Epithelial-to-mesenchymal transition correlates with gefitinib resistance in NSCLC cells and the liver X receptor ligand GW3965 reverses gefitinib resistance through inhibition of vimentin
OncoTargets and Therapy
epithelial-to-mesenchymal transition correlates with gefitinib resistance in NSCLC cells and the liver X receptor ligand GW3965 reverses gefitinib resistance through inhibition of vimentin
Yong hu 1
Jialan Zang 0
Xiaobing Qin 1
Dali Yan 1
Jianzhong Wu 1
Ji-Feng Feng 1
0 Department of Oncology, The First Hospital of Harbin City , Harbin, Heilongjiang , People's Republic of China
1 Research Center for Clinical Oncology, Affiliated Cancer Hospital of Nanjing Medical University, Jiangsu cancer hospital and Jiangsu institute of Cancer Research , Nanjing, Jiangsu
The role of epithelial-to-mesenchymal transition in cancer drug resistance is increasingly acknowledged. We examined whether epithelial-to-mesenchymal transition affects gefitinib resistance in non-small cell lung cancer (NSCLC) cells. Cell viability was detected by CCK-8 assay, VIM expression levels were determined by quantitative real-time polymerase chain reaction. Western blot and immunocytochemistry were performed to determine the protein expression level of vimentin. We observed morphologic differences between gefitinibsensitive and -insensitive cells. Compared with the sensitive parental cell line, HCC827, vimentin expression levels were increased in HCC827 cells with acquired gefitinib resistance. Vimentin expression was also markedly upregulated in cells with intrinsic gefitinib resistance, and upregulated vimentin expression was correlated with gefitinib sensitivity. Our previous study demonstrated that coadministration of gefitinib and GW3965 resulted in decreased cell proliferation and induced apoptosis. Therefore, we investigated the relationship among GW3965, vimentin, and gefitinib resistance in NSCLC cells by analysis of the expression of vimentin in cells treated with a combination of gefitinib and GW3965. Gefitinib treatment led to increased levels of intracellular vimentin, while combined treatment with gefitinib and GW3965 resulted in decreased vimentin expression levels through reduction of gefitinib drug resistance in NSCLC cells. Overall, these findings suggest that vimentin expression is associated with sensitivity to gefitinib, and our study highlights the potential usefulness of the drug, GW3965, for reversal of gefitinib resistance through inhibition of vimentin expression.
gefitinib resistance; LXR; EMT; GW3965; vimentin
open access to scientific and medical research
O r i g i n a l r e s e a r c h
Lung cancer is the leading cause of cancer-related mortality,1 and tobacco use is the
main risk factor for lung cancer development.2 The majority of patients with non-small
cell lung cancer (NSCLC) are diagnosed at an advanced stage because the disease can
be symptomless in its early stages.
Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) consist
of a large number of different small-molecule EGFR antagonists which have been used
for the treatment of advanced NSCLC. EGFR-TKIs are well tolerated and have
effective antitumor activity. Gefitinib is an orally administered selective EGFR-TKI that
can inhibit proliferation of NSCLC cells. EGFR-mutated lung cancer responds well to
gefitinib; however, all patients eventually develop resistance to the drug.3 Therefore,
exploring the mechanisms of acquired gefitinib resistance could lead to changes in
lung cancer therapy and potential clinical benefits. Recent
research has revealed that smoking abolishes the
therapeutic effects of EGFR-TKIs in NSCLC patients with EGFR
mutations.4 Current smokers showed lower disease control
rates and objective response rates and shorter
progressionfree and overall survival, compared with former smokers and
never smokers when they received EGFR-TKI therapy.5 The
8 stimulation of epithelial–mesenchymal transition (EMT) by
-120 cigarette smoking has been observed in lung cancer.6 For
l-Ju some patients, acquired resistance to EGFR inhibitors is
n31 associated with the phenomenon of EMT.7
o21 EGFR has an important role in lung cancer tumor
pro.519 gression and metastasis, a key indicator of poor prognosis.
..23 Furthermore, the EMT process is receiving increasing
atten123 tion due to its role in acquired drug resistance. This study
/yb focused on the link between EMT and drug resistance.8 EMT
.com is a complex process, which is primarily characterized by
ss downregulation of markers commonly expressed in epithelial
rvpee l.yno cells, such as E-cadherin, and increased expression of
mesen.dow lsue chymal markers, such as N-cadherin and vimentin.9 EMT has
/ww ano an important role in tumor invasion and metastasis. A recent
ttsp rspe in vitro study suggested a molecular and phenotypic
h ro tion between EMT and chemoresistance.10 The acquisition
from F of EMT features by tumors resistant to EGFR inhibition
deda has been described previously, both in preclinical studies
lon and in investigations using clinical samples.11 E-cadherin
dow expression is significantly elevated following treatment of
ryap EGFR-TKI-resistant cell lines with TKIs. A previous study
ehT indicated that restoration of E-cadherin expression increased
and sensitivity to EGFR-TKIs.12 EMT progression is
charactertseg ized by the loss of proteins involved in cell junctions, such as
raT E-cadherin,13 and the gain of mesenchymal markers, such as
cno vimentin.9 Genes affected by EMT include those associated
O with adherens junctions, such as E-cadherin (CDH1, CDH2,
and CDH12), β-catenin (CTNNB1), mesenchymal markers
such as VIM, and genes that regulate EMT gene expression,
such as ZEB1, ZEB2, and SNAI1, SNAI3.13 Upregulation of
mesenchymal-associated proteins, such as vimentin, is a
signature of gefitinib resistance.13
Vimentin is a member of the intermediate filament
protein family. Together with microtubules and actin filaments,
vimentin is a component of the cytoskeleton. Vimentin
expression is typically involved in cell adhesion, migration,
apoptosis, and cell signal transduction. The research of Xu et
al indicated that vimentin can modulate and control cellular
processes, such as migration, and possibly also proliferation.14
High expression of vimentin is significantly associated with
cell invasiveness. Furthermore, vimentin is expressed at
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significantly higher levels in drug-resistant cancer cells,
including paclitaxel-resistant prostate cancer, GR malignant
glioma,15 and tamoxifen-resistant breast cancer.16
Liver X receptor (LXR) belongs to the nuclear receptor
family of ligand-dependent transcription factors, and ligands
of LXR, such as TO901317 or GW3965, regulate cholesterol,
glucose, and fatty acid metabolism in human cancer cells.17
Recent studies demonstrate that LXR activation can
regulate tumor cell death,18 and our previous study indicated
that LXR ligands, such as TO901317 and GW3965, can
reverse acquired gefitinib resistance in NSCLC cells through
inhibition of AKT phosphorylation;19 however, the effect of
LXR inhibition on EMT has not been previously reported.
The goal of this study was to determine the mechanism
by which EMT contributes to acquired gefitinib resistance
and to test whether GW3965 can reverse GR cell growth by
inhibition of the EMT mesenchymal marker, vimentin. This
work could highlight novel potential strategies to reverse
acquired gefitinib resistance in NSCLC.
Reagents and suppliers were as follows: gefitinib (AstraZeneca
UK Limited, Macclesfield, Cheshire, UK); CCK-8 cell
viability kit (Dojindo, Kumamoto, Japan); GW3965
(SigmaAldrich Co., St Louis, MO, USA); penicillin–streptomycin
solution (Thermo Fisher Scientific, Waltham, MA, USA);
antibodies against vimentin, E-cadherin, β-actin, and EGFR
(Cell Signaling Technology, Danvers, MA, USA); cell lines,
HCC827, H1299, H1975, H1650, and H358 (Shanghai
Institutes for Biological Sciences, China Academy of Cell
HCC827 GR cells were generated from HCC827
(gefitinibsensitive) cells and maintained in the Affiliated Cancer
Hospital of Nanjing Medical University. This was achieved
by exposure of HCC827 cells to increasing concentrations
of gefitinib, as previously described.20 All cells were
cultured in RPMI 1640 medium supplemented with 10%
heat-inactivated FBS (Wisent Inc. Montreal, QC, Canada)
and 1% penicillin–streptomycin solution.
Drug sensitivity assay
Cells were seeded in 96-well plates in 100 µL media at a
density of 5×103 per well. After 24 h of incubation, cells
were treated with indicated concentrations of gefitinib (1, 5,
10, 20, and 40 µM for resistant cell lines; 1, 5, 10, 20, and
40 nM for sensitive cell lines). GW3965 (5 µM) and
gefitinib (5 µM) were used to treat cells separately or in
combination, as previously described.19 After incubation for
72 h, CCK-8 assay reagent (0.5 mg/mL) was mixed with
RPMI 1640 medium, added to cells, and the plates then
read at 450 nm on an enzyme-linked immunosorbent assay
plate reader to determine cell viability. Three individual
experiments were performed.
Observation of morphologic changes
Cells were seeded into six-well flat-bottomed plates. Briefly,
cells were synchronized by culture in serum-free RPMI
1640 medium for 1 h before the experiment and then washed
twice with ice-cold phosphate-buffered saline (PBS). After
synchronization, the cell morphology was analyzed under
an inverted microscope. Three individual experiments were
RNA isolation and quantitative real-time
polymerase chain reaction (RT-PCR)
Total RNA samples were extracted from sorted cells
using TRIzol reagent (Invitrogen) according to the
manufacturer’s protocol. Total RNA was quantified using a
spectrophotometer (NanoDrop Technologies). The mRNA
levels were determined by quantitative RT-PCR and each
sample was analyzed in triplicate. Complementary DNA was
synthesized using RT Master Mix (Takara, Dalian, People’s
Republic of China) according to the manufacturer’s
instructions. Quantitative PCR was performed using SYBR Green
PCR Mix (Roche, Mannheim, Germany) on a StepOnePlus
RT PCR system (Applied Biosystems, Foster City, CA,
USA). Primer sequences are presented in Table 1. Gene
expression levels were calculated using the ΔΔCt method.
Three individual experiments were performed.
Transwell cell migration assay
A total of 5×104 cells were seeded into the top chamber
of polycarbonate transwell filters. Transwell inserts were
incubated at 37°C for at least 12 h before seeding cells.
Primer sequence 5′ to 3′
After incubation at 37°C for 24 h, the cells that adhered to
the inserts and those inside the upper chamber were
carefully wiped with cotton swabs. The cells that had migrated
to the lower membrane surface were fixed with 70%
methanol, stained with 0.1% crystal violet, and ten random 100×
magnification fields per well were counted.
Protein extraction and Western blot
Proteins were extracted using RIPA lysis buffer (Beyotime
Biotechnology, Jiangsu, People’s Republic of China),
resuspended in SDS-containing buffer (Beyotime Biotechnology,
Jiangsu, People’s Republic of China), and denatured at 100°C
for 10 min. Protein lysates were separated by 10%
SDSpolyacrylamide gel electrophoresis gels for 90 min at 110 V
and then transferred to polyvinylidene difluoride membranes
(EMD Millipore, Billerica, MA, USA). Membranes were
blocked with 5% bovine serum albumin and then incubated
with primary antibodies (1:1000) at 4°C overnight. Next, the
membranes were incubated with secondary antibody for 2 h
at room temperature. Bands were detected by
chemiluminescence with BeyoECL Plus kits (Beyotime, People’s Republic
of China). Three individual experiments were performed.
Cells were fixed in 4% paraformaldehyde for 30 min and
then incubated with primary antibodies (1:1000). Cells were
then incubated with horseradish peroxidase-conjugated IgG
antibody (Cell Signaling Technology, Danvers, MA, USA)
for 1 h at room temperature. Then, the cells were incubated
with anti-rabbit biotin-conjugated secondary antibody for
1 h, followed by treatment with Vectastain ABC reagent for
30 min at 4°C, and then stained with DAB and hematoxylin.
Images were captured using a light microscope imaging
system, and the selected regions of interest were outlined
manually. Three individual experiments were performed.
The results of all experiments are expressed as mean ± standard
deviation of at least three separate tests. Statistical
comparisons between groups were performed using Student’s
Analysis of gefitinib sensitivity
We classified the cell lines into those that were insensitive
(H1299, H1975), sensitive (HCC827), and moderately
sensitive (H358) to gefitinib-mediated growth inhibition
in vitro. Sensitivity was defined as .50% in vitro growth
inhibition at a gefitinib concentration of 5 µmol/L. IC50
values of gefitinib for the H1299, H1975, and H358 cell
lines are presented in Table 2. Next, we established cell lines
with acquired gefitinib resistance. After a continuous
treatment with gefitinib at gradually increasing concentrations,
HCC827/GR cell lines grew steadily in culture medium
containing gefitinib. Next, we compared the drug sensitivity
of the HCC827/GR cell lines to that of the parental (HCC827)
cells. IC50 values of HCC827/GR cell lines are presented
in Table 2. The results indicate that GR cell lines were
successfully established from parental gefitinib-sensitive
Acquisition of gefitinib resistance
is associated with changes in cell
morphology and migration
GR cell lines exhibited significant morphologic differences
rvpee l.yno compared with sensitive cells (Figure 1A). GR cells typically
.dow lsue showed varying degrees of shrinkage, blebbing, aggregation,
/w no and lack of obvious boundaries, whereas gefitinib-sensitive
ttp pe cells had clear, regular boundaries. Cell lines with both
intrinohm roF sic and acquired gefitinib resistance exhibited morphologic
frd changes consistent with EMT, consistent with the fact that
odea GR cells acquire migratory properties, contributing to cancer
lnw metastasis. The mesenchymal phenotype of these resistant
ydo cells was confirmed by their increased capacity for migration.
rape We observed that resistant cells acquired increased
migradhT tory ability, as determined by transwell migration assays
sna (Figure 1B). Compared with sensitive HCC827 cells and
trge moderately sensitive H358 cells, the GR H1299 and H1975
aoT NSCLC cell lines with morphology consistent with EMT
cnO displayed higher migration capability. Furthermore, our data
demonstrate that cells with acquired gefitinib resistance also
showed moderately increased migration capacity, despite
the poor migration ability of the parental HCC827 cell line
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(Figure 1B). These results reveal that the phenotype
associated with intrinsic or acquired gefitinib resistance involves
EMT, resulting in higher migration capacity. Our results
demonstrate a potential link between migration capacity and
Altered expression of the epithelial and
mesenchymal cell marker, vimentin, is
correlated with sensitivity to gefitinib
To confirm EMT in GR cells, we performed genome-wide
mRNA expression analysis of the parental HCC827 and
the GR HCC827/GR-8-1 cells, and EMT markers with
altered expression were subject to further analysis. A total
of 18 mRNAs, including the EMT-related gene VIM, were
upregulated in the GR HCC827/GR-8-1 cell line relative to
gefitinib-sensitive HCC827 cells (Figure 2A). In addition,
the GR cell lines H1299 and H1975 exhibited substantial
upregulation of VIM. Similarly, insensitive cell lines,
including HCC827/GR-8-1, HCC827/GR-8-2, and HCC827/
GR-8-12, appeared to have undergone EMT, as judged
by VIM expression. Hence, consistent with the observed
morphologic changes, GR cells showed upregulation of the
mesenchymal marker vimentin. As shown in Figure 2B, VIM
expression levels were dramatically higher in GR than in
parental cells. These results suggest that gefitinib resistance
is associated with EMT.
To better understand the mechanisms of acquired gefitinib
resistance, we examined differences in protein expression
of mesenchymal markers between gefitinib-sensitive
and -insensitive NSCLC lines. An obvious difference in
expression of vimentin was observed between gefitinib-sensitive
and -resistant NSCLC cells (Figure 2C). The expression
of vimentin was elevated in GR H1299 and H1975 cells,
whereas relatively low levels of vimentin expression were
observed in gefitinib-sensitive NSCLC lines. The substantial
upregulation of vimentin associated with gefitinib resistance
was subsequently studied in HCC827 parental/resistant cell
line pairs, revealing that vimentin protein was significantly
upregulated in HCC827/GR-8-1, HCC827/GR-8-2, and
HCC827/GR-8-12 cells compared with the parental
gefitinibsensitive cell line. The majority of cell lines analyzed retained
the expression of E-cadherin. Hence, although the insensitive
cells maintained expression of some epithelial cell proteins,
they also began to express mesenchymal proteins.
We conducted further investigation of the expression
levels of vimentin in gefitinib-sensitive and -insensitive
NSCLC lines by immunohistochemistry. A marked increase
in expression of mesenchymal markers, including vimentin,
was observed in the GR lines, H1299, H1975, H358, and
HCC827/GRs (Figure 2D). In addition, the ratio of
mesenchymal vimentin to epithelial E-cadherin was enhanced in
cells with acquired gefitinib resistance, compared with their
GW3965 treatment alleviates gefitinib
sensitivity and is associated with
suppression of vimentin expression
In our previous study, we demonstrated that GW3965 can
reverse GR cell growth of HCC827/GR cells.19 Treatment
of HCC827/GR cells with a combination of gefitinib (5 µM)
and GW3965 (5 µM) led to significant suppression of cell
viability.19 In lung cancer, EMT is not required for
metastasis, but can contribute to chemoresistance.21 A recent study
revealed that the EMT phenotype is associated with both
intrinsic and acquired resistance to EGFR-specific TKIs in
NSCLC cell lines.22 Upon induction of EMT, HCC827 cells
become significantly more resistant to EGFR-TKI.7
In this study, EMT-induced cells were found to be
insensitive to treatment with gefitinib, while cotreatment
with GW3965 resensitized the GR cells. The exposure of
HCC827 cells to gefitinib for several days resulted in the
expected EMT, as assessed by increased vimentin expression
(Figure 3). We next sought to determine the mechanism of
drug resistance following EMT, and tested the possibility
that recombinant GW3965 could alleviate gefitinib
resistance. HCC827/GR-8-1 cells were exposed to gefitinib
and GW3965, either in combination or separately, in 72 h
viability assays. We found that vimentin was significantly
overexpressed in the gefitinib-treated group, compared with
the control group. GW3965 alone had no effect on vimentin
levels, while treatment of HCC827/GR-8-1 cells with
that combined treatment with GW3965 can reverse acquired
GW3965 (5 µM) and gefitinib (5 µM) indicated that GW3965
drug resistance in HCC827/GR-8-1 cancer cells by inhibition
decreased vimentin levels in the presence of gefitinib,
sugof the gefitinib-induced increase in vimentin expression.
gesting that GW3965 inhibits EMT in gefitinib-treated
HCC827/GR-8-1 cells. Moreover, GW3965 inhibition of
vimentin expression in HCC827/GR-8-1 cells was
doseThe EGFR-TKI, gefitinib, is used as a second- or third-line
dependent in the presence of gefitinib. These findings suggest
treatment for patients with mutant EGFR.23 Despite frequent
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Figure 3 Western blots demonstrating that the GR cell line, HCC827/GR-8-1,
demonstrated markedly increased levels of vimentin, and that treatment with
gefitinib led to the complete absence of β-catenin protein expression.
Notes: However, after treatment with a combination of GW3965 and gefitinib, the
expression level of vimentin was significantly reduced and that of β-catenin clearly
increased. GW3965 did not affect the expression of E-cadherin. No changes in EGFR
protein levels were observed in cultured cells exposed to 5 µmol/mL gefitinib.
Abbreviations: EGFR, epidermal growth factor receptor; GR, gefitinib-resistant.
dramatic responses to gefitinib, nearly all patients develop
resistance and undergo disease progression.22 Acquisition of
drug resistance is a common phenomenon during tumor
progression, and can arise through a wide range of mechanisms,
including changes in drug efflux or drug metabolism and
specific mutations of drug targets, among others.
Data suggest that EMT may be a general biologic switch
rendering NSCLC sensitive or insensitive to EGFR
inhibition. Our study revealed that the expression of epithelial- and
mesenchymal-associated proteins differs between
gefitinibsensitive and -resistant NSCLC cell lines. Genes associated
with mesenchymal proteins, such as vimentin, were highly
expressed in NSCLC cell lines resistant to gefitinib, while
they exhibited low expression levels in sensitive lines.
The GR cell model established in our laboratory provided
us with a unique opportunity to study the contribution of EMT
to acquired EGFR-TKI resistance. Our results demonstrate
that long-term exposure of NSCLC cells to gefitinib results in
the acquisition of phenotypic features associated with tumor
EMT. In summary, sensitive cells remaining after long-term
exposure to gefitinib exhibited an EMT phenotype
accompanied by acquired drug resistance. This was demonstrated by
the substantial upregulation of markers, including vimentin,
typically associated with the mesenchymal phenotype in
tumor cells resistant to gefitinib. Our results show that the
expression levels of the vimentin protein and its encoding
gene are significantly increased in GR cells. These results
indicated that vimentin could be associated with the
mechanism underlying acquired gefitinib resistance.
In the context of gefitinib resistance, we demonstrated
that GW3965 can reverse mesenchymal tumor features and
resensitize tumor cells to the cytotoxic effects of gefitinib.
We previously determined that GW3965 can enhance the
inhibitory effects of gefitinib on the growth of GR HCC827/
GR-8-1, and our results demonstrated that HCC827/GR-8-1
cells treated with gefitinib at different concentrations in the
presence of GW3965 at 5 µM had a reduced IC50 for gefitinib.
These data suggest that GW3965 has synergistic effects
with gefitinib in HCC827-8-1 cells.19 During treatment with
gefitinib, the HCC827/GR-8-1 cells exhibit upregulation
of E-cadherin. E-cadherin expression is also significantly
elevated following treatment with TKIs. A previous study
indicated that restoring E-cadherin expression increases
sensitivity to EGFR-TKIs.12 However, although the cell lines
were gefitinib resistant, treatment with the drug was still able
to induce upregulation of E-cadherin expression.
Moreover, gefitinib-treated HCC827/GR-8-1 cells
exhibited significantly upregulated expression of vimentin.
Together with other recent studies, which suggest an
association between EMT and drug resistance,24 our results
confirm the association of vimentin expression with gefitinib
resistance in NSCLC cell lines. We also assessed the effects
of GW3965 combined with gefitinib. Combination treatment
led to a reduction of vimentin upregulation stimulated by
gefitinib in HCC827/GR-8-1, and restoration of β-catenin
expression, which was ablated by gefitinib treatment. The
decreased expression of vimentin is likely to contribute to
the restoration of drug sensitivity; however, the
combination of the two drugs was also associated with maintenance
of E-cadherin upregulation, which is also associated with
decreased resistance to gefitinib.
A recent report showed that LXR ligands can be used
in combined therapeutics with other anticancer drugs.25
However, it remains unclear how GW3965 interferes with
EMT. Considering the marked changes in vimentin
expression after treatment with gefitinib in resistant cells, there may
be a threshold for the function of LXR agonists. LXR ligands
trigger cell signaling pathway regulation, which may also
participate in this process. Meanwhile, as a drug affecting lipid
metabolism, GW3965 may interfere with lipid-dependent
processes, such as cholesterol pump effusion, to influence
cell signaling. Here, we explored the ability of GW3965 to
reverse drug resistance in GR cells. Understanding of the
specific role of GW3965 in this context will require further
In conclusion, GR human NSCLC cell lines were
established and found to exhibit dysregulated expression of
vimentin, which may have a prominent role in gefitinib
resistance of NSCLC cells. This study investigated the resistance
mechanisms of NSCLC and provided a theoretical basis for the
reversal of gefitinib resistance through inhibition of vimentin
using LXR ligands, such as GW3965. Until recently, the
majority of data connecting EMT with drug resistance was primarily
derived from in vitro studies;26 therefore, additional research
will be required in the future to confirm the applicability of
these findings in vivo and, ultimately, their clinical utility.
This work was supported by grants from the National Nature
Science Foundation of China (81372396).
The authors report no conflicts of interest in this work.
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