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ORIGINAL ARTICLE
Reduced expression of PTEN and increased PTEN
phosphorylation at residue Ser380 in gastric cancer
tissues: A novel mechanism of PTEN inactivation
Zhen Yang a,1,2,3,4, Xiao-Gang Yuan a,2, Jiang Chen a,2, Shi-Wen Luo a,b,4,
Zhi-Jun Luo c,4, Nong-Hua Lu a,1,4,∗
a
Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, 330006 Jiangxi, PR China
Center for Laboratory Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, 330006 Jiangxi, PR China
c
Department of Biochemistry, Boston University School of Medicine, MA 02118 Boston, USA
b
Summary
Aim: PTEN is a tumor suppressor gene in different cancers. This study was to determine the
protein expression of PTEN and phosphorylation of PTEN (p-PTEN) at residue Ser380 in different
histology specimens of gastric tissues.
Methods: A total of 179 tissue specimens of normal gastric mucosa, chronic gastritis, intestinal
metaplasia, dysplasia, and gastric cancer were recruited for immunohistochemical analysis of
PTEN and p-PTEN expression. Four gastric cancer AGS, MKN-45, MKN-28, and SGC-7901 cell lines
and a non-cancerous gastric GES-1 cell line were used to detect expression of PTEN and p-PTEN
protein using Western blot.
Results: Expression level of PTEN protein was significantly decreased in gastric cancer tissues compared to normal gastric mucosa, chronic gastritis, intestinal metaplasia and dysplasia
(P < 0.05). In contrast, p-PTEN protein level was significantly increased in intestinal metaplasia,
dysplasia and gastric cancer compared to normal gastric mucosa and chronic gastritis (P < 0.05).
However, there was no any association of PTEN and p-PTEN expression with clinicopathological
characteristics from gastric cancer patients. Moreover, the ratio of p-PTEN and PTEN was higher
in gastric cancer cell lines than that of the non-malignant cells.
Conclusions: This study demonstrated that aberrant expression of PTEN and p-PTEN at residue
Ser380 was early event that could contribute to gastric carcinogenesis, and that PTEN phosphorylation at residue Ser380 could be a mechanism for PTEN inactivation.
© 2012 Elsevier Masson SAS. All rights reserved.
∗
1
2
3
4
Corresponding author. Tel.: +86 791 88692705; fax: +86 791 88623153.
E-mail address: [email protected] (N.-H. Lu).
Designed the study.
Performed the study.
Analyzed the data.
Drafted the manuscript.
2210-7401/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.clinre.2012.03.002
Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at
residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012),
doi:10.1016/j.clinre.2012.03.002
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2
Z. Yang et al.
Introduction
Gastric cancer is the fourth most common cancer worldwide, with an estimated one million new cases in 2007.
Gastric cancer is the second leading cause of cancer-related
death in men and the fourth among women resulting in
approximately 800,000 deaths in 2007, indicating that it is
still one of the most significant health burdens in the world,
although the incidence and mortality of gastric cancer have
declined [1,2]. To date, surgical resection remains the only
curative means to improve survival of patients with gastric
cancer [3], while chemotherapy has limited effects and is
usually served to palliatively relieve patients’ symptoms and
increase survival time. Like most other cancers, the underlying mechanism responsible for gastric cancer development
and progression is still poorly understood. Therefore, it is
necessary to investigate the mechanisms of gastric carcinogenesis and to develop novel strategies to prevent
gastric cancer. For this purpose, our research objective
focuses on PTEN (phosphatase and tensin homolog deleted
on chromosome ten), a tumor suppressor gene identified in
1997 by three independent groups using slightly different
strategies [4—6]. PTEN encodes a dually functional phosphatase with lipid and protein phosphatase activities and is
involved in regulation of a variety of signaling transduction
pathways [7—10], which are critical in cell differentiation,
apoptosis, adhesion, and mobility as well as reproduction.
PTEN is frequently inactivated in different human cancers
[4,5,11]. Recent studies have indicated that loss or reduced
expression of PTEN commonly occurs in gastric cancers
due to genetic or epigenetic changes, such as mutation,
loss of heterozygosity (LOH), and promoter hypermethylation [12—16]. However, it remains to be defined whether
other mechanisms account for inactivation of PTEN and how
PTEN inactivation promotes carcinogenesis. Indeed, several
recent studies have demonstrated that PTEN protein can
be regulated by a variety of other mechanisms, including
phosphorylation, oxidation, lipid ligands and protein binding partners. Among these, phosphorylation is the most
important mechanism of post-translational modification of
PTEN. To date, there are multiple phosphorylation sites in
PTEN protein, such as Ser380, Thr382, and Thr383, which
leads to a loss of phosphatase activities or a gain of PTEN
stability [17,18], and in turn results in loss of tumor suppressor function and increased cancer susceptibility. Among
the phosphorylation sites of PTEN protein, Ser380 is more
critical for regulation of PTEN function [17]. Development
of gastric cancer is a multistep process from normal gastric
mucosa to chronic gastritis, intestinal metaplasia, dysplasia, and to invasive cancer [19]. In this study, we aimed to
determine expression of PTEN vs. phosphorylated PTEN (pPTEN) at residue Ser380 in different stages of gastric lesions
and to gain an insight into the role of PTEN protein and most
importantly, p-PTEN in gastric cancer development.
Patients and methods
Patients
Gastric tissue samples were collected from patients who
underwent a gastroduodenoscopy at the First Affiliated
Hospital of Nanchang University from January 2007 to
September 2009. A total of 179 patients were enrolled
in this study, which included 20 cases of normal gastric mucosa, 21 chronic gastritis, 40 intestinal metaplasia,
48 dysplasia (including 33 low-grade and 15 high-grade
intraepithelial neoplasia), and 50 gastric cancers. The clinical characteristics of these patients are summarized in
Table 1 and there was no significant difference in the age
and gender distribution among these groups. The clinicopathological characteristics were also obtained from 43 of
50 patients with gastric carcinoma who underwent gastrectomy. This study was approved by the ethics committee of
the First Affiliated Hospital of Nanchang University.
Histological examinations
All biopsies or surgical specimens from patients with
different gastric lesions were taken from the gastric antrum
and individual lesions from patients. The tissues used for
histological analysis were fixed in 10% formaldehyde in Ca2+
and Mg2+ -free phosphate buffered saline (PBS) overnight at
4 ◦ C before paraffin embedding. Paraffin sections of 4 ␮m
were cut with a microtome and stored at room temperature.
Pathologic diagnosis and classification were made according
to the criteria of the World Health Organization [20] and the
updated Sydney system [21].
Immunohistochemistry
Paraffin sections were prepared form these gastric tissues
or biopsy specimens. For immunohistochemistry, these tissue sections were first dewaxed in xylene and sequentially
dehydrated in 100%, 95%, and 85% ethanol. The sections
were immunostained using the PV-9000 Polymer Detection
System (Zhongshan Golden-bridge, Beijing, China) according
to the standard staining protocol. Briefly, the sections
were first washed in PBS and endogenous peroxidase was
blocked using 3% H2 O2 . After the specimens were incubated with the primary antibody overnight at 4 o C (rabbit
polyclonal anti-human PTEN [ab31392] and monoclonal antihuman p-PTEN [Ser 380] [ab76431], diluted to 1:150, Abcam,
Cambridge, UK), they were washed with PBS again, followed by incubation with polymer helper for 30 min and
polyperoxidase-anti-rabbit IgG for 30 min. After washing
three times with PBS, the sections were further incubated
with 3,3-diaminobenzidin (DAB, Zhongshan Golden-bridge)
for developing positive color. The negative control sections
were just incubated with PBS without the primary antibodies. After that, the sections were counterstained with
hematoxylin and mounted with coverslips.
Review and scoring of immunostained sections
The stained sections were reviewed and scored under a light
microscope by two pathologists without knowing the clinicopathological data. The concordance rates were generally
high and data with any grading discrepancies were rereviewed and discussed by them to make a final score.
Epithelial cells stained positively with yellow or brown color
in the nuclear and/or cytoplasm were defined as positive
Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at
residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012),
doi:10.1016/j.clinre.2012.03.002
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Reduced expression of PTEN and increased PTEN phosphorylation in gastric cancer tissues
1
1
1
1
2
2
2
3
3
4
0
0
0
0
0
0
0
0
6
2
1
9
5.0
9.5
82.5
60.4
44.0
48.6
Cell lines and culture
1
2
27
27
21
78
+++
19
19
7
19
28
92
The immortalized gastric epithelial mucosa cell line GES-1,
established by Beijing Institute for Cancer Research (Beijing,
China), was a kind gift from Prof. Y Ke of Beijing Institute
for Cancer Research. Human gastric cancer SGC7901 (moderately differentiated), MKN28 (moderately differentiated),
MKN45 (poorly differentiated) and AGS (poorly differentiated) cell lines were kind gifts from Prof. DM Fan of
Xijing Hospital (Xi’an, China). These cell lines were cultured at 37 o C in an atmosphere of 5% CO2 in Dulbecco
modified Eagle’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum, 100 unit penicillin, and
100 ␮g/mL streptomycin (Gibco BRL, Life Technologies, New
York, USA).
1
1
1
1
2
2
2
3
3
4
85.0
95.2
67.5
72.9
40.0
66.5
4
2
4
4
1
15
4
8
8
15
9
44
9
10
15
16
10
60
20
21
40
48
50
179
Normal gastric mucosa1
Chronic gastritis2
Intestinal metaplasia3
Dysplasia4
Gastric cancer5
Overall
PR: positive rate.
a P < 0.05 was considered as statistically significant.
3
1
13
13
30
60
12/8
10/11
21/19
26/22
31/19
100/79
±
±
±
±
±
±
n
50.1
54.2
54.9
57.4
54.0
54.7
14.5
14.7
12.3
12.5
12.3
13.8
(M/F)
(mean ± SD)
−
Sex
Age
PTEN
+
++
+++
PR (%)
P
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
2:
3:
4:
5:
3:
4:
5:
4:
5:
5:
0.685
0.167
0.410
0.001a
0.048a
0.185
0.000a
0.458
0.017a
0.001a
−
p-PTEN
+
++
PR (%)
P
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
2:
3:
4:
5:
3:
4:
5:
4:
5:
5:
0.583
0.000a
0.000a
0.002a
0.000a
0.000a
0.005a
0.010a
0.000a
0.098
staining. Each section was chosen, reviewed, and scored
from five randomly selected fields (magnification of × 200).
In each microscopic field from the representative areas,
100 immunoreactive cells were assessed and quantified as
the percentage and then averaged from the five fields
for % of immunostaining, i.e. 0, < 5.0%; 1, 5.1—25.0%; 2,
25.1—50.0%; 3, 50.1—75.0%; and 4 > 75.0%. Moreover, the
staining intensity was also semi-quantitatively assessed as
0, no staining; 1, weak staining; 2, moderate staining;
and 3, strong staining. After that, the integrals of the
‘‘area × intensity’’ were calculated, based on which the
overall expression levels of the proteins in the section as
follows: negative (−), score 0—2; weak positive (+), score
3—5; moderate positive (++), score 6—8; and strong positive
(+++), score 9—12.
Protein extraction and Western blotting
Group
Differential expression of PTEN and p-PTEN at different histology of gastric tissue specimens.
Table 1
3
Approximately 5 × 106 cells at log growth phase were collected and lysed in a buffer containing 0.5% Lubrol-PX,
50 mM KCl, 2 mM CaCl2 , 20% glycerol, 50 mM Tris-HCl (pH
7.4), and 0.1% protease and 1% phosphatase inhibitors
(Sigma-Aldrich, Saint-Louis, MO, USA). The concentrations of protein sample were measured based on the
BCA’s method (Pierce Biotechnology, Rockford, IL, USA).
Sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (4 ×) sample buffer (250 mM Tris-HCl, pH6.8,
4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% ␤mercaptoethanol) was added to the protein samples and
in turn denatured by boiling for 5 min in a water bath.
Samples containing 50 ␮g of protein were analyzed on 10%
SDS-PAGE gels and electroblotted onto nitrocellulose membranes (Whatman GmbH, Dassel, Germany). After blocking
with 5% nonfat milk in Tris-buffered saline (TBS) containing
0.1% Tween-20 (TBST), the blots were incubated overnight
with the primary antibody at 4 o C (i.e., a rabbit monoclonal anti-human PTEN [#9559] at a dilution of 1:1,000 from
Cell Signaling Biotechnology [Danvers, MA, USA] or a monoclonal anti-human p-PTEN [Ser 380] [ab76431] antibody at a
dilution of 1:1,000 from Abcam [Cambridge, UK], or a rabbit polyclonal anti-human ␤-actin antibody [sc-1615-R] at
1:1,000 dilution from Santa Cruz Biotechnology [Santa Cruz,
CA, USA]). In the next day, HRP-conjugated goat anti-rabbit
IgG (diluted 1:10,000, Zhongshan Golden-bridge) was used
as a secondary antibody and incubated with the membrane
Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at
residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012),
doi:10.1016/j.clinre.2012.03.002
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Z. Yang et al.
Figure 1 Immunohistochemical staining of PTEN and p-PTEN protein in different histology of gastric tissues. Magnification at × 200.
A—E. The expression of PTEN in normal gastric mucosa (A), chronic gastritis (B), intestinal metaplasia (C), dysplasia (D), and gastric
cancer (E). F—J. The expression of p-PTEN in normal gastric mucosa (F), chronic gastritis (G), intestinal metaplasia (H), dysplasia
(I), and gastric cancer (J).
Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at
residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012),
doi:10.1016/j.clinre.2012.03.002
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Reduced expression of PTEN and increased PTEN phosphorylation in gastric cancer tissues
5
at 4 o C for 4 h. The reactions were subjected to incubation with the enhanced chemiluminescence (ECL) detection
system (Pierce Biotechnology, Rockford, IL, USA) and then
exposed to X-ray film for visualizing the positive bands.
Statistical analysis
Data are summarized as mean ± standard deviation (SD)
or percentage of control. The Chi2 test (SPSS v. 16.0 for
windows; SPSS, Inc., Chicago, IL, USA) was performed to
evaluate difference in categorical variables, such as gender among different defined groups. The one-way ANOVA
(SPSS v.16.0) was used to determine the differences, such
as patients’ ages in numerical variables among the groups.
Kruskal-Wallis test or Mann-Whitney test (SPSS v.16.0) was
used to determine the differences in numerical variables
between differently defined groups. A P value of less than
0.05 was considered statistically significant.
Results
Differential expression of PTEN and p-PTEN in
different gastric lesions
Immunohistochemically, PTEN protein was primarily
expressed in the cytoplasm of epithelial cells, with
occasionally expression in the nuclei. The immunohistochemical data of PTEN expression was then analyzed
semi-quantitatively. The data showed that weak to strong
expression of PTEN was observed in 85.0%, 95.2%, 67.5%,
72.9%, and 40.0% of normal gastric mucosa, chronic
gastritis, intestinal metaplasia, dysplasia, and gastric
cancer, respectively. PTEN expression level was significantly decreased in gastric cancer compared to the normal
gastric mucosa, chronic gastritis, intestinal metaplasia, or
dysplasia (P < 0.05; Fig. 1 and Table 1). In addition, there
was significant difference in PTEN expression between
the tissue specimens of chronic gastritis and intestinal
metaplasia (P < 0.05; Fig. 1 and Table 1).
Moreover, p-PTEN was expressed in the cytoplasm of positive cells (Fig. 1). Weak to strong expression of p-PTEN
was observed in 5.0%, 9.5%, 82.5%, 60.4%, and 44.0% of
normal gastric mucosa, chronic gastritis, intestinal metaplasia, dysplasia, and gastric cancer, respectively, indicating
that p-PTEN expression level was significantly increased in
intestinal metaplasia, dysplasia, and gastric cancer compared to the normal gastric mucosa and chronic gastritis
(P < 0.05; Fig. 1 and Table 1). Again, there was significant
difference in p-PTEN expression between intestinal metaplasia and dysplasia or gastric cancer (P < 0.05; Fig. 1 and
Table 1).
Nevertheless, our data obtained from 43 cases of gastric
cancer do not show an obvious association between PTEN or
p-PTEN levels and clinicopathological grades (Table 2).
Figure 2 Expression of p-PTEN and PTEN in non-cancerous and
cancerous gastric cell lines by Western blot. A. Western blot.
These cell lines were grown for two days and then subjected to
total cellular protein isolation and Western blot analysis of PTEN
and p-PTEN expression. B. Quantitative data of A. Immunoblots
of PTEN and p-PTEN were scanned and the ratio of p-PTEN to
PTEN densitometric units was expressed as % of ␤-actin. * P < 0.05
compared with GES-1.
levels of both PTEN and p-PTEN protein were higher in gastric cancer cell lines (i.e., SGC-7901, MKN-28, MKN-45, and
AGS) than in non-cancerous GES-1 cells (Fig. 2A). Moreover, further analysis of data on Western blots revealed
that the PTEN phosphorylation and PTEN ratio was higher
in gastric cancer cell lines than that of non-malignant GES1 cells, e.g., 2.00 ± 0.37 (P < 0.05), 5.60 ± 0.88 (P < 0.05),
6.15 ± 0.72 (P < 0.05), and 3.20 ± 0.29 (P < 0.05) folds of
MKN-28, MKN-45, SGC-7901, and AGS, respectively to the
ratio of GES-1 (Fig. 2B).
Discussion
Expression of PTEN and p-PTEN in non-malignant
and malignant gastric cell lines
Next, we examined PTEN and p-PTEN expression in noncancerous and cancerous gastric cell lines and found that
In the present study, expression of PTEN protein was shown
to be progressively lost during gastric carcinogenesis from
normal gastric mucosa via chronic gastritis, intestinal metaplasia, and dysplasia to gastric cancer, whereas p-PTEN
Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at
residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012),
doi:10.1016/j.clinre.2012.03.002
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Z. Yang et al.
Table 2
Association between PTEN and p-PTEN expression and clinicopathological data from gastric cancer patients.
Characteristics
n
Overall score of the protein expression
PTEN
p-PTEN
−
+
++
+++
PR (%)
Gender
Male
Female
23
20
15
11
6
4
2
4
0
1
34.8
45.0
Age (years)
≥ 55
< 55
22
21
14
12
4
6
3
3
1
0
36.4
42.9
Location
Antrum
Body and cardia
24
19
14
12
7
3
2
4
1
0
41.7
36.8
Gross type
(Borrmann)
I + II
III + IV
Differentiation
Well and
moderately
Poorly and
undifferentiatedly
Invasive depth
Above submucosa
Muscularis propria
Below subserosa
TNM
I + II
III + IV
Lymph node
metastasis
+
−
P
−
+
++
13
15
7
4
3
1
0
0
43.5
25.0
13
15
5
6
4
0
0
0
40.9
28.6
15
13
5
6
4
0
0
0
37.5
31.6
+++
PR (%)
0.323
0.192
0.835
0.242
0.922
0.458
0.659
6
37
3
23
2
8
1
5
0
1
50.0
37.8
23
16
4
3
0
20
10
6
3
7
4
32
5
3
18
1
0
9
12
31
9
17
1
9
0.381
3
25
2
9
1
3
0
0
50.0
32.4
30.4
17
4
2
0
26.1
1
50.0
11
7
2
0
45.0
1
1
4
0
0
1
28.6
25.0
43.8
5
2
21
1
1
9
1
1
2
0
0
0
28.6
50.0
34.4
2
4
0
1
25.0
45.2
8
20
2
9
2
2
0
0
33.3
35.5
0.205
1 vs. 2: 1.000
1 vs. 3: 0.520
2 vs. 3: 0.649
0.241
0.322
17
9
8
0
6
2
0
1
45.2
25.0
1 vs. 2: 0.509
1 vs. 3: 0.895
2 vs. 3: 0.422
0.923
0.487
31
12
P
0.161
18
10
10
1
3
1
0
0
41.9
16.7
PR: positive rate.
protein was progressively increased from normal gastric
mucosa to intestinal metaplasia, and taking into account
total PTEN reduction, was sustained high expression in
dysplasia and gastric cancer. Moreover, these data were
also confirmed in gastric cancer cell lines compared to
the non-cancerous gastric cells. Nevertheless, the aberrant
expression of PTEN and p-PTEN protein was not associated with any of the clinicopathological data we collected,
such as gender, tumor differentiation, stages, and metastasis of gastric cancer. The current data demonstrates that
the altered PTEN protein expression is an early event and
may contribute to gastric cancer development. Detection
of altered PTEN and p-PTEN at residue Ser380 expression
could serve as a biomarker for diagnosis or risk prediction of
gastric cancer.
Our current findings confirm a previous observation
reported by Yang et al. [22] that expression of PTEN protein
was progressively decreased from normal gastric mucosa to
gastric cancer. In their study, expression of PTEN protein was
lower in the diffuse-type of gastric cancer than that of the
intestinal-type gastric cancer and was conversely associated
with lymph node metastasis of gastric cancer and poor and
undifferentiated cancer. The reason why we were unable
to show any association of PTEN expression with clinicopathological data might be because of the small sample size.
However, in accordance with our study, two previous studies
showed that the loss of PTEN expression was not associated
with clinicopathological data, such as tumor size, differentiation, and lymph node metastasis of gastric cancers [16,23].
The function of PTEN protein is to dephosphorylate
phosphatidylinositol second messengers, leading to blocking PI3 K signaling [18]. Phosphorylation of the three residues
(Ser380, Thr382, and Thr383) within the tail of PTEN protein
increases protein stability and inhibits PTEN phosphatase
activity [17]. Thus, previous studies have reported that
phosphorylation of PTEN protein at Ser380, Thr382, and
Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at
residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012),
doi:10.1016/j.clinre.2012.03.002
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Reduced expression of PTEN and increased PTEN phosphorylation in gastric cancer tissues
Thr383 results in the loss of tumor suppressor function of
PTEN [17,18], and Ser380 phosphorylation is more critical in
inactivation of PTEN protein [17]. In keeping with this, we
showed both reduction of PTEN abundance and increases in
its phosphorylation at Ser380, which might contribute to the
loss of PTEN function in cells, although other phosphorylation sites of PTEN protein are also worthwhile studying and
future development of antibodies will aid in these studies.
Our investigation of PTEN expression revealed that the
expression levels in cancer cell lines were higher than
that of normal gastric cells, which is in contrast to cancer specimens. The reason of this discrepancy remains to
be determined, but it might be attributed to the following;
first, we did not analyze enough normal gastric cell lines and
second, it is possible that cultured cancer cell lines somehow
overcome the suppressive mechanism of expression. Nevertheless, after the ratio of phosphorylated to total PTEN
was measured, we found that ratio was increased, which is
consistent with the data obtained from human cancer tissue specimens. Hence, this suggests that detection of the
increased PTEN phosphorylation is more reliable than that
of total of PTEN in determining malignancy of gastric cells.
Based on the data observed in the present study, we concluded that alterations in the expression of PTEN and p-PTEN
at residue Ser380 during gastric carcinogenesis are an early
event. It is true that their alterations first occur in lesions of
chronic gastritis, intestinal metaplasia, and dysplasia. Thus,
evaluation of changes in expression of PTEN and p-PTEN
proteins at residue Ser380 may be a useful biomarker for
prediction of the malignant transformation of non-cancerous
lesions. In a future study, we will expand the sample size to
confirm our findings.
However, there is a discrepancy in this study, i.e., PTEN
phosphorylation at residue Ser380 could increase PTEN protein stability, but our data showed reduced expression
of total PTEN protein. The reason for this discrepancy is
unknown. However, previous studies suggested that loss
or reduced expression of PTEN protein commonly occurs
in gastric cancers due to genetic or epigenetic changes,
such as mutation, LOH, and gene promoter hypermethylation [12—16]. We speculate that a combination of these
mechanisms may lead to reduction in PTEN expression, while
increased activity of some kinases is able to phosphorylate
the remaining PTEN protein and keep PTEN protein more
stable in the early stage of gastric carcinogenesis (such
as chronic gastritis and intestinal metaplasia). Reduction
of PTEN expression mainly occurs in intestinal metaplasia, whereas PTEN phosphorylation mainly starts to occur
in chronic gastritis. Once the alterations have occurred, the
altered expression of PTEN protein is sustained, which is why
expression of PTEN was shown to be progressively lost during
gastric carcinogenesis, whereas p-PTEN protein progressively increased from normal gastric mucosa to intestinal
metaplasia. However, further studies incorporating these
independent mechanisms are needed.
In conclusion, this study demonstrated that PTEN expression is progressively reduced from normal gastric mucosa
to gastric cancer, whereas p-PTEN protein increases in the
transition from normal gastric mucosa to intestinal metaplasia, and is sustained at high levels in dysplasia and
gastric cancer, indicating that alteration of PTEN and p-PTEN
at residue Ser380 protein may contribute to gastric caner
7
development. This study also supports the notion that phosphorylation of PTEN at Ser380 is a mechanism by which PTEN
inactivated during gastric carcinogenesis.
Disclosure of interest
The authors declare that they have no conflicts of interest
concerning this article.
Acknowledgements
This work was supported in part by grants from the
National Natural Science Foundation of China (Grant Number: 81060038) and Graduate Innovative Foundation of
Jiangxi Province, China (Grant Number: YC10A020). We
thank Medjaden Bioscience Limited for assisting in the
preparation of this manuscript.
References
[1] Garcia M, Jemal A, Ward EM, Center MM, Hao Y, Siegel RL,
et al. Global cancer facts & figures 2007. Atlanta, GA: American
cancer society, 2007.
[2] Parkin DM. International variation. Oncogene 2004;23:
6329—40.
[3] Jemal A, Siegal R, Xu J, Ward E. Cancer statistics, 2010. CA
Cancer J Clin 2010;60:277—300.
[4] Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI,
et al. PTEN, a putative protein tyrosine phosphatase gene
mutated in human brain, breast, and prostate cancer. Science
1997;275:1943—7.
[5] Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH,
et al. Identification of a candidate tumour suppressor gene,
MMAC1, at chromosome 10q23. 3 that is mutated in multiple
advanced cancers. Nat Genet 1997;15:356—62.
[6] Li DM, Sun H. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by
transforming growth factor beta. Cancer Res 1997;57:2124—9.
[7] Tamura M, Gu J, Takino T, Yamada KM. Tumor suppressor PTEN
inhibition of cell invasion, migration, and growth: differential
involvement of focal adhesion kinase and p130Cas. Cancer Res
1999;59:442—9.
[8] Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C,
Sasaki T, et al. Negative regulation of PKB/Akt-dependent cell
survival by the tumor suppressor PTEN. Cell 1998;95:29—39.
[9] Gu J, Tamura M, Yamada KM. Tumor suppressor PTEN
inhibits integrin- and growth factor-mediated mitogenactivated protein (MAP) kinase signaling pathways. J Cell Biol
1998;143:1375—83.
[10] Tamura M, Gu J, Matsumoto K, Aota S, Parsons R, Yamada KM.
Inhibition of cell migration, spreading, and focal adhesions by
tumor suppressor PTEN. Science 1998;280:1614—7.
[11] Sansal I, Sellers WR. The biology and clinical relevance
of the PTEN tumor suppressor pathway. J Clin Oncol
2004;22:2954—63.
[12] Wang JY, Huang TJ, Chen FM, Hsieh MC, Lin SR, Hou MF,
et al. Mutation analysis of the putative tumor suppressor gene
PTEN/MMAC1 in advanced gastric carcinomas. Virchows Arch
2003;442:437—43.
[13] Byun DS, Cho K, Ryu BK, Lee MG, Park JI, Chae KS, et al.
Frequent monoallelic deletion of PTEN and its reciprocal association with PIK3CA amplification in gastric carcinoma. Int J
Cancer 2003;104:318—27.
Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at
residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012),
doi:10.1016/j.clinre.2012.03.002
+Model
CLINRE-244; No. of Pages 8
ARTICLE IN PRESS
8
Z. Yang et al.
[14] Li YL, Tian Z, Wu DY, Fu BY, Xin Y. Loss of heterozygosity on
10q23.3 and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions. World J Gastroenterol
2005;11:285—8.
[15] Kang YH, Lee HS, Kim WH. Promoter methylation and silencing
of PTEN in gastric carcinoma. Lab Invest 2002;82:285—91.
[16] Hino R, Uozaki H, Murakami N, Ushiku T, Shinozaki A, Ishikawa
S, et al. Activation of DNA methyltransferase 1 by EBV latent
membrane protein 2A leads to promoter hypermethylation of
PTEN gene in gastric carcinoma. Cancer Res 2009;69:2766—74.
[17] Vazquez F, Ramaswamy S, Nakamura N, Sellers WR. Phosphorylation of the PTEN tail regulates protein stability and function.
Mol Cell Biol 2000;20:5010—8.
[18] Torres J, Pulido R. The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications
for PTEN stability to proteasome-mediated degradation. J Biol
Chem 2001;276:993—8.
[19] Correa P. Human gastric carcinogenesis: a multistep and
multifactorial process–First American cancer society award
lecture on cancer epidemiology and prevention. Cancer Res
1992;52:6735—40.
[20] Hamilton SR, Aaltonen LA. Pathology and genetics of tumors of
the digestive system. World Health Organization classification
of tumors. Lyon: IARCP; 2000.
[21] Dixon MF, Genta RM, Yardley JH, Correa P. Classification and
grading of gastritis. The updated Sydney system. International
workshop on the histopathology of gastritis, Houston 1994. Am
J Surg Pathol 1996;20:1161—81.
[22] Yang L, Kuang LG, Zheng HC, Li JY, Wu DY, Zhang SM, et al. PTEN
encoding product: a marker for tumorigenesis and progression
of gastric carcinoma. World J Gastroenterol 2003;9:35—9.
[23] Park GS, Joo YE, Kim HS, Choi SK, Rew JS, Park CS, et al. Expression of PTEN and its correlation with angiogenesis in gastric
carcinoma. Korean J Gastroenterol 2005;46:196—203.
Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at
residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012),
doi:10.1016/j.clinre.2012.03.002