Expression of GRP78 predicts taxane-based therapeutic resistance and

recurrence of human gastric cancer

Lei Yang
a,b
, Shuyun Yang
c
, Jibin Liu
d
, Xiaolin Wang
e
, Jianmei Ji
b
, Yongfeng Cao
b
, Kun Lu
f
,
Jianhong Wang
b
, Yong Gao
a,

a
Department of Oncology and Tumor Institute, Shanghai East Hospital Afliated to Tongji University School of Medicine, Shanghai 200120, People's Republic of China
b
Department of Medical Oncology, Nantong University Afliated Tumor Hospital, Jiangsu, Nantong 226361, People's Republic of China
c
Department of Pathology, Nantong University Afliated Tumor Hospital, Jiangsu, Nantong 226001, People's Republic of China
d
Biological Center, Nantong University Afliated Tumor Hospital, Jiangsu, Nantong 226001, People's Republic of China
e
Department of General Surgery, Nantong University Afliated Tumor Hospital, Jiangsu, Nantong 226361, People's Republic of China
f
The Department of Endocrinology, Tongji University School of Medicine, Shanghai Tenth People's Hospital, Shanghai 200072, People's Republic of China
a b s t r a c t a r t i c l e i n f o
Article history:
Received 20 January 2013
and in revised form 19 February 2014
Available online 3 March 2014
Keywords:
Glucose-regulated protein 78
Gastric cancer
Proliferation
Prognosis
Cancer cells adapt to chronic stress in the tumor microenvironment by inducing the expression of glucose-
regulated protein 78 (GRP78), a major endoplasmic reticulum chaperone with Ca
2+
-binding and antiapoptotic
properties. The effect in and potential role of its expression in progression of and prognosis for gastric cancer
(GC) are unclear. In the present study, we investigated the clinical value of GRP78 expression in judgment of
the severity of and prognosis for GC in a retrospective cohort study of 160 patients who underwent D2 radical
gastrectomy and adjuvant chemotherapy. GRP78 expression was detected using immunohistochemistry. The re-
lationships of GRP78 expression with age, sex, differentiation, invasion depth, disease stage, lymph node metas-
tasis, and time to recurrence (TTR) were analyzed. The GRP78 expression was higher in tumors from patients
with deep tumor inltration, with poor differentiation, at late disease stages, and with lymph node metastasis
than that in tumors from patients without. Also, GRP78 positivity was associated with short TTR (hazard ratio
[HR], 1.75; 95% condence interval [CI], 1.074.85; P = 0.041). Subgroup analysis revealed that the HR in the
GRP78-high group increased signicantly in patients who did not receive taxane-containing regimens (HR,
2.21; 95% CI, 1.237.36; P = 0.038). In contrast, in the patients who received taxane-based chemotherapy, the
association between GRP78 positivity and increased risk of recurrence was not statistically signicant (HR,
1.16; 95% CI, 0.812.98; P = 0.111). In the patients with GRP78 expression, those who underwent taxane-
containing chemotherapy had longer median TTRs than did those who did not undergo this treatment
(P = 0.017). Downregulation of GRP78 expression markedly inhibited proliferation of the GC cells at the G1
phase, whereas GRP78 overexpression promoted cell-cycle progression. These ndings suggest that GRP78
overexpression promotes GC cells proliferation and is an independent indicator of poor prognosis for GC.
2014 Elsevier Inc. All rights reserved.
Introduction
Gastric cancer (GC) is the world's second leading cause of cancer
mortality behind lung cancer despite a sharp worldwide decline in
both its incidence and mortality rate since the second half of the 20th
century. Adjuvant chemotherapy after complete resection of GC
improves survival durations; however, standard therapeutic strategies
result in unnecessary treatment and side effects in large numbers of pa-
tients (Meyer and Wilke, 2011). Current adjuvant treatment strategies
for GC are based primarily on grouped risk assessments mainly accord-
ing to tumor stage, histological grade, and lymph node status. Despite
the benets, the 5-year survival rate in patients who undergo it is
still less than 30% (Nagini, 2012). Thus, the need for identication of
additional novel predictive factors for chemoresponsiveness of GC is a
critical one.
The endoplasmic reticulum (ER) plays an important role in regula-
tion of the synthesis, folding, and targeting of secretory and membrane
proteins. Oxidative stress, glucose deprivation, chemical toxicity, alter-
ations in intracellular Ca
2+
levels, blockade of glycosylation, and hypox-
ia induce ER stress, in which expression of several ER stress-responsive
Experimental and Molecular Pathology 96 (2014) 235241
Abbreviations: CI, condence interval; ER, endoplasmic reticulum; GC, gastric cancer;
GRP78, glucose-regulated protein 78; HR, hazard ratio; TTR, time to recurrence.
Corresponding author at: Department of Oncology and Tumor Institute, Shanghai East
Hospital, 150 Jimo Road, Shanghai 200120, The People's Republic of China. Fax: +86 21
58798999.
E-mail address: yonggao.53@gmail.com (Y. Gao).
http://dx.doi.org/10.1016/j.yexmp.2014.02.011
0014-4800/ 2014 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
Experimental and Molecular Pathology
j our nal homepage: www. el sevi er . com/ l ocat e/ yexmp
genes, including glucose-related protein 78 (GRP78), is activated.
Eukaryotic cells adapt to ER stress and thereby survive via translational
attenuation, limiting further accumulation of misfolded proteins, tran-
scriptional activation of genes encoding ER-resident chaperones, and
the ER-associated degradation pathway to restore the protein folding
capacity. If eukaryotic cells are exposed to prolonged or strong ERstress,
they are destroyed via apoptosis (Malhi and Kaufman, 2011; Ye et al.,
2010). GRP78 is ubiquitously expressed in the ER and assists in protein
folding and assembly. Consequently, GRP78 is considered as an ER
molecular chaperone.
GRP78 was discovered in the late 1970s as cellular protein, whose
expression is induced by glucose starvation and that is almost 50%
homologous with heat shock protein 70. Although GRP78 is localized
constitutively within the ER and performs normal physiological func-
tions at moderate levels of expression, its expression may be induced
under certain pathological conditions, such as tumor growth and toxic
damage. Tumor cells are often confronted with oxygen deprivation
and nutrient stress, even with extensive angiogenesis, inducing expres-
sion of GRP genes (Misra et al., 2011). Investigators have demonstrated
that GRP78 is expressed at high levels in a variety of malignancies, such
as breast and hepatocellular carcinoma, lung and prostate cancer, and,
more recently, GC (Navin and Hicks, 2011; Zhang et al., 2006;
Daneshmand et al., 2007; Miao et al., 2013). Invitro studies demonstrat-
ed that GRP78expressionis alsochemoprotective (Li and Lee, 2006; Lee,
2007). Persistent expression of proteins that facilitate cell survival, such
as GRP78, may be a central feature of adaptation of tumor cells to ER
stress (Rutkowski et al., 2007). In support of this, in some cases,
GRP78 expression is associated with tumor development and growth
and correlated with tumor resistance to certain forms of chemotherapy
(Lee, 2007). In particular, GRP78 expression is known to protect tu-
mors against certain DNA-damaging agents, including the topo-
isomerase II inhibitors etoposide and doxorubicin (Adriamycin), the
topoisomerase I inhibitor camptothecin, and the alkylating agent
cisplatin(Pyrko et al., 2007). Induction of GRP78 expression may be a de-
fense mechanismfor the survival of cancer cells exposed to certain stress
conditions. Recent studies indicated a potential role for altered GRP78
expression and function in tumor development and progression
(Fu and Lee, 2006; Misra et al., 2011). GRP78 expression is induced
during embryonic development, and authors have widely reported
this induction in human cancer cells (Mao et al., 2004; Jin et al.,
2012). GRP78 promotes the survival and chemoresistance of both
proliferating and dormant cancer cells (Fasano et al., 2012; Mao et al.,
2004).
The effect of GRP78 expression in GC cells and its potential role in
progression of and prognosis for this cancer remains unclear. Herein
we report the results of GRP78 expression in a retrospective cohort
study of 160 GC patients. This is the rst reported study to demonstrate
that GRP78 expression is a prognostic factor for GC. Specically, this
study revealed that high-level GRP78 expression predicts short time
to recurrence (TTR) in GC patients who undergo D2 radical gastrectomy
and adjuvant chemotherapy.
Materials and methods
Patient samples
From January 2006 to May 2010, 237 patients with stage IIII GC
underwent D2 radical gastrectomy at the Afliated Tumor Hospital of
Nantong University in Nantong, People's Republic of China. Of these
patients, 160 received adjuvant chemotherapy and were selected for
this retrospective study. Their demographic and clinical information
were abstracted from hospital records. According to the 2002 American
Joint Committee on Cancer staging criteria, the patients had stage I, II, or
III tumors. The collection and use of the tissue specimens from the pa-
tients were approved and the need for informed consent fromthe donors
was waived by the Institutional ReviewBoards of both the Shanghai East
Hospital and the Afliated Nantong Tumor Hospital.
Immunohistochemistry
Four-micron sections of parafn-embedded, formalin-xed tissue
samples obtained from the study patients were stained for an anti-
GRP78 antibody using the standard immunohistochemical method
with a commercial kit (Beijing Zhongshan Golden Bridge Biotechnology
Co., Beijing, People's Republic of China), according to the instructions of
the manufacturer. 3,3-diaminobenzidine chromogenic examination of
the tissue sections using a 3,3-diaminobenzidine substrate kit (Beijing
Zhongshan Golden Bridge Biotechnology Co.), and observation of
staining under a microscope were carried out. The staining solution
in which the rst antibody was replaced with normal IgG was used
as a control. Stained plasma cells were used as positive controls.
Immunohistochemically stained slides containing tissue sections
obtained from each subject were reviewed by a pathologist who
was blinded to all clinical data. The staining score was based on the
percentage of positive cells and staining intensity. Specically, the
percentage of positive cells was divided into ve grades (percentage
scores): b10% (0), 1025% (1) , 2550% (2) , 5075% (3) , and
N75% (4) . The intensity of staining was divided into four grades
(intensity scores): no staining (0), light brown (1), brown
(2), and dark brown (3). GRP78 staining positivity was deter-
mined by the formula: overall scores = percentage score intensity
score. GRP78 staining was classied into four groups: negative,
weak, moderate and strong. The overall score of 3 was dened as
weak or negative, of N3 to 6 as moderate, and of N6 as strong. For
survival analysis, the weak and moderate staining patients were de-
ned as GRP78-low, and the strong staining patients as GRP78-
high. To determine the reader reproducibility of immunohisto-
chemical evaluation of the sections for GRP78, a random sample of
50 slides was chosen and re-evaluated by the same pathologist
without knowledge of the previous results. The coefcient was
used to evaluate the agreement between the two evaluations. The
coefcient for the two evaluations was 0.75 (95% condence interval
[CI], 0.610.97), indicating substantial agreement according to the
LandisKoch criteria for the reproducibility.
Follow-up and statistical analyses
TTR was calculated from the date of surgery until the date of docu-
mented recurrence. For patients who did not have a recurrence by the
time of the last follow-up report (death or last contact at the hospital
or with the treating physician), TTR was censored at the last follow-up
date. Associations of demographic and clinical characteristics with
GRP78 expression were evaluated using contingency tables and the
2
test. Associations between TTR and GRP78 expression or other potential
prognostic factors were evaluated using KaplanMeier plots and the
Cox proportional hazards model. All reported P values are two-sided
and based on the likelihood ratio test associated with the Cox model.
To determine whether the association between GRP78 expression and
TTR was independent of other potential prognostic factors, stratication
was performed according to each prognostic factor, and the relationship
between GRP78 expression and TTR according to type of chemotherapy
was evaluated. The test of interaction among those factors was per-
formed by introducing an interaction term into the Cox proportional
hazards model.
Cell culture, plasmid construction, and cell transfection
The GC cell line SGC-7901 was grew in 5% CO
2
in 1640 medium
supplemented with 10% fetal bovine serum at 37 C. To determine the
effects of enforced GRP78 expression on this cell line, the GRP78-
expressing plasmid pcDNA3.1-GRP78 was used for gene transfection.
236 L. Yang et al. / Experimental and Molecular Pathology 96 (2014) 235241
In addition, GRP78 vector-based RNA interference plasmid pSilence-
GRP78 short hairpin RNA was used to knock down endogenous GRP78
expression in SGC-7901 cells. Lipofectamine 2000 reagent (Invitrogen,
Carlsbad, CA) was used for SGC-7901-cell transfection according to the
manufacturer's instructions. Empty vectors were used for mock trans-
fections in enforced GRP78-expression and -knockdown assays, respec-
tively. Stable SGC-7901 cells with overexpression or downregulated
expression of GRP78 were harvested and then investigated after G418
selection of the cells for 14 days.
Cell-viability and cell-cycle analyses
SGC-7901-cell viability was assessed using a Cell Proliferation Kit II
(XTT; Roche Applied Science, Indianapolis, IN) for 5 days according to
the manufacturer's instructions. This experiment was performed intrip-
licate for each indicated time point. The phases of the cells in the cell
cycle were determined using uorescence-activated cell sorter analysis
of propidium iodide-stained cells with a FACSCalibur ow cytometer
(Becton Dickinson, San Jose, CA). The cells were synchronized via
serum starvation for 24 h and stimulated with Dulbecco's modied
Eagle's medium containing 10% serum for 12 h before harvesting. The
cells were then washed with phosphate-buffered saline and xed with
cold 70% ethanol for at least 1 h. Prior to ow cytometric analysis, the
cells were incubated with 200 l of DNase-free RNaseA at 200 g/ml
and propidium iodide at 1 mg/ml at 37 C for 30 min. A total of 10
4
cells from each indicated sample were analyzed. Cell-cycle analysis
was performed using the ModFit LT software program (version 3.0;
Verity Software House, Topsham, ME).
Results
GRP78 expression in human gastric tumor specimens
All of the GC patients with tumor specimens available for GRP78
analysis underwent D2 radical gastrectomy. For the sake of simplicity,
we classied their tumors into GRP78-low and GRP78-high groups
based on the overall GRP78 staining intensity and percentage of cells
stained. Thus, the low group included tumors that stained weakly for
GRP78 and/or had limited areas of staining, whereas the tumors in the
high group exhibited moderate to strong staining. As expected for an
ER protein, GRP78 staining was limited primarily to the perinuclear/
cytoplasmic region of tumor cells. Plasma cells had high levels of expres-
sion of GRP78, facilitating immunoglobulin chain assembly (Terasaki and
Reese, 1994). All of the tissue sections contained plasma cells, and these
cells' generally uniform high levels of immunoreactivity with the anti-
GRP78 antibody made them ideal internal positive controls. Representa-
tives of negative/weak, moderate and strong staining of GRP78 protein
in gastric tumors are shown in Fig. 1. Of the 237 patients who underwent
50 200
W
e
a
k
/
n
e
g
a
t
i
v
e
M
o
d
e
r
a
t
e
S
t
r
o
n
g
Fig. 1. Different expression levels of GRP78 in GC tissue. Sections of human gastric tumor specimens were prepared and subjected to immunohistochemical staining with a specic anti-
GRP78 antibody. Representative photos with various expression levels of GRP78 were shown.
237 L. Yang et al. / Experimental and Molecular Pathology 96 (2014) 235241
D2 radical gastrectomy, 163 (68.8%) exhibited GRP78-positive staining
before initiation of chemotherapy (Table 1).
Association of GRP78 expression with clinical characteristics
As shown in Table 1, the expression of GRP78 protein was closely
correlated with prognostic factors suchas inltration depth, histological
grade, disease stage, and lymph node status but was not correlated with
sex or age (P N 0.05). Increased GRP78 expression was positively corre-
lated with inltration depth, poor differentiation, a late disease stage,
and lymph node metastasis. GRP78 expression was signicantly
correlated with inltration depth (P = 0.005), differentiation level
(P = 0.000), and disease stage (P = 0.004). Also, the GRP78 expres-
sion rate was higher in the patients with lymph node metastasis than
in those without it (P b 0.001).
Association between GRP78 expression and TTR
Among the 160 subjects who received adjuvant chemotherapy,
those high for GRP78 expression had an increased likelihood of
recurrence and a short TTR (hazard ratio [HR], 1.75; 95% CI, 1.074.85;
P = 0.041) (Fig. 2A; Table 2). In a multivariate analysis, the strength
of the association between GRP78 expression and increased likelihood
of recurrence remained the same after adjustment for tumor stage,
and histological grade according to the propensity score. Post-hoc
analysis of GRP78 staining and TTR in the patients according to chemo-
therapy regimen revealed two strong trends. First, the association
between expression of GRP78 and risk of recurrence was not statistical-
ly signicant in patients who received taxane-containing (paclitaxel or
docetaxel) chemotherapy (HR, 1.16; 95% CI, 0.812.98; P = 0.111)
(Fig. 2B; Table 2). Furthermore, adjustment for tumor stage, and histo-
logical grade according to the propensity score produced similar results.
In contrast, in the patients who did not undergo taxane-containing
chemotherapy, the association between expression of GRP78 and risk
of recurrence was statistically signicant (HR, 2.21; 95% CI, 1.237.36;
P = 0.038) (Fig. 2C; Table 2). Second, in the group of patients with
GRP78 high expression, the patients receiving taxane-containing
chemotherapy hadlonger TTRs thandidthose not receiving this therapy
(P = 0.017) (Fig. 2D). In agreement with these results for taxane-
containing treatment, patients with GRP78 expression who underwent
adjuvant taxane-containing chemotherapy appeared to have a lower
risk of recurrence than did patients who did not undergo this adjuvant
treatment.
GRP78 promotes GC cell proliferation
Because GRP78 expression was positively correlated with differenti-
ation, disease stage, lymph node status, and GC prognosis, we next
examined the effect of GRP78 expression on the viability of the GC cell
line SGC-7901 by using an XTT assay to test the hypothesis that GRP78
plays a role in GC progression. Downregulation of GRP78 expression
markedly inhibited SGC-7901 cell proliferation at the G1 phase but did
not affect proliferation of cells transfected with an empty vector control
(Fig. 3A). Conversely, enforced expression of GRP78 markedly increased
cell proliferation at the G1 phase (Fig. 3B). In ow cytometric analysis,
we observed that downregulation of GRP78 expression caused a great
accumulation of SGC-7901 cells but not cells transfected with a control
vector at the G1 phase (Fig. 3C). On the other hand, the numbers of
GRP78-depleted cells at the S and G2-M phases were markedly lower
Table 1
Relationship between expression of GRP78 and clinical parameters of gastric cancer.
Item Groups n High GRP78 P

Age 60 years 144 99

N60 years 93 64 0.991
Gender Male 169 121 0.140
Female 68 42
Inltration depth Mucosa and submucosa layer 34 18 0.005
Proper muscle 46 26
Serosa 157 119
Differentiation Well 42 11 0.000
Moderate 62 38
Poorly 133 114
Stage (TNM) I 26 7 0.004
II 65 44
III 146 112
Lyn metastasis No 93 38
YES 144 125 0.000
Fisher's Exact Test, statistically signicant (P b 0.05).
A B
C D
GRP78(-)
(n=54)
GRP78(+)
(n=106)
All patient: p=0.041 Taxane group: p=0.112
GRP78(-)
(n=24)
GRP78(+)
(n=52)
GRP78(-)
(n=30)
GRP78(+)
(n=54)
No Taxane group: p=0.038 GRP78(+) group: p=0.017
Taxane
(n=52)
No Taxane
(n=54)
Time after surgery (months) Time after surgery (months)
Time after surgery (months) Time after surgery (months)
0 20 40 60 80 0 20 40 60 80
0 20 40 60 80 0 20 40 60 80
R
e
c
u
r
r
e
n
c
e

f
r
a
c
t
i
o
n
0
.25
.50
.75
1.00
0
.25
.50
.75
1.00
0
.25
.50
.75
1.00
0
.25
.50
.75
1.00
R
e
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u
r
r
e
n
c
e

f
r
a
c
t
i
o
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c
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c
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f
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a
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e
c
u
r
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n
c
e

f
r
a
c
t
i
o
n
Fig. 2. KaplanMeier analyses of the prognostic signicance of GRP78 for GC patients treated with or with taxane. Survival differences between patients with high and lowGRP78 expres-
sion (A); between those patients who received taxane treatment with high and low GRP78 expression (B); between those patients not receiving taxane treatment with high and low
GRP78 expression (C); and between those patients with high GRP78 expression with or without taxane treatment (D).
238 L. Yang et al. / Experimental and Molecular Pathology 96 (2014) 235241
than those of cells transfected with a vector control. Enforced expression
of GRP78 in SGC-7901 cells resulted in a much lower number of these
cells at the G1 phase than that of cells transfected with a control vector
but higher numbers of SGC-7901 cells at the S and G2-M phases
(Fig. 3D). These data demonstrated that GRP78 expression increases
the proliferation of GC cells.
Discussion
In the present study, we observed a higher GRP78 expression in
tumors frompatients with deep tumor inltration, with poor differenti-
ation, at late disease stages, and with lymph node metastasis than that
in tumors from patients without. Also, GRP78 positivity was associated
Time (hr) Time (hr)
2
0
4
6
8
0 24 48 72
4
0
8
12
16
0 24 48 72
R
e
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a
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G
r
o
w
t
h

R
a
t
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R
e
l
a
t
i
v
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G
r
o
w
t
h

R
a
t
e
Control
Knockdown
Control
Overexpression
A B
N
u
m
b
e
r
DNA content DNA content DNA content DNA content
C D
N
u
m
b
e
r
Control Knockdown Control Overexpression
Control Knockdown Control Overexpression
40
0
60
80
100
20
40
0
60
80
100
20
C
e
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s

i
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c
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c
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(
%
)
C
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l
s

i
n

c
e
l
l

c
y
c
l
e

(
%
)
Fig. 3. Inuences of altered expression of GRP78 on GC cells growth in vitro. GC cells were either transfected with GRP78 expression (Overexpression) or GRP78 siRNA (Knockdown).
Cell growth was determined by cell counting (A & B); and cell cycle was determined by FACS analysis (C & D). *P b 0.05 as determined by student t test.
Table 2
Relative risk of recurrence in gastric cancer patients associated with high or low GRP78 expression.
Groups n Number of recurrence Univariate analysis Multivariate analysis
a
HR (95% CI) P
b
HR (95% CI) P
b
Overall
Low 54 20 1.00 1.00
High 106 68 1.75 (1.074.85) 0.041 1.73 (1.034.90) 0.048
Taxanes
Low 24 9 1.00 1.00
High 52 23 1.16 (0.812.98) 0.111 1.19 (0.843.02) 0.231
No-taxane
Low 30 11 1.00 1.00
High 54 45 2.21 (1.237.36) 0.038 2.20 (1.197.73) 0.040
a
Stratied analysis using propensity score (based on tumor inltration depth, grade, stage and lymph node status) divided into quintiles.
b
Ps from likelihood ratio test based on Cox model.
239 L. Yang et al. / Experimental and Molecular Pathology 96 (2014) 235241
with short TTR. Subgroup analysis revealed that the HR in the GRP78-
high group increased signicantly in patients who did not receive
taxane-containing regimens. In contrast, in the patients who received
taxane-based chemotherapy, the association between GRP78 positivity
and increased risk of recurrence was not statistically signicant. In the
patients with GRP78 high expression, those who underwent taxane-
containing chemotherapy had longer TTRs than did those who did not
undergo this treatment). Experimentally, downregulation of GRP78
expression markedly inhibited proliferation of the GC cells at the G1
phase, whereas GRP78 overexpression promoted cell-cycle progression.
Our ndings suggest that GRP78 overexpression is involved in the pro-
liferation of GC cells and is an independent indicator of poor prognosis
for GC.
Prior studies have reported elevated GRP78 expression in cases of a
number of solid cancers (Lee, 2007; Ma and Hendershot, 2004), but
none have reported on a study of GRP78 expression in gastric tumor
specimens. The present study is the rst to analyze GRP78 expression
and its possible associations with clinicopathological data and prognosis
in GC patients, particularly chemotherapeutic responses. Our study
demonstrated that GRP78 is expressed at relatively high levels in gastric
tumors. Specically, 68.8% of tumor specimens collected from patients
before they underwent adjuvant chemotherapy were positive for
GRP78 expression. This suggests that the gastric tumor microenviron-
ment induces ERstress andGRP78 expression. Althoughthe mechanism
for GRP78-low phenotype in GC patients remains to be determined,
rapid tumor growth and, perhaps, inadequate vascularization may
create a tumor microenvironment with hypoxia, glucose deprivation,
and acidosis, which in turn results in chronic ER stress, inducing
GRP78 expression. In addition, increased glycolytic activity in malignant
gastric tumor cells may contribute to ER stress (Lee, 2007; Kandala and
Srivastava, 2012). One previous study demonstrated that increased lac-
tate dehydrogenase levels, which are indicative of increased glycolytic
activity, are common in metastatic melanomas (Hersey and Zhang,
2008). Whether properties of GC cells other than increased glycolytic
activity predispose patients to ER stress remains unknown. Further-
more, authors reported that ER stress in tumor cells was induced at
early stages of melanoma development by transfection of melanocytes
with an oncogenic form of HRAS (HRASG12V) (Denoyelle et al.,
2006). We found that increased GRP78 expression was positively corre-
lated with poor differentiation, a late disease stage, and lymph node me-
tastasis. Therefore, we believe that GRP78 is a novel cell-cycle-regulating
protein capable of promoting cell proliferation. In agreement with that,
we found that overexpression of GRP78 greatly increased the prolifera-
tion of the GC cell line. Furthermore, the number of GRP78-reduced
SGC-7901 cells at the S and G2-M phases was much lower than that of
cells transfected with an empty vector control, demonstrating that
GRP78 is an essential component of cell-proliferation pathways.
Thus, GRP78 may be a biomarker of the malignancy of GC.
Resistance of GC to chemotherapy is a major obstacle to successful
treatment of it after radical gastrectomy. Recent studies suggested that
GRP78 expression enhances the chemosensitivity of melanoma and
breast cancer cells (Hersey and Zhang, 2008; Jiang et al., 2009; Lee
et al., 2011), suggesting that GRP78 is protumorigenic and therefore
may reduce TTR. Consistently, we appear to be the rst group to observe
a strong association between GRP78 expression and TTR in patients
withGCwhoundergo radical gastrectomy. Specically, GRP78positivity
seemed to be associated with increased risk of recurrence in patients
who underwent D2 radical gastrectomy and chemotherapy. However,
different treatment regimens could obviously contribute to the hetero-
geneity of this association.
Furthermore, we observed two potentially important interactions
between GRP78 expression and treatment with taxanes. In the subset
of patients who did not undergo taxane-containing chemotherapy, the
association between GRP78 positivity and increased risk of recurrence
was statistically signicant, suggesting a strong association between
GRP78 expression and chemoresistance. In contrast, in the subset of
patients who received taxane-containing chemotherapy, the associa-
tion between GRP78 positivity and increased risk of recurrence was
not statistically signicant. In addition, patients with GRP78 expression
who underwent taxane-containing chemotherapy had longer median
TTRs than did those who did not undergo this chemotherapy. Therefore,
taxane-containing chemotherapy may be a good choice for GC patients
with GRP78 expression. Although we observed these effects in a
relatively small number of patients, they suggest that taxanes diminish
or even reverse the effect of GRP78 expression on chemotherapy-
resistance mechanisms in GC cells. By preventing polymerization of
new microtubules, treatment with taxanes blocks ER elongation and
movement, which are required for maintenance of its unique subcellu-
lar structure (Fisher, 1994). Thus, taxane-based treatment apparently
can alter or interfere with GRP78 function, such as inhibition of the
translation and degradation of misfolded proteins, by disrupting the
ER structure and inhibiting GRP78 transcription (Fu and Lee, 2006).
In conclusion, our study demonstrated that GRP78 may be a novel
biomarker for prediction of chemoresistance and an independent
predictor of poor prognosis in GC patients. These results warrant conr-
mation in larger clinical studies and in patients with other types of
cancer.
Conict of interest statement
The authors declare that there are no conicts of interest.
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