Published OnlineFirst February 19, 2016; DOI: 10.1158/1541-7786.MCR-15-0480
Molecular
Cancer
Research
Oncogenes and Tumor Suppressors
Reciprocal Negative Regulation between EGFR
and DEPTOR Plays an Important Role in the
Progression of Lung Adenocarcinoma
Xuefeng Zhou1, Jialong Guo2, Yanmei Ji3, Gaofeng Pan1, Tao Liu2,
Hua Zhu4, and Jinping Zhao1
Abstract
The epidermal growth factor receptor (EGFR) activates downstream mTOR phosphorylation to promote the progression of
many different tumor types, thus making it a prime therapeutic
target. However, the role of DEP domain-containing mTORinteracting protein (DEPTOR), a natural mTOR inhibitor,
remains unclear in this process. Here, it is reported that EGFR
expression is significantly increased in tumors of lung adenocarcinoma patients and is negatively correlated with the expression
of DEPTOR. Activation of EGFR signaling, by EGF, in A549 lung
adenocarcinoma cells (overexpressing EGFR) significantly
enhanced the function of the mTOR autoamplification loop,
consisting of S6K, mTOR, CK1a, and bTrCP1, which resulted in
downregulation of DEPTOR expression. Gefitinib, a specific EGFR
inhibitor, stimulated DEPTOR accumulation by downregulating
the function of the mTOR autoamplification loop. Furthermore, a
series of assays conducted in DEPTOR knockout or ectopic
expression in A549 cells confirmed that DEPTOR inhibited proliferation, migration, and invasion as well as the in vivo tumor
growth of lung adenocarcinoma. Importantly, tumor progression
mediated by EGFR ectopic expression was diminished by transfection with DEPTOR. This study uncovers the important inhibitory role of DEPTOR in lung adenocarcinoma progression and
reveals a novel mechanism that EGFR downregulates DEPTOR
expression to facilitate tumor growth.
Introduction
In lung adenocarcinoma, EGFR is overexpressed in more than
39% and has emerged as a leading target for the treatment of
patients with adenocarcinoma (10–12). Some TKIs, like gefitinib,
significantly improved survival and prognosis of patients with
lung adenocarcinoma, while some patients acquired resistance
after initial response to TKIs or even had intrinsic resistance
without EGFR mutations (13). To date, the possible mechanisms
that underlie the primary resistance of these drugs have been
supposed as follows: (i) a result of somatic mutations occurring
within the tyrosine kinase domain of EGFR (14, 15), the phosphorylation of EGFR triggered several downstream signal transduction cascades, such as the MAPK, Akt/mTOR, and JNK pathways, which almost totally inhibited by gefitinib, while mutated
receptors sustain a hyperactivated downstream signaling (16–18);
(ii) activation of other signaling pathways alternative to EGFR,
such as the insulin-like growth factor 1 receptor (IGF-1R) and
vascular endothelial growth factor receptor (VEGFR; ref. 19), and
resulted in the upregulation of downstream signaling pathways
such as AKT/mTOR (20–23). Therefore, it is of great significance to
further explore the downstream pathways of EGFR and thus
develop novel therapeutic strategies for patients with lung
adenocarcinoma.
The mammalian target of rapamycin (mTOR) is a highly conversed serine-threonine kinase that is located in the PI3K/AKT
pathway and is frequently hyperactivated in tumors by mutations
in upstream signaling factors (e.g., EGFR, VEGFR, and PI3K
regulators; refs. 23, 24). Various mTOR inhibitors have been used
in trials for cancer therapeutic (25, 26). DEP domain-containing
mTOR-interacting protein (DEPTOR), as a naturally occurring
inhibitor of mTOR, usually acts as a tumor suppressor by blocking
As one of the most common cell-surface receptors that specifically bind with extracellular protein ligands of the epidermal
growth factor (EGF) family, the epidermal growth factor receptor
(EGFR) belongs to the ErbB family of receptors and is involved in
different types of diseases, including age-related diseases, autism
spectrum disorders, and cancers (1–3). In light of the confirmed
evidence that EGFR plays a pivotal role in tumorigenesis, drug
resistance, relapse, and the metastasis of various cancers (4–7),
some of EGFR tyrosine kinase inhibitors (TKIs) or monoclonal
antibodies have been developed and entered clinical trials as
cancer therapeutics, such as afatinib, gefitinib, and erlotinib
(www.fda.gov; refs. 8, 9).
1
Department of Thoracic and Cardiovascular Surgery, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, P.R. China. 2Department of
Cardiothoracic Surgery, Taihe Hospital, Hubei University of Medicine,
Shiyan, Hubei, P.R. China. 3Department of Intensive Care Unit, Taihe
Hospital, Hubei University of Medicine, Shiyan, Hubei, P.R. China.
4
Department of Surgery, Davis Heart and Lung Research Institute,
The Ohio State University Wexner Medical Center, Columbus, Ohio.
X. Zhou and J. Guo have contributed equally to this article.
Corresponding Authors: Hua Zhu, Department of Surgery, Davis Heart and Lung
Research Institute, The Ohio State University Wexner Medical Center, 460 West
12th Avenue, BRT396, Columbus, OH 43210. Phone: 614-292-2130; Fax: 614-2922130; E-mail: [email protected]; and Jinping Zhao, Department of Thoracic
and Cardiovascular Surgery, Zhongnan Hospital of Wuhan University, Wuhan,
Hubei 430071, P.R. China. E-mail: [email protected]
doi: 10.1158/1541-7786.MCR-15-0480
2016 American Association for Cancer Research.
Implications: DEPTOR acts as a tumor suppressor by limiting
EGFR-driven lung adenocarcinoma progression. Mol Cancer Res;
14(5); 448–57. 2016 AACR.
448 Mol Cancer Res; 14(5) May 2016
Downloaded from mcr.aacrjournals.org on June 16, 2017. © 2016 American Association for Cancer Research.
Published OnlineFirst February 19, 2016; DOI: 10.1158/1541-7786.MCR-15-0480
EGFR and DEPTOR in Lung Cancer Progression
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r2 = 0.6247
P < 0.0001
n = 51
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EGFR
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DEPTOR
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DEPTOR
b-Actin
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Relative DEPTOR expression
Relative EGFR expression
C
tro
l
B
10
Tu
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or
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A
Figure 1.
DEPTOR expression is negatively correlated with EGFR in human lung adenocarcinoma tissues. mRNA expression of EGFR (A) and DEPTOR in all 51 samples of
DDCt
patients with lung adenocarcinoma was detected by qRT-PCR. GAPDH was used as an internal control and the data were analyzed by the 2
method. C,
Spearman correlation analysis of EGFR with DEPTOR in human lung adenocarcinoma tissues, data derived from A and B. D, representative expression of EGFR and
DEPTOR in non-cancerous adjacent tissue and tumor tissue was detected by Western blotting, b-actin was used as the internal control. , P < 0.0001.
the activation of mTOR to inhibit cell proliferation, invasion, and
survival effect of AKT (27, 28). However, under certain circumstances, DEPTOR could act as an oncogene by relieving the
feedback inhibition of mTOR, activation of mTOR enhances the
downstream S6K expression, which could negatively regulate AKT
via downregulating expression of IRS-1/2 (27, 29). More importantly, mTOR could also cooperate with S6K or CK1a to enhance
bTrCP to degrade the expression of DEPTOR and thus generate an
autoamplification loop to promote its own full activation (27, 30).
Although the potential role of DEPTOR as an oncogene or a
tumor suppressor has been investigated in different types of
tumors (28, 31–33), and DEPTOR could inhibit proliferation of
lung adenocarcinoma cells (34), it has not been previously tested
whether and how DEPTOR plays a role in EGFR-induced tumor
progression of lung adenocarcinoma. In this study, we showed
that DEPTOR expression is significantly reduced in patients with
lung adenocarcinoma and negatively correlated with EGFR
expression. Activation of EGFR signaling with EGF in EGFR over-
expressing A549 cells dramatically inhibited DEPTOR expression
by enhancing the function of the mTOR autoamplification loop,
which mainly responds to the feedback degradation of DEPTOR.
Furthermore, DEPTOR knockout by using the CRISPR/Cas9 system significantly promoted tumor growth both in vivo and in vitro.
And, more importantly, tumor progression induced by EGFR
ectopic expression was significantly inhibited by simultaneously
transfected with DEPTOR-overexpressing plasmid. Taken together, our results reveal a novel mechanism that EGFR used to
facilitate tumor growth and DEPTOR may be a novel therapeutic
option for the treatment of human lung adenocarcinoma.
Materials and Methods
Patients and tissue samples
The study was approved by the Ethical Review Board for
research in the Zhongnan Hospital of Wuhan University. Fiftyone patients diagnosed as lung adenocarcinoma were given
Table 1. Clinicopathologic features of lung adenocarcinoma patients
Characteristic
Total
Sex
Male
Female
Median age (years, range)
Smoking status
Current or former smoker
Non-smoker
TNM stage
I
II
III
IV
Lymph node metastasis
Yes
No
www.aacrjournals.org
Cases (%)
51
DEPTOR
mRNA expression
0.52 0.28
P
EGFR
mRNA expression
P
0.9667
35 (68.6%)
16 (31.4%)
60.2 (39–78)
0.52 0.29
0.53 0.27
32 (62.7%)
19 (37.3%)
0.53 0.30
0.50 0.25
8 (15.7%)
16 (31.4%)
11 (21.5%)
16 (31.4%)
0.90 0.15
0.68 0.17
0.41 0.19
0.25 0.11
30 (58.8%)
21 (41.2%)
0.36 0.20
0.76 0.19
0.8607
3.24 1.79
3.34 1.94
0.7153
<0.0001
<0.0001
0.7682
3.33 1.85
3.18 1.81
1.38 0.40
2.01 0.46
3.44 0.47
5.37 1.50
4.31 1.67
1.80 0.56
<0.0001
<0.0001
Mol Cancer Res; 14(5) May 2016
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449
Published OnlineFirst February 19, 2016; DOI: 10.1158/1541-7786.MCR-15-0480
Zhou et al.
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2B
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BE
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Tu
49
A5
1
I-H
-9
NC
PC
EGFR
DEPTOR
b-Actin
EGFR
BE
Cell culture
The normal human lung epithelial cell line BEAS-2B and lung
adenocarcinoma cell line A549, PC-9 and NCI-H1975 were
obtained from Shanghai Bogoo Biotechnology Limited, and
cultured in RPMI-1640 medium (Gibco) supplemented with
10% fetal bovine serum (Gibco) at 37 C in a humidified atmosphere containing 5% CO2. BEAS-2B cells were cultured in BEBM
medium according to ATCC protocols.
Immunohistochemistry
Biopsy samples were fixed in 4% formalin and embedded in
paraffin. Tissue slices were cut into 5-mm thick for H&E staining
and examined under a microscope. Immunohistochemistry was
performed using the Vectastain ABC Kit (Rabbit IgG, Vector
Laboratories; ref. 34). Rabbit primary antibodies for DEPTOR
(1:500), EGFR (1:200), phospho-EGFR (Tyr1068, 1:50) were
purchased from Abcam. Slices were developed with DAB and
counterstained with hematoxylin.
Quantitative real-time PCR (qRT-PCR)
Total RNA of tumor tissues or cells was extracted using TRIzol
(Invitrogen) according to the manufacturer's instructions. RNA
was reversely transcribed into cDNA using the First-Strand cDNA
Synthesis Kit (Toyobo). The mRNA expression of EGFR and
DEPTOR was quantified using a real-time RT-PCR with the SYBR
Green real-time PCR Master Mix kit (Toyobo). The following
primers were used: EGFR, 50 -TGATAGACGCAGATAGTCGCC-30
and 50 -TCAGGGC ACGGTAGAAGTTG-30 (35); DEPTOR, 50 -TTTGTGGTGCGAGGAAGTAA-30 and 50 -CATTG CTTTGTGTCATTCTGG-30 ; GAPDH, 50 -CTCTCTGCTCCTCCTGTTCGAC-30 and
50 -TGAGCGATGTGGCTCGGCT-30 (31). The ABI StepOne Plus
0.6
0.3
0.0
-2
B
A5
49
NC PC
I-H -9
19
75
0
informed consent according to institutional guidelines. Biopsy
specimens and paired adjacent normal tissues of all patients were
acquired and immediately stored at liquid nitrogen until use. The
patients' characteristics are summarized in Table 1.
450 Mol Cancer Res; 14(5) May 2016
Relative DEPTOR mRNA expression
2
0.9
AS
4
1.2
BE
6
AS
DEPTOR
8
-2
B
A5
49
NC PC
I-H -9
19
75
pEGFR
D
Relative EGFR mRNA expression
C
Figure 2.
The expression of EGFR and DEPTOR
in tumor tissues and cell lines of
human lung adenocarcinoma. A,
representative immunohistochemistry
images of lung adenocarcinoma
samples that stained with EGFR, EGFR
phosphorylation, and DEPTOR
antibodies. B, protein expression of
EGFR and DEPTOR was detected in
three human lung adenocarcinoma cell
lines, including A549, PC-9, and NCIH1975, as well as normal human lung
epithelial cells, BEAS-2B. mRNA
expression of EGFR (C) and DEPTOR
(D) in these cell lines was detected by
qRT-PCR. Data presented as Mean
SD; n 3 independent experiments.
, P < 0.0001. Scale bar, 100 mm.
(Applied Biosystems) was used to perform the amplification
reaction. Each experiment was performed in triplicate. And the
date was analyzed by the 2DDCt method.
Western blotting
Total proteins of tissue samples were extracted by using the
Total Protein Extraction Kit (Millipore). As for cultured tumor
cells, the cells were lysed with RIPA Lysis Buffer (Beyotime).
Equivalent proteins (30 mg per sample) were electrophoresed in
SDS-polyacrylamide gel and transferred onto polyvinylidene
difluoride (PVDF) membranes (Millipore). The PVDF membranes were blocked with 5% nonfat milk in TBST buffer for 1
hour and incubated overnight at 4 C with primary antibodies.
The following rabbit antibodies were used: DEPTOR (Abcam),
EGFR (Abcam), EGFR (Tyr1068; Abcam), mTOR, mTOR
(Ser2448), CK1a, bTrCP1, S6K, S6K (Thr389), and b-actin (all
from Cell Signaling Technology). Signals were detected using
ECL detection reagent (Millipore) following the manufacturer's
instructions.
Establishment of stably transfected cells
Full-length EGFR and DEPTOR were cloned into the vehicle
vector pcDNA3.1 to generate EGFR and DEPTOR overexpressing
plasmids, as we previously described (1, 27, 31). A549 cells were
transiently transfected with the above three plasmids using Lipofectamine 2000 according to the manufacturer's instructions
(Invitrogen) and retained in a medium containing 10% FBS and
1 mg/mL puromycin to select the stable transfected cells.
DEPTOR CRISPR/Cas9 Knockout
A549 cells (2 105) in 3 mL antibiotic-free medium were
plated in 6-well plate. Twenty-four hours later, after cells reach
80% confluency, DEPTOR CRISPR/Cas9 KO Plasmid and DEPTOR HDR Plasmid (Cat. No. sc-402253 and sc-402253-HDR
from Santa Cruz Biotechnology, Inc.) were mixed at equivalent
ratio and then cotransfected into A549 cells using Lipofectamine
Molecular Cancer Research
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Published OnlineFirst February 19, 2016; DOI: 10.1158/1541-7786.MCR-15-0480
EGFR and DEPTOR in Lung Cancer Progression
FR
G
Figure 3.
EGFR activation enhances the function
of the mTOR autoamplification loop to
inhibit DEPTOR expression. A, protein
expression of EGFR, pEGFR, and
DEPTOR was detected in EGFR stable
overexpressing A549 cells. EGFR
overexpressing A549 cells was
constructed as described in Materials
and Methods, and then was subjected to
Western blotting analysis with indicated
antibodies. B, EGFR overexpressing
A549 cells were stimulated with EGF (20
ng/mL) for 1 hour, gefitinib (5 mmol/L)
for 4 hours, DMSO used as a solvent
control. Then, cell lysates were subjected
to Western blotting analysis with
indicated antibodies. The treatment of C
and D was the same as in B; the
expression of proteins that consist of the
mTOR autoamplification loop was
detected by Western blotting.
A
NA
D
pc
E
A-
DN
pc
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C
pEGFR
EG
FR
EGFR
DEPTOR
b-Actin
D
+E
FR
i
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B
FR
EG
ef
G
+E
FR
EG
+G
FR
EG
pEGFR
EGFR
FR
EG
EG
+G
FR
EG
pmTOR
pmTOR
mTOR
mTOR
CK1a
CK1a
p-S6K
p-S6K
S6K
S6K
bTrCP1
bTrCP1
b-Actin
b-Actin
b
i
tin
ib
in
it
ef
F
DEPTOR
b-Actin
Cell proliferation assay
A cell proliferation assay was carried out with a Cell Counting
Kit-8 (Beyotime; ref. 36). A549 or stable transfected cells were
plated in 96-well plates at approximately 2 103 cells per well.
The numbers of cells per well were detected by the absorbance
(450 nm) of reduced WST-8 at the indicated time points. The
absorbance (450 nm) was measured by using SpectraMax i3x
microplate reader (Molecular Devices).
Xenograft mouse model
Six-to-eight-week-old female BALBc/nude mice were purchased from the Laboratory Animal Center of Wuhan University (Wuhan, China) and maintained in cages under sterile
conditions with a specific pathogen-free environment. A549
cells, EGFR-overexpressing A549 cells, or EGFR/DEPTOR double overexpressing cells (1 106) were injected subcutaneously
within a volume of 100 mL in the flank at the time of inoculation (39). Tumor size was measured by external caliper
measurement every three days after injection, and tumor volume was calculated as 1/2 (tumor length) (tumor width)2
(40). Mice were sacrificed, and tumors were dissected and
measured on day 21 after inoculation.
Wound-healing assay
Cell migration was determined by a wound-healing assay, and
cells were seeded into 12-well plate until reached 90% confluence.
Cell monolayers were carefully scratched using a sterile 200-mL
pipette tip, and the cellular debris was subsequently removed by
washing with PBS to form wound gaps (37). The wound location
was marked, and cells were photographed at 0 and 24 hours to
measure the cell migration ability by using an IX70 microscope
(Olympus GmbH).
Statistical analysis
All data are presented as mean SD and analyzed by using
Graphpad Prism V.5.00 software (GraphPad Software). A Spearman correlation test was used to assess the association between
mRNA expression of EGFR and DEPTOR in tumor tissues. A
comparison between two groups for statistical significance was
performed with an unpaired Student t test. For more groups, oneway ANOVA followed by Neuman–Keuls post hoc test was used.
P < 0.05 was considered statistically significant.
Invasion assay
Cell invasion was determined by the Transwell assay. A total of
3 104 cells in 0.5% FBS medium were transferred on the top of
the Matrigel-coated invasion chambers (8-mm pore; Corning
Costar), and 10% FBS medium was added in the lower chamber.
After 48 hours of incubation, noninvasion cells were removed by
wiping with a cotton swab. The invading cells that had invaded the
bottom side of the membrane were then fixed with 4% formaldehyde and stained with 40 ,6-dia-midino-2-phenylindole (DAPI;
ref. 38). The numbers of invasive cells were obtained by counting
five fields (100) per membrane and represented the average of
three independent experiments.
Results
2000 according to the manufacturer's instructions. Forty-eight
hours after transfection, aspirate the medium and replace with
fresh medium containing puromycin (1 mg/mL) to select the
stable transfected cells.
www.aacrjournals.org
mRNA expression of EGFR negatively correlates with DEPTOR
in lung adenocarcinoma tissues
The abnormal hyperactivity of EGFR and its downstream
mTOR pathways is the leading cause of resistance to TKIs; furthermore, mTOR inhibitors could effectively control the growth
of lung adenocarcinoma even after acquiring resistance to TKIs
(13, 41, 42). In order to further unveil the downstream regulatory
molecule mechanisms involved in EGFR/mTOR activation, we
simultaneously analyzed mRNA expression of EGFR and DEPTOR, a naturally occurring inhibitor of mTOR, in tumor tissues of
patients with lung adenocarcinoma. As shown in Fig. 1A, the
Mol Cancer Res; 14(5) May 2016
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451
Published OnlineFirst February 19, 2016; DOI: 10.1158/1541-7786.MCR-15-0480
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Zhou et al.
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pEGFR
EGFR
24 h
DEPTOR
b-Actin
D
20
0
N
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D
pc
Hours
96
72
48
24
50
mRNA level of EGFR is significantly upregulated in lung carcinoma tissues than in adjacent normal compartment, and DEPTOR is downregulated in tumor tissues (Fig. 1B). In addition,
this result is further confirmed by Western blotting (Fig. 1D)
and immunohistochemistry analysis (Fig. 2A). Next, we then
examined potential correlativity between aberrant EGFR and
DEPTOR expression, Pearson correlation assay revealed that
mRNA expression of EGFR significantly and negatively correlated with DEPTOR expression in tumor tissues (r2 ¼ 0.6247,
P < 0.0001; Fig. 1C). As DEPTOR has been reported to inhibit
tumor cell growth of lung adenocarcinoma (34), these findings
suggest that there may be a link between EGFR and DEPTOR to
regulate tumor growth.
EGFR activation inhibits DEPTOR expression via enhancing the
function of the mTOR autoamplification loop
Next, we detected EGFR and DEPTOR expression in three lung
adenocarcinoma cell lines, including A549, PC-9, and NCIH1975. Compared with normal human lung epithelial cells,
BEAS-2B, protein (Fig. 2B) and mRNA (Fig. 2C) expression of
EGFR were significantly increased in these tumor cells, and
DEPOR expression was decreased at different levels (Fig. 2B and
D). As A549 cells occupy the lowest level of DEPTOR expression
and a relative higher level of EGFR expression, we then used A549
cells in our following experiments.
A549 cells were transfected with pcDNA3.1-EGFR to construct
stable EGFR overexpressing cell line, and in comparison with
empty vector, overexpression of EGFR significantly decreased
452 Mol Cancer Res; 14(5) May 2016
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50
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A pcD
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-D N
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EP A
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60
200
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150
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pc
Wound closure (%)
E 100
200
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pcDNA
pcDNA-DEPTOR
Control
DEPTOR knockout
A pcD
-D N
EP
EP A
TO
T
R Co OR
kn nt
oc ro
ko l
ut
300
Number of invaded cells/field
B
Figure 4.
DEPTOR inhibits proliferation,
migration, and invasion of human lung
adenocarcinoma cells. A, DEPTOR
ectopic expression A549 cells and
knockout cells were constructed as
described in Materials and Methods,
and then cell lysates were subjected to
Western blotting analysis with DEPTOR
antibody. b-Actin was used as a loading
control. B, cell proliferation of these cell
lines was examined with a Cell Counting
Kit-8 assay. C, cellular migration was
examined by using a wound-healing
assay, and migration was assessed after
24 hours. D, cellular invasion was
evaluated in Transwell chamber that
precoated with Matrigel, and then
stained with DAPI after 24 hours of
incubation. E, quantification of wound
closure. Histogram represents the
wound width as the mean SD of the
percentage of the closure of original
wound in triplicate plates. F,
quantification of invaded cells.
Histogram represents the invaded cells
of six random fields. Data presented as
Mean SD, and similar results were
obtained in three experiments. , P <
0.01; , P < 0.0001. Scale bar, 50 mm.
DEPTOR expression in A549 cells (Fig. 3A). Previous studies
revealed that EGF could induce the activation of EGFR kinase
activity, and Gefitinib is selective EGFR TKI that usually used to
block the EGFR signaling (43, 44). Herein we treated EGFR
overexpressing A549 cells with these two reagents to test the effect
of EGFR activation on DEPTOR expression. Our data showed that
EGF (20 ng/mL) strongly stimulated the phosphorylation of
EGFR and inhibited the expression of DEPTOR, while gefitinib
(5 mmol/L) inhibited the phosphorylation of EGFR and significantly enhanced DEPTOR expression (Fig. 3B).
As studies have identified that S6K, CK1a, and bTrCP1 could be
cooperated with mTOR to generate an autoamplification loop
and thus trigger the degradation of DEPTOR (27, 30), we then
detected the expression of these proteins with the presence of EGF
or gefitinib. EGF significantly enhanced the phosphorylation of
mTOR and S6K, as well as the expression of CK1a and bTrCP1
(Fig. 3C). Furthermore, blocking the activation of EGFR with
gefitinib showed the opposite function on these four proteins'
expression with EGF (Fig. 3D). Thus, we confirmed that EGFR
activation enhances the function of mTOR autoamplification
loop to downregulate DEPTOR expression.
DEPTOR suppresses cellular proliferation, migration, and
invasion of lung adenocarcinoma cells
Given that EGFR inhibited DEPTOR expression in A549 cells, we
next investigated the potential roles of DEPTOR in lung adenocarcinoma development. First, we constructed two stable A549 cell
lines, DEPTOR overexpressing cells and DEPTOR knockout cells,
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Published OnlineFirst February 19, 2016; DOI: 10.1158/1541-7786.MCR-15-0480
EGFR and DEPTOR in Lung Cancer Progression
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/V
FR
FR
EG
EG
O
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FR
FR
EG
EG
E
/D
FR
EG
0h
EGFR
DEPTOR
24 h
b-Actin
48 h
120
B
D
90
80
60
40
20
24
Time (h)
by using plasmid transfection or DEPTOR CRISPR/Cas9 system
(Fig. 4A). Compared with empty vector control, overexpression of
DEPTOR significantly decreased cell proliferation (Fig. 4B) and
resulted in reduced abilities of migration (Fig. 4C and D) and
invasion (Fig. 4D and F). However, in DEPTOR knockout cells,
these tumor-related activities were all significantly enhanced as
compared with those of parental control cells (Fig. 4B–F). Thus,
these results confirmed that DEPTOR acts as a tumor suppressor in
the tumorigenesis of lung adenocarcinoma.
DEPTOR ectopic expression suppresses EGFR-driven
tumorigenesis
Because EGFR has been found to widely overexpress and
promote tumor progress in lung adenocarcinoma (15, 17), we
consider whether DEPTOR ectopic expression could reverse the
tumor- promoting effects of EGFR. EGFR overexpressing A549
cells were then further stably transfected with pcDNA-DEPTOR
plasmid. As shown in Fig. 5A, pcDNA-DEPTOR plasmid transfection significantly elevated DEPTOR expression in EGFR overexpressing A549 cells. Simultaneously enforcing DEPTOR expression in EGFR ectopic expression A549 cells markedly decreased
EGFR expression and its phosphorylation level. Analysis of
tumor-related activities further revealed that DEPTOR inhibited
the cell proliferation (Fig. 5B), migration (Fig. 5C and E), and
invasion (Fig. 5D and F) of EGFR ectopic expression in A549 cells.
Thus, these results demonstrated that ectopic expression of DEP-
200
150
100
50
0
EG FR/ FR
FR Vec
/D to
EP r
TO
R
100
F
EG
Hours
EGFR
EGFR/Vector
EGFR/DEPTOR
120
48
EG
96
72
48
0
EGFR
EGFR/Vector
EGFR/DEPTOR
24
30
Wound closure (%)
E
Number of invaded cells/field
60
0
www.aacrjournals.org
/
FR
EG
C
P
DE
pEGFR
Cell growth rate (%)
Figure 5.
DEPTOR ectopic expression suppresses
EGFR-mediated tumor-promoting
effects in human lung adenocarcinoma
cells. A, DEPTOR and EGFR double
overexpressing A549 cells were
constructed, and then cell lysates were
subjected to Western blotting analysis
with indicated antibodies; b-actin was
used as a loading control. B, cell
proliferation of three cell lines was
examined with a Cell Counting Kit-8
assay. C, cellular migration was
examined by using a wound-healing
assay, and migration was assessed after
24 and 48 hours. D, cellular invasion was
evaluated in the Transwell chamber that
precoated with Matrigel, and then
stained with DAPI after 24 hours of
incubation. E, quantification of wound
closure. Histogram represents the
wound width as the mean SD of the
percentage of the closure of original
wound in triplicate plates. F,
quantification of invaded cells.
Histogram represents the invaded cells
of six random fields. Data presented as
Mean SD, and similar results were
obtained in three experiments.
, P < 0.01; , P < 0.0001. Scale bar,
50 mm.
A
ec
/V
TOR reverses EGFR-driven tumorigenesis in lung adenocarcinoma cells.
DEPTOR provides feedback promotion on protein degradation
of EGFR
We noted that ectopic expression of DEPTOR significantly
suppressed EGFR expression and its phosphorylation (Figs. 4A
and 5A). To determine if protein synthesis was involved
in DEPTOR-induced suppression of EGFR, cycloheximide (5
mg/mL) was used to inhibit protein synthesis, which at this dose
has been shown to have an inhibitory effect on protein translation
(45, 46). As shown in Fig. 6A and B, ectopic expression of DEPOR
significantly promoted the degradation of EGFR protein. To
further confirm the role of DEPTOR on mRNA expression of
EGFR, the mRNA expression levels of EGFR in DEPTOR overexpression or knockout cells were detected, and there is no
detectable difference in EGFR mRNA expression in response to
DEPTOR expression (Fig. 6C). Next, actinomycin D (5 mg/mL;
refs. 47, 48) was used to block mRNA transcription and then EGFR
mRNA half-life was evaluated. Followed with actinomycin D
treatment, no significant difference was seen in the rate of EGFR
mRNA decay in response to DEPTOR expression (Fig. 6D). These
results revealed that DEPTOR provides a feedback promotion on
protein degradation of EGFR and does not influence EGFR mRNA
transcription. So there is a reciprocal negative regulation between
DEPTOR and EGFR expression.
Mol Cancer Res; 14(5) May 2016
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453
Published OnlineFirst February 19, 2016; DOI: 10.1158/1541-7786.MCR-15-0480
Zhou et al.
A
Cycloheximide (5 mg/mL)
Control
min
30
0
DEPTOR-knockout
pcDNA-DEPTOR
60
0
120
30
60
0
120
30
60
120
EGFR
b-Actin
1.0
0.8
0.6
0.4
0.2
TO
tro
EP
Control
pcDNA-DEPTOR
DEPTOR-knockout
60
40
20
0
2
4
6
8
Hours after actinomycin D administration
TO
N
R
A
-k
-D
C
D
EP
D
-k
R
TO
EP
D
pc
no
EP
-D
A
N
80
0
on
t
TO
ck
ou
R
l
tro
on
C
D
pc
100
0.0
l
0.0
1.2
Degredation of EGFR mRNA (%)
0.5
D
t
1.0
C
ck
ou
60 min
120 min
R
0 min
30 min
no
Relative EGFR protein expression
1.5
Relative EGFR mRNA expression
B
Figure 6.
DEPTOR increases EGFR protein degradation, but not inhibits EGFR mRNA transcription. A, normal A549 cells (control), DEPTOR ectopic expression A549 cells and
knockout cells were treated with cycloheximide (5 mg/mL), and then cell lysates at different time points were subjected to Western blotting analysis
with EGFR antibody; b-actin was used as a loading control. B, relative expression level of EGFR was quantified by ImageJ software. C, mRNA expression of EGFR in
these three cell lines that cultured at normal condition was detected by qRT-PCR. D, similar to A, three cell lines were treated with 5 mg/mL actinomycin
D, and then total mRNA was extracted at 1, 2, 4, 6, and 8 hours following the addition of actinomycin D. EGFR mRNA expression was quantified by qRT-PCR, and then
the degradation percentage of mRNA in each time point was calculated by comparing with normal condition. Data presented as Mean SD (n ¼ 3).
, P < 0.01; , P < 0.0001.
DEPTOR ectopic expression suppresses EGFR-driven tumor
growth in vivo
The inhibitory effect of DEPTOR on the growth of lung adenocarcinoma cells in vivo was evaluated by using nude mouse
models. As pcDNA empty vector stable transfection showed no
detectable influence on EGFR and DEPTOR expression, as well as
the proliferation, migration and invasion of A549 cells (Fig. 5), we
used normal A549 cells as a control. EGFR overexpressing A549
cells and EGFR/DEPTOR double overexpressing A549 cells were
injected into the flank of nude mice. The volume of tumor
xenografts was monitored every 3 days. At day 21, mice were
sacrificed, and the tumor xenograft of each mouse was dissected
(Fig. 7A). As compared with normal control (214 66 mm3),
EGFR overexpressing A549 cells significantly promoted the
growth of xenografts in nude mice (570 132 mm3), while
simultaneously transfected with EDPTOR overexpressing (169 65 mm3) almost totally abrogated EGFR-enhanced tumor growth
(Fig. 7B), as well as tumor weights (Fig. 7C). Taken together, these
in vivo tumor growth results were in agreement with our in vitro
results and indicated that DEPTOR is a pivotal tumor suppressor
454 Mol Cancer Res; 14(5) May 2016
that was downregulated by the EGFR signaling pathway to promote tumor growth of lung adenocarcinoma.
Discussion
The EGFR signaling pathway plays a critical role in promoting
cancer cell growth and survival, which serves as an attractive
therapeutic target, is attracting even more and more attention
(49). EGFR tyrosine kinase inhibitors (TKI), such as gefitinib,
which operate by repression of EGFR oncogenic signaling, have
proven to provide a significant response and survival benefit for
patients with lung cancer. However, all responders eventually
acquire chemotherapy resistance (15, 50), and lung cancer is still
estimated to be responsible for nearly one in five (1.59 million
deaths, 19.4% of the total) of death from cancer worldwide
(http://globocan.iarc.fr/Default.aspx). Because we found DEPTOR upregulated in response to gefitnib treatment (Fig. 3B), it
would be interesting to test the role of DEPTOR in gefitnib
resistance, the exact molecular mechanisms of which we are still
unclear about.
Molecular Cancer Research
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Published OnlineFirst February 19, 2016; DOI: 10.1158/1541-7786.MCR-15-0480
EGFR and DEPTOR in Lung Cancer Progression
EGFR
EGFR/DEPTOR
C
800
Control
EGFR
EGFR/DEPTOR
700
600
0.5
Tumor weight (g)
500
400
300
200
100
0.4
0.3
0.2
0.1
R
EG
FR
/D
EP
TO
l
EG
tro
21
18
9
12
15
6
3
0
Day after inoculation
FR
0.0
0
on
B
Tumor volume (mm3)
Figure 7.
DEPTOR ectopic expression suppresses
EGFR-driven in vivo tumor growth of
human lung adenocarcinoma cells. Six-toeight-week-old female BALBc/nude mice
were subcutaneously injected with normal
A549 cells (control), EGFR overexpressing
A549 cells or EGFR/DEPTOR double overexpressing cells in the flank of mice
5
(1 10 ) in a volume of 100 mL at the time of
inoculation. A, the mice were sacrificed and
the tumors were dissected on the 21th day
after inoculation. B, tumor size was
measured by using external caliper every 3
days after injection. C, tumors in different
groups were weighted. Data presented as
mean SD; six mice for each group.
, P < 0.01; , P < 0.0001.
Control
C
A
One of the downstream pathways controlled by EGFR involves
mTOR, a proto-oncogene activated in various cancers (51). The
kinase mTOR is a critical target of EGFR signaling, linking growth
factor abundance to cell growth and proliferation (52). DEPTOR,
as a natural negative regulator of mTOR, has been reported to
involve in sensitive to apoptosis gene (SAG)-induced lung tumor
formation and may inhibit cell growth of lung adenocarcinoma
(34); furthermore, loss of DEPTOR increased drug resistance to
EGFR TKIs of lung adenocarcinoma cells (53). Thus, we questioned whether DEPTOR serves as a tumor suppressor and EGFR
downregulates DEPTOR expression in lung adenocarcinoma.
To answer this question, we first detected the mRNA expression
of EGFR and DEPTOR in clinical samples from patients with lung
adenocarcinoma. Our results showed that EGFR expression
increases with TNM stage and lymph node metastasis, while
DEPTOR shows a contrary tendency (Table 1), and there is
significant negative correlation between EGFR and DEPTOR
expression (Fig. 1). In the EGFR signaling cascade, ligand stimulation promotes EGFR phosphorylation and then leads to receptor internalization and downstream signaling activation (54). In
lung adenocarcinoma patients, we also observed that EGFR/
pEGFR is partially expressed in the cytoplasm that DEPTOR
mainly located (Fig. 2A).
Next, we chose human lung adenocarcinoma A549 cell lines to
conduct our study and cloned EGFR overexpressing A549 cells.
Compared with parental cells, EGFR ectopic expression significantly decreased DEPTOR expression; this could be further
enhanced with the presence of EGF, EGFR ligand. More importantly, DEPTOR expression was enhanced with the stimulation of
gefitinib. The following molecular analysis further identified that
EGFR activation enhanced mTOR and S6K phosphorylation,
CK1a and bTrCP1 expression. Of these four molecules, S6K is
www.aacrjournals.org
a direct downstream molecule of mTOR (55) and consists of the
autoamplification loop of mTOR to degrade DEPTOR expression
(27, 30). CK1a and bTrCP1 are involved in promoting tumor
progression and have been found to act synergistically with EGFR
to inhibit cell expansion of lung cancer (56, 57), but how EGFR
activation stimulates CK1a and bTrCP1 expression is still
unknown and needs to be identified in our future study.
The role of DEPTOR expression in the progression of human
malignancies is still controversial; this may be because the regulation of DEPTOR is complicated in different individuals and
tissues (28, 31), as two opposite results were obtained in the same
myeloma cells (31, 58). In lung adenocarcinoma, there is still no
direct evidence that DEPTOR regulates tumor progression. By
constructing two stable A549 cell lines, namely, DEPTOR overexpression or knockout, we first demonstrated that DEPTOR acts
as a tumor suppressor in the tumorigenesis of lung adenocarcinoma, as DEPTOR inhibited cell proliferation, migration, invasion, and in vivo tumor growth. Furthermore, simultaneous
knockin DEPTOR in EGFR overexpressing A549 cells suppressed
EGFR expression and markedly abolished EGFR-driven tumorigenesis of lung adenocarcinoma. Accompanied with the observation that DEPTOR expression provides a feedback promotion
on EGFR protein degradation, more detailed molecular mechanisms need to be investigated in the future.
Of course, there are still some potential problems with our
current study. For example, it is well known that CRISPR/Cas9mediated gene editing has off-target effects. Although we believe
that the guide RNA sequence has been tested by the commercial
provider for our study, there might still be chances that our guide
RNA can target non-DEPTOR sequence in human genome. We
will take this into consideration in future studies. Inspired by two
elegant studies from leading scientists in the CRISPR/Cas9
Mol Cancer Res; 14(5) May 2016
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455
Published OnlineFirst February 19, 2016; DOI: 10.1158/1541-7786.MCR-15-0480
Zhou et al.
research field (59, 60), we will try to use modified Cas9 nucleases
with high fidelity in our future studies, with the hope of minimal
off-target effects.
In conclusion, our study reveals a novel link between EGFR and
DEPTOR, and that EGFR promotes DEPTOR degradation by
enhancing the function of the mTOR autoamplification loop,
including increased mTOR and S6K phosphorylation, as well as
CK1a and bTrCP1 expression. Furthermore, we also provide solid
experimental evidence that DEPTOR plays a tumor suppressive
role in lung adenocarcinoma cells, and ectopic expression of
DEPTOR could suppress EGFR-driven tumor progression.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): X. Zhou, J. Guo, G. Pan, T. Liu
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): X. Zhou, J. Guo, Y. Ji, H. Zhu, J. Zhao
Writing, review, and/or revision of the manuscript: X. Zhou, J. Guo, Y. Ji,
H. Zhu, J. Zhao
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): X. Zhou, J. Guo, G. Pan, T. Liu, J. Zhao
Study supervision: X. Zhou, J. Guo, J. Zhao
Grant Support
This work was supported by the National Natural Science Foundation of
China (grant no. 81101775) and American Heart Association Grant
12SDG12070174.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Authors' Contributions
Conception and design: X. Zhou, J. Guo, H. Zhu, J. Zhao
Development of methodology: X. Zhou, J. Guo, H. Zhu
Received December 14, 2015; revised February 10, 2016; accepted February
10, 2016; published OnlineFirst February 19, 2016.
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