volume 7 | Issue 3 | e34071
study showed that the down-regulation of E-cadherin by the
PI3K/Akt pathway contributes to the progression to the invasive
phenotype . In this study, we show that LGC-derived MPSC1
cells express lower levels of E-cadherin and higher levels of N-
cadherin than SBOT cells, suggesting that EMT may contribute to
the progression from SBOT to invasive LGC.
Figure 4. EGF induces Snail, Slug and ZEB1 expression in SBOT3.1 and SBOT4-LT cells, but not in MPSC1 and ILGC cells. A, Cells were
treated with 50 ng/ml EGF for 24 hr, and the mRNA levels of Snail, Slug, Twist and ZEB1 were analyzed by RT-qPCR. B, SBOT3.1 cells were treated with
AG1478 (10 mM) in the presence or absence of 50 ng/ml EGF for 24 hr, and mRNA levels were analyzed by RT-qPCR. C, SBOT3.1 cells were transfected
with control siRNA (si-Ctrl) or EGFR siRNA (si-EGFR) for 48 hr and then treated with 50 ng/ml EGF for 48 hr. The protein levels of Snail, Slug and ZEB1
were analyzed by western blot. The results are expressed as the mean 6 SEM of at least three independent experiments. *p,0.05 compared with
Ctrl.#p,0.05 compared with EGF or EGF in si-Ctrl.
Figure 5. EGF activates ERK1/2 and Akt pathways in SBOT3.1 cells. SBOT3.1 and MPSC1 cells were treated with 50 ng/ml EGF for the
indicated durations. Phosphorylation of ERK1/2, p38 MAPK and Akt were determined by western blot using antibodies specific for phosphorylated,
activated forms of ERK1/2 (p-ERK1/2), p38 MAPK (p-p38) and Akt (p-Akt). Membranes were stripped and reprobed with antibodies to total ERK1/2,
p38 MAPK and Akt.
EGF Induces SBOT Cell Invasion by Stimulating EMT
PLoS ONE | www.plosone.org7 March 2012 | Volume 7 | Issue 3 | e34071
In the present study, our data demonstrate that in SBOT
cells, ERK1/2 and Akt mediated the EGF-induced down-
regulation of E-cadherin expression, whereas only ERK1/2 was
involved in EGF-induced N-cadherin expression. Down-regu-
lation of E-cadherin is mainly due to the up-regulation of Snail,
Slug, Twist, ZEB1 and other transcription factors that repress
E-cadherin . We show here that the expression of Snail, Slug
and ZEB1, but not Twist, was increased by EGF treatment in
SBOT cells. Recent studies have shown that Twist and ZEB1
not only repress E-cadherin expression but also induce the
expression of N-cadherin [45,46]. Treatment with LY294002
did not block the EGF-induced up-regulation of N-cadherin,
which may be due to the lack of an inhibitory effect of
LY294002 on ZEB1 expression. Nevertheless, both the ERK1/
2 and PI3K/Akt pathways were involved in EGF-induced
SBOT cell migration and invasion. These results are consistent
with our previous finding that E-cadherin, but not N-cadherin,
plays an important role in the regulation of SBOT cell invasion
In summary, this study demonstrates that EGFR is expressed in
cultured SBOT and LGC cells and that treatment with EGF
induces cell migration and invasion by activating EMT in SBOT
cells, which may play an important role in the progression from
SBOT to invasive LGC. In addition, this study suggests that there
may be E-cadherin-independent mechanisms that mediate the
EGF-induced cell migration and invasion in LGC cells.
Conceived and designed the experiments: NA PCKL. Performed the
experiments: JCC. Analyzed the data: JCC. Contributed reagents/
materials/analysis tools: JCC. Wrote the paper: JCC NA PCKL.
Figure 6. EGF induces cadherin switch through ERK1/2 and Akt activation in SBOT3.1 cells. A, SBOT3.1 cells were treated for 48 hr with
PD98059 (20 mM) or LY294002 (20 mM) in the presence or absence of 50 ng/ml EGF. E-cadherin and N-cadherin mRNA (left panel) and protein (right
panel) levels were analyzed by RT-qPCR and western blot, respectively. B, SBOT3.1 cells were treated for with PD98059 (20 mM) or LY294002 (20 mM)
in the presence or absence of 50 ng/ml EGF and Snail, and the Slug, Twist and ZEB1 mRNA levels were analyzed by RT-qPCR. C, SBOT3.1 cells were
treated with 50 ng/ml EGF in combination with PD98059 (20 mM) or LY294002 (20 mM). D, MPSC1 cells were treated with 50 ng/ml EGF in
combination with PD98059 (20 mM) SB203580 (10 mM) or LY294002 (20 mM). After treatment, cells were seeded into un-coated (migration) and
Matrigel-coated (invasion) transwell inserts. After 24 hr (migration) and 48 hr (invasion) incubation, non-invading cells were wiped from the upper
side of the filter and the nuclei of invading cells were stained with Hoechst 33258. Results are expressed as the mean 6 SEM of at least three
independent experiments. *p,0.05 compared with Ctrl.#p,0.05 compared with EGF.
EGF Induces SBOT Cell Invasion by Stimulating EMT
PLoS ONE | www.plosone.org8 March 2012 | Volume 7 | Issue 3 | e34071
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