Overexpression of GPR39 contributes to malignant development of human esophageal squamous cell carcinoma.

Abstract

By using cDNA microarray analysis, we identified a G protein-coupled receptor, GPR39, that is significantly up-regulated in ESCC. The aim of this study is to investigate the role of GPR39 in human esophageal cancer development, and to examine the prevalence and clinical significance of GPR39 overexpression in ESCC.
The mRNA expression level of GPR39 was analyzed in 9 ESCC cell lines and 50 primary ESCC tumors using semi-quantitative RT-PCR. Immunohistochemistry was used to assess GPR39 protein expression in tissue arrays containing 300 primary ESCC cases. In vitro and in vivo studies were done to elucidate the tumorigenic role of GPR39 in ESCC cells.
We found that GPR39 was frequently overexpressed in primary ESCCs in both mRNA level (27/50, 54%) and protein level (121/207, 58.5%), which was significantly associated with the lymph node metastasis and advanced TNM stage (P < 0.01). Functional studies showed that GPR39 has a strong tumorigenic ability. Introduction of GPR39 gene into ESCC cell line KYSE30 could promote cell proliferation, increase foci formation, colony formation in soft agar, and tumor formation in nude mice. The mechanism by which amplified GPR39 induces tumorigenesis was associated with its role in promoting G1/S transition via up-regulation of cyclin D1 and CDK6. Further study found GPR39 could enhance cell motility and invasiveness by inducing EMT and remodeling cytoskeleton. Moreover, depletion of endogenous GPR39 by siRNA could effectively decrease the oncogenicity of ESCC cells.
The present study suggests that GPR39 plays an important tumorigenic role in the development and progression of ESCC.

Full text

Page 1

RESEARCH ARTICLE Open Access
Overexpression of GPR39 contributes to
malignant development of human esophageal
squamous cell carcinoma
Fajun Xie1†, Haibo Liu1†, Ying-Hui Zhu1, Yan-Ru Qin3, Yongdong Dai1, Tingting Zeng1, Leilei Chen2, Changjun Nie1,
Hong Tang4, Yan Li1, Li Fu1,2*, Xin-Yuan Guan1,2*
Abstract
Background: By using cDNA microarray analysis, we identified a G protein-coupled receptor, GPR39, that is
significantly up-regulated in ESCC. The aim of this study is to investigate the role of GPR39 in human esophageal
cancer development, and to examine the prevalence and clinical significance of GPR39 overexpression in ESCC.
Methods: The mRNA expression level of GPR39 was analyzed in 9 ESCC cell lines and 50 primary ESCC tumors
using semi-quantitative RT-PCR. Immunohistochemistry was used to assess GPR39 protein expression in tissue
arrays containing 300 primary ESCC cases. In vitro and in vivo studies were done to elucidate the tumorigenic role
of GPR39 in ESCC cells.
Results: We found that GPR39 was frequently overexpressed in primary ESCCs in both mRNA level (27/50, 54%)
and protein level (121/207, 58.5%), which was significantly associated with the lymph node metastasis and
advanced TNM stage (P < 0.01). Functional studies showed that GPR39 has a strong tumorigenic ability.
Introduction of GPR39 gene into ESCC cell line KYSE30 could promote cell proliferation, increase foci formation,
colony formation in soft agar, and tumor formation in nude mice. The mechanism by which amplified GPR39
induces tumorigenesis was associated with its role in promoting G1/S transition via up-regulation of cyclin D1 and
CDK6. Further study found GPR39 could enhance cell motility and invasiveness by inducing EMT and remodeling
cytoskeleton. Moreover, depletion of endogenous GPR39 by siRNA could effectively decrease the oncogenicity of
ESCC cells.
Conclusions: The present study suggests that GPR39 plays an important tumorigenic role in the development and
progression of ESCC.
Background
Esophageal squamous cell carcinoma (ESCC), the major
histological form of esophageal cancer, is one of the most
aggressive malignancies with poor prognosis in the
world, especially in the Northern part of China [1]. Like
other types of solid tumors, the development of ESCC is
also the accumulation of the abnormal expression of
oncogenes and tumor suppressor genes (TSGs). Several
genetic alterations have been associated with the develop-
ment of ESCC including mutations of p53 and p16,
amplification of cyclin D, c-myc, and EGFR, as well as
allelic loss on chromosomes 3p, 5q, 8p, 9p, 9q, 13q, 17p,
18q, and 21q [2-5]. Our previous studies have character-
ized the common deletion regions at 3p and candidate
TSGs within frequently deleted regions including PLCD1
and PCAF [6,7]. However, many genes associated with
the development and progression of ESCC have not been
characterized. To better understand the molecular
mechanisms that underlie the ESCC development and
progression, cDNA microarray was used to compare the
gene expression profiles between 10 primary ESCC
tumors and their paired non-tumorous tissues.
Among the 185 up-regulated genes, one gene named
GPR39 drew our attention. GPR39 belongs to the G
* Correspondence: gracelfu@hkucc.hku.hk; xyguan@hkucc.hku.hk
† Contributed equally
1State Key Laboratory of Oncology in Southern China, Cancer Center, Sun
Yat-Sen University, Guangzhou, PR China
Full list of author information is available at the end of the article
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
© 2011 Xie et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Page 2

protein-coupled receptors (GPCRs) superfamily, which is
the largest family of cell-surface molecules involved in
signal transmission. It has been reported that GPR39
plays an important role in the regulation of gastrointest-
inal and metabolic function [8]. GPR39 receptor is now
thought to be activated by Zn2+signals and may have
other, as yet unidentified, cognitive ligands [9]. More-
over, GPR39 receptor also displays a strong ligand-
independent signaling activity through Ga12/13as well as
Gaq[10,11]. A recent study suggests that overexpression
of GPR39 may inhibit cell death induced by oxidative
stress, endoplasmic reticulum (ER) stress, and activation
of the caspase by Bax overexpression [12]. Emerging evi-
dence indicates that G protein-coupled receptors are
crucial players in cancer progression and metastasis
[13,14], however, the role of GPR39 in cancer develop-
ment remains unclear. In this study, we studied GPR39
expression pattern in ESCC. The tumorigenic function
of GPR39 was demonstrated by both in vitro and in vivo
assays. The tumorigenic mechanism of GPR39 was also
addressed. In addition, the clinical significance of
GPR39 overexpression in ESCC was investigated.
Methods
ESCC cell lines and specimens
Chinese ESCC cell line HKESC1 was kindly provided by
Professor Srivastava (Department of Pathology, The
University of Hong Kong, Hong Kong, China), and two
Chinese ESCC cell lines (EC18 and EC109) were kindly
provided by Professor Tsao (Department of Anatomy,
The University of Hong Kong). Six Japanese ESCC cell
lines (KYSE30, KYSE140, KYSE180, KYSE410, KYSE510
and KYSE520) [15] were obtained from DSMZ
(Braunschweig, Germany), the German Resource Centre
for Biological Material. Fifty pairs of primary ESCCs and
their surrounding non-tumorous esophageal tissues
were collected immediately after surgical resection at
Linzhou Cancer Hospital (Henan, China). Samples used
in this study were approved by the Committees for Ethi-
cal Review of Research involving Human Subjects at
Zhengzhou University and Sun Yat-Sen University.
Semiquantitative RT-PCR
Total RNA was extracted from cell lines and frozen
ESCC tissues using the Trizol reagent (Invitrogen, Carls-
bad, CA) according to the manufacture’s instruction.
Reverse transcripation of total RNA (2 μg) was done
using SuperScript II reverse transcriptase (Invitrogen,
Carlsbad, CA), and cDNA was subjected to PCR for a 30-
cycle amplification with primers for GPR39Fw: 5’-GC
CACCGGGGTCTCACTTGC-3’ and GPR39Rv: 5’-GGC
CGCAGCCATGATCCTCC-3’. GAPDH (Fw: 5’-CATGA
GAAGTATGACAACAGCCT; Rv: 5’-AGTCCTTCCAC
GATACCAAAGT) was used as an internal control.
Tissue Microarrays (TMA) and Immunohistochemistry
(IHC)
A total of 300 formalin-fixed and paraffin-embedded
ESCC tumor specimens were kindly provided by Linz-
hou Cancer Hospital (Henan, China). TMAs containing
300 pairs of primary ESCC tumor samples and their
corresponding nontumourous tissues were constructed
as described previously [16]. Standard streptavidin-
biotin-peroxidase complex method was used for IHC
staining [16]. Briefly, TMA section was deparaffinized,
blocked with 10% normal rabbit serum for 10 min, and
incubated with rabbit anti-human GPR39 polyclonal
antibody (Abcam, 1:100 dilution) overnight at 4°C. The
TMA section was then incubated with biotinylated goat
anti-rabbit immunoglobulin at a concentration of 1:100
at 37°C for 30 min. All of the IHC staining results were
reviewed independently by two pathologists. Positive
expression of GPR39 was defined as the brown staining
in the cytoplasm. The staining results for GPR39 were
scored semiquantitatively. Intensity was estimated in
comparison to the control and scored as follows: 0,
negative staining; 1, weak staining; 2, moderate staining;
and 3, strong staining. Scores representing the percen-
tage of tumor cells stained positive were as follows: 0,
<1% positive tumor cells; 1, 1-10%; 2, 10-50%; 3,
50-75%; and 4, >75%. A final score was calculated by
adding the scores for percentage and intensity, resulting
in scores of 0 and 2-7. A score of 0 was considered
negative; 2-3 was considered weak; 4-5 was considered
moderate; and 6-7 was considered strong. For statistical
analysis, 0-3 were counted as low expression of GPR39,
while 4-7 were counted as overexpression of GPR39.
Tumorigenic function of GPR39
To test the tumorigenic function of GPR39, full-length
GPR39 was PCR amplified, subcoloned into pcDNA3.1
(+) vector (Invitrogen, Carlsbad, CA) and stably trans-
fected into ESCC cell line KYSE30. Stable GPR39-
expressing clones (GPR39-c1 and GPR39-c4) were
selected for further study. Empty-vector transfected
KYSE30 cells (Vec-30) were used as control.
For foci formation assay, 1 × 103GPR39-expressing
cells or Vec-30 cells were seeded into 6-well plate. After
7 days culture, surviving colonies (>50 cells/colony)
were counted with 1% crystal violet staining. Triplicate
independent experiments were performed. Colony for-
mation in soft agar was performed by growing 1 × 104
cells in 0.4% Seaplague agar on a base of 0.6% agar in a
6-well plate. After 3 weeks, colonies consisted of more
than 80 cells were counted and expressed as the means
± SD of triplicate within the same experiment. To per-
form cell growth assay, GPR39-expressing cells and con-
trol Vec-30 cells were seeded in 96-well plate at a
density of 800 cells per well. The cell growth rate was
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
Page 2 of 12

Page 3

measured using cell counting kit-8 kit (Dojindo, Japan)
according to the manufacturer’s instruction. Triplicate
independent experiments were done.
Flow cytometry assay
GPR39-c4 or Vec-30 cells were cultured in DMEM
medium containing 10% FBS. Serum was withdraw from
the culture medium when cells were 70% confluent.
After 72 hrs, 10% FBS was added in the medium for an
additional 8 hrs, Cells were fixed in 70% ethanol, stained
with propidium iodide, and DNA content was analyzed
by Cytomics FC (Beckman Coulter, Fullerton, CA).
Tumor formation in nude mice
For in vivo experiment, stable GPR39-expressing
KYSE30 cells or control Vec-30 cells (1 × 106) in
200 μL serum-free DMEM (Life Technologies) were
injected s.c. into the right and left flank of 4 week-old
nude mice (5 mice for GPR39-c1 cells and 5 for
GPR39-c4 cells), respectively. The tumor volume was
calculated by the formula V = 0.5 × L × W2[17]. All
experiments were done in accordance with institutional
standard guidelines of Sun Yat-Sen University for ani-
mal experiments.
Migration and invasion assays
For cell migration assay, GPR39-c4 cells or Vec-30 cells
were grown to confluence and then mechanically
scratched with a sterile pipette tip. Cells were rinsed
with PBS and grown in culture medium for additional
24 hrs. The cell motility in terms of wound closure was
measured by photographing at three random fields at
time points 0 and 24 hr. For invasion assay, GPR39-c4
cells or Vec-30 cells were starved with serum free med-
ium for 24 hrs before the assay. Cells (5 × 104) were
suspended in 0.5 ml serum-free medium and loaded on
the upper compartment of invasion chamber coated
with Matrigel (BD Biosciences). The lower compartment
was filled with complete medium as chemoattractant.
After 24 hrs, invasive cells were fixed, stained, and
counted under a microscope. Triplicate independent
experiments were done.
F-actin staining
Cells grown on coverslips were washed three times in
PBS, fixed in 4% paraformaldehyde for 20 min, and per-
meabilized with 0.1% Triton X-100 for 10 min. Cells
were then stained with rhodamin-labeled phalloidin
(Molecule Probes) in PBS containing 1% bovine serum
albumin at room temperature for 30 min. After addi-
tional PBS washes, cells were counterstained with DAPI
and photographed with a Leica DMRA fluorescence
microscope (Rueil-Malmaison, France).
RNA interference
Small interfering RNA (siRNA) (20 μM) against GPR39
(s6073; Ambion) was transfected into KYSE180 cells in
6-well plates using Lipofectamine 2000 Reagent (Invitro-
gen) according to the manufacturer’s instructions. At
48 hrs after transfection, the effects of gene silencing
were measured via RT-PCR.
Western blot analysis
Western blot analysis was performed with the standard
method with antibodies to GPR39, N-cadherin and
GAPDH (Abcam, Cambridge Science Park, Cambridge,
UK), cyclin D1, p21, CDK4 and CDK6 (Cell Signalling
Technology, Frankfurt, Germany), and E-cadherin
(Santa Cruz Biotechnology, Santa Cruz, CA).
Statistical analysis
Statistical analysis was performed with the SPSS stan-
dard version 16.0 (SPSS Inc., Chicago, IL). The relation-
ship between the expression of GPR39 protein and
clinicopathologic characteristics was assessed by c2test.
Results expressed as mean ± SD were analyzed using
the Student t test. Differences were considered signifi-
cant when P < 0.05.
Results
GPR39 is frequently overexpressed in ESCC
Semi-quantitative RT-PCR was used to study the expres-
sion status of GPR39 in 50 primary ESCCs and 9 ESCC
cell lines. Compared with their paired non-tumorous tis-
sues, overexpression of GPR39 was detected in 27/50
(54%) of primary ESCCs (Figure 1A). Overexpression of
GPR39 was also frequently detected in ESCC cell lines
(HKESC1, KYSE140, KYSE180, KYSE410, KYSE510 and
KYSE520; Figure 1B). GPR39 expression in protein level
was further studied in 300 primary ESCCs by IHC using
a tissue microarray. Informative IHC results were
obtained from 207 pairs of ESCCs. Non-informative sam-
ples included lost samples, unrepresentative samples,
samples with too few tumor cells, and samples with inap-
propriate staining; such were not used in data complica-
tion. The expression of GPR39 in normal epithelial cells
was always negative or weak whereas strong positive
staining of GPR39 was observed in 121/207 (58.5%) of
informative ESCCs (Figure 1C).
Clinical significance of GPR39 overexpression in ESCC
The correlation between the GPR39 overexpression and
clinicopathologic features of ESCC including age (≤60
versus >60), gender (male versus female), tumor inva-
sion (T stage: tumor depth; T3, T4 versus T1, T2),
lymph nodes metastasis (N stage; N0 versus N1), TNM
stage (I, IIa versus IIb, III-IV), was studied (Table 1).
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
Page 3 of 12

Page 4

Figure 1 Overexpression of GPR39 in ESCC. GPR39 was frequently overexpressed in primary ESCCs (A) and ESCC cell lines (B) detected by RT-
PCR. For primary ESCCs, expression of GPR39 in tumor tissues (T) was compared with their paired non-tumorous tissues (N). Normal esophageal
tissue was used as a normal control. 18S rRNA was used as an internal control. (C) Representative of GPR39 expression in a pair of ESCC (right)
and adjacent normal tissue (left) detected by immunostaining with anti-GPR39 antibody (brown). The slide was counterstained with hematoxylin
(original magnification × 200).
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
Page 4 of 12

Page 5

The results showed that overexpression of GPR39 was
significantly associated with lymph node metastasis (P =
0.008) and advanced clinical stage (P = 0.004). No corre-
lation was observed between GPR39 overexpression and
age (P = 0.735), gender (P = 0.887), tumor differentia-
tion (P = 0.846) and tumor invasion (P = 0.085).
Tumorigenic function of GPR39
To investigate the tumorigenic potential of GPR39,
GPR39-expression vector was stably transfected into
KYSE30 cells with silenced GPR39. GPR39 mRNA and
protein expression in these clones were confirmed by
RT-PCR and Western blot analysis (Figure 2A). The
tumorigenic function of GPR39 was assessed by both
in vitro and in vivo assays including foci formation,
colony formation in soft agar, cell growth rate assays
and tumor xenograft experiment. Foci formation assay
showed that the frequency of foci formation was signif-
icantly increased (P < 0.01) in GPR39-transfectants
compared with control cells (Figure 2B). A similar
result was shown in soft agar assay (P < 0.01, Figure
2C). Cell growth assay also revealed that the cell
growth rates in GPR39-c1 and GPR39-c4 cells were
significantly enhanced by GPR39 compared with Vec-
30 cells (P < 0.01, Figure 2D). To further explore the
in vivo tumorigenic ability of GPR39, tumor formation
in nude mice was tested by injection of GPR39-c1 cells
(n = 5) or GPR39-c4 cells (n = 5), whereas Vec-30
cells were used as controls. Tumor formation was
observed in all tested animals. The results showed that
the tumor growth curve of GPR39-overexpressing cells
was significantly increased compared to Vec-30 cells
(P < 0.01, Figure 2E).
GPR39 promotes G1/S transition
To explore the mechanism underlying growth promo-
tion by GPR39, the cell cycle distributions of GPR39-c4
and Vec-30 cells were determined by flow cytometry.
Before treatment, the percentage of GPR39-c4 cells in
G1 phase was obviously reduced in comparison with
Vec-30 cells (38.37 ± 1.02% versus 45.87 ± 0.47%, P <
0.05; Figure 3A). After 3 days’ serum starvation followed
by addition of 10% serum for 8 hrs, the percentage of
cells in S phase was significantly increased in GPR39-c4
cells compared to Vec-30 cells (26.43 ± 0.71% versus
8.97 ± 0.31%, P < 0.05; Figure 3A), suggesting that
GPR39 was able to promote G1/S transition. To reveal
the potential molecular mechanism of GPR39 in cell
cycle promotion, expressions of several key cell cycle
regulators including p21, cyclin D1, CDK4 and CDK6
were compared between GPR39-c4 and Vec-30 cells.
Increased expression of cyclin D1 and CDK6, but not
p21 and CDK4, were detected in GPR39-c4 (Figure 3B).
GPR39 enhances cell motility and invasiveness of ESCCs
As the TMA result showed that overexpression of GPR39
was closely associated with ESCC metastasis, the effects
of GPR39 on cell migration and invasion were studied by
wound-healing and cell invasion assays. Wound-healing
assay showed that that the ectopic expression of GPR39
could significantly increase cell migration ability in
GPR39-transfected cells compared with empty-vector
control (P < 0.05, Figure 3C). Matrigel invasion assay also
found that the ectopic expression of GPR39 could signifi-
cantly enhanced the invasiveness of ESCC cells, as
demonstrated by a significant increase in the number of
invaded cells (P < 0.01, Figure 3D), in GPR39-transfected
cells compared with empty-vector control.
GPR39 induces partial epithelial-mesenchymal transition
(EMT)
In this study, we found that the cell morphology
changed obviously after the transfection of GPR39.
Table 1 Association between GPR39 expression and
clinical characteristics of ESCC patients (n = 207)
Clinicopathologic
characteristics
GPR39 expression no. (%)
P
OverexpressionLow
expression
Age (y)
≤60
>60
69 (59.5)
52 (57.1)
47 (40.5)
39 (42.9)
0.735
Sex
Male
Female
68 (57.6)
53 (59.6)
50 (42.4)
36 (40.4)
0.887
Tumor location
Upper
Middle
Lower
Tumor cell differentiation
Well
Moderate
Poor
Tumor invasion (T)
T1
T2
T3
T4
Lymph node metastasis (N)
N0
N1
TNM stage
I
IIa
IIb
III-IV
22 (53.7)
82 (59.4)
16 (61.5)
19 (46.3)
56 (40.6)
10 (38.5)
0.762
15 (53.6)
76 (58.9)
30 (60.0)
13 (46.4)
53 (41.1)
20 (40.0)
0.846
2 (25)
44 (65.7)
75 (57.3)
1 (100)
6 (75)
23 (34.3)
56 (42.7)
0 (0)
0.085
59 (50.4)
62 (68.9)
58 (49.8)
28 (31.1)
0.008*
1 (14.3)
57 (52.3)
15 (75.0)
48 (67.6)
6 (85.7)
52 (47.7)
5 (25.0)
23 (32.4)
0.004*
* Statistically significant (P < 0.05).
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
Page 5 of 12

Page 6

Figure 2 Tumorigenic function of GPR39 in ESCC cells. (A) Expression of GPR39 in GPR39-transfected KYSE30 cells was confirmed by RT-PCR
(left) and Western blot analysis (right). c1 and c4 are two independent GPR39-expressing clones. Vec-30 represents empty vector-transfected
KYSE30 cells. (B) Representative of foci formation in monolayer culture. Quantitative analyses of foci numbers were shown in the right panel.
Values were the mean ± SD of at least three independent experiments. **P < 0.01; independent Student’s t-test. (C) Representative of colony
formation in soft agar. Percentage of colonies formed was summarized in the right panel. Values were the mean ± SD of at least three
independent experiments. **P < 0.01. (D) Growth curves of GPR39-expressing cells were compared with Vec-30 cells by cell growth assay. The
results were expressed as mean ± SD of at least three independent experiments. **P < 0.01. (E) Tumor growth curves of GPR39-expressing cells
in nude mice were compared with Vec-30 cells by tumor xenograft experiment. The average tumor volume of GPR39-expressing cells vs Vec-30
cells was expressed as mean ± SD in 10 inoculated sites for each group of cells. **P < 0.01. (F) Representative examples of tumors formed in
nude mice following injection of GPR39-expressing KYSE30 cells (right) and Vec-30 cells (left).
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
Page 6 of 12

Page 7

GPR39-transfected cells showed spindle shape and fibro-
blastic changes in monolayer culture, whereas empty
vector-transfected cells, like KYSE30 parental cells, kept
their cobblestone-like phenotype (Figure 4A). To deter-
mine whether the effect of GPR39 on cell motility was
associated with EMT, expressions of several epithelial
markers (E-cadherin, N-cadherin) and mesenchymal
markers (vimentin, and fibronectin) were compared
between GPR39-c4 and Vec-30 cells by RT-PCR and
Western blot analysis. The results showed that E-
cadherin was obviously down-regulated in GPR39-c4
cells; however, no obvious difference was observed in
the expression of N-cadherin, vimentin and fibronectin
between GPR39-c4 and Vec-30 cells (Figure 4B). These
findings indicated that GPR39 increased cell motility
was partially through the EMT.
Figure 3 GPR39 promotes G1/S transition and enhances cell motility. (A) DNA content between GPR39-expressing cells and control Vec-30
cell were compared by Flow-cytometry. Untreated, cells were cultured in DMEM medium with 10% FBS; Withdraw serum, cells were cultured in
DMEM medium without serum for 3 days; Add serum, cells were cultured again in DMEM medium with 10% FBS for 8 hr. (B) Expression of p21,
cyclin D1, CDK4, and CDK6 were compared between GPR39-expressing cells (c4) and control Vec-30 cells by Western blot analyses. GAPDH was
used as loading control. (C) The effect of GPR39 on cell migration was determined by wound-healing assay. During a period of 16 hr, the
spreading speed of GPR39-expressing cells along the wound edge was faster than that in control Vec-30 cells. (D) Representative images showed
the GPR39-expressing cells and Vec-30 cells that invaded through the matrigel. Number of invaded tumor cells was quantified in the right panel.
Columns, mean of triplicate experiments; **P < 0.01.
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
Page 7 of 12

Page 8

Figure 4 GPR39 promotes cell mobility and invasion by inducing partial EMT and remodeling cytoskeleton. (A) Representatives of cell
morphology of GPR39-expressing cells and Vec-30 cells (original magnification × 200). (B) Expressions of epithelial markers E-cadherin and
mesenchymal markers fibronectin, N-cadherin, and vimentin, were compared by RT-PCR or Western blotting analysis between GPR39-expressing
cells and Vec-30 cells. GAPDH was used as loading control. (C) Representative images of F-actin staining. Formation of lamellipodia (indicated by
arrows) was stimulated by GPR39 compared to control cells (magnification × 400).
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
Page 8 of 12

Page 9

Overexpression of GPR39 induced lamellipodia formation
To further explore the molecular mechanism of GPR39
in regulating cancer invasion and metastasis, the role of
GPR39 in the polymerized actin was investigated by
phalloidin staining. The results showed that GPR39-
expressing cells exhibited enhanced lamellipodia forma-
tion compared with control cells (Figure 4C), indicating
that GPR39 could induce cytoskeleton remodeling to
facilitate esophageal cancer cell migration and invasion.
Silencing GPR39 expression by RNA interference (RNAi)
ESCC cell line KYSE180, which expresses a high level of
endogenous GPR39, was used in the siRNA experiment.
Two siRNAs targeting GPR39 (GPR39-si1 and GPR39-
si2) were tested and the efficiency of GPR39 gene silen-
cing was detected by RT-PCR. The result showed that
the GPR39-si1 had a better silencing effect (Figure 5A).
Silencing of GPR39 resulted in a significant inhibition of
the cell growth rate (P < 0.01, Figure 5B) and migration
(Figure 5C). DNA content analysis by flow cytometry
showed that GPR39-si1 was able to inhibit the cell cycle
at the G1/S checkpoint (Figure 5D). The percentage of
cells in the S phase was significantly reduced in GPR39-
si1-treated cells (27.23 ± 1.26%) compared with that in
control-si-treated cells (35.13 ± 1.12%; P < 0.05). These
findings further supported that the tumorigenic function
of GPR39 was through its role in promoting cell prolif-
eration and motility.
Discussion
Many G protein-coupled receptors (GPCRs) have been
found to play critical roles in the development and pro-
gression of cancer, including malignant transformation
[18,19], tumor growth and survival [20,21], as well as
invasion and metastasis [22,23]. Herein, we report that
one of the G protein-coupled receptors, GPR39, is fre-
quently overexpressed in human ESCC. To our knowl-
edge, this is the first illustration that GPR39 contributes
to the development and progression of ESCC. In the
present study, the tumorigenic function of GPR39 was
demonstrated by both in vitro and in vivo assays. Func-
tional studies showed that GPR39 could effectively pro-
mote ESCC cancer cell growth, increase foci formation
and colony formation and enhance tumor formation in
nude mice. A recent study suggested that zinc could be
a ligand capable of activating the GPR39 receptor [11].
Interestingly, zinc deficiency along with its associated
increased cell proliferation can be tumorigenic in the rat
esophagus [24,25]. Our study also provided evidence
that ectopic expression of GPR39 increased ESCC
cancer cell growth, indicating involvement of the GPR39
receptor in the tumorigenesis of esophageal cancer.
However, whether GPR39 signaling is activated by zinc
in esophageal carcinogenesis needs to be further
investigated. Further study revealed that overexpression
of GPR39 in esophageal cancer cells KYSE30 promoted
G1/S phase transition. We showed for the first time that
GPR39 controls cell cycle progression through the acti-
vation of CDK6 and its activating protein, cyclin D1.
G1/S phase transition is a major checkpoint for cell
cycle progression and cyclin D1-CDK6 complex is one
of the critical positive regulators during this transition
[26,27]. On the other hand, we found that silencing of
GPR39 expression could inhibit tumorigenicity in
KYSE180 cells through the cell cycle arrest at G1/S
checkpoint.
Another interesting finding of this study is the pro-
moting effect of GPR39 on tumor metastasis in ESCC.
Our data showed that overexpression of GPR39 could
promote cell motility and invasiveness of ESCC cells
in vitro. This mirrored the findings of GPR39 overex-
pression in human ESCC samples and its association
with advanced clinical stage and lymph node metastasis
of ESCC. Conversely, when we knocked down the endo-
genous GPR39 by RNAi in ESCC cells, the mobility of
ESCC cells was significantly reduced, suggesting that
GPR39 is closely involved in ESCC invasion and metas-
tasis. Moreover, the observation of overexpression of
GPR39 resulting in cell morphological alteration pro-
moted us to further investigate its effect on EMT. We
found that GPR39 has some impact on the EMT as
shown by decreasing the epithelial molecule E-cadherin,
an event critical in tumour invasion and a ‘master’ regu-
lator of EMT. E-cadherin provides a physical link
among adjacent cells and is crucial for the establishment
and maintenance of polarity and the structural integrity
of epithelia. Indeed, due to the physical and functional
link between E-cadherin based complexes and cytoskele-
tal components, a change in the E-cadherin mediated
adhesiveness leads to rearrangement of the cytoskeleton
[28]. In view of this, we further explored the role of
GPR39 in reorganization of the actin cytoskeleton. As
expected, our result showed that GPR39 led to signifi-
cant alterations on cytoskeleton by inducing the lamelli-
podia formation in GPR39-transfected ESCC cells. This
finding was consistent to previous studies that some G
protein-coupled receptors (GPCRs) were able to pro-
mote actin reorganization and result in cell shape
changes and enhanced cell migration [13,29], indicating
that GPR39 might directly alter the cytoskeleton to
favor the tumor cell invasion and metastasis in ESCC.
In this study, we have also provided evidence that tar-
geting of GPR39 with specific RNAi will reduce the
oncogenic characteristics of ESCC tumor cells. To date,
some G protein-coupled receptors (GPCRs) provide
important practical options for preclinical research, clin-
ical trials, and cancer treatment [30]. Therefore, consid-
eration should be given to the development of novel
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
Page 9 of 12

Page 10

Figure 5 Silencing of GPR39 expression suppresses tumorigenic ability of GPR39. (A) GPR39 expression was efficiently decreased by the
treatment of siGPR39 by RT-PCR. Relative expression level was measured by densitometer and summarized in the right panel. **P < 0.01. (B)
Growth curve of KYSE180 cells treated with GPR39 siRNA was compared with control siRNA treated cells by cell growth assay. **P < 0.01. (C) Cell
migration assay was used to compare the frequency of migratory cells between KYSE180 cells treated with control siRNA and GPR39 siRNA.
(D) DNA content between control siRNA and GPR39 siRNA treated cells were compared by Flow-cytometry. *P < 0.05.
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
Page 10 of 12

Page 11

therapeutics targeting GPR39 for use in GPR39-expres-
sing ESCC tumors.
Conclusions
In summary, our findings demonstrate that GPR39 plays
an important role in ESCC development and progres-
sion via promoting cell proliferation, enhancing cell
motility and invasiveness, regulating cytoskeleton and
inducing EMT. A better understanding of the molecular
mechanism of GPR39 in ESCC development and pro-
gression would provide novel therapeutic strategies to
ESCC cancer patients.
Abbreviations
EMT: epithelial mesenchymal transition; ESCC: esophageal squamous cell
carcinoma; GPCR: G protein-coupled receptor; siRNA: small interfering RNA;
TMA: tissue microarray; TSG: tumor suppressor gene; L: length; V: volume; W:
width.
Acknowledgements
This work was supported by Grants from National Natural Science
Foundation of China (30772475, 30700820 and 30971606), Sun Yat-Sen
University “Hundred Talents Program” (85000-3171311), Grant from the Major
State Basic Research Program of China (2006CB910104), Research Fund for
the Doctoral Program of Higher Education of China (20070558272) and
Research Grant Council Central Allocation (HKUST 2/06C).
Author details
1State Key Laboratory of Oncology in Southern China, Cancer Center, Sun
Yat-Sen University, Guangzhou, PR China.2Department of Clinical Oncology,
University of Hong Kong, Hong Kong, PR China.3Department of Clinical
Oncology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou, PR
China.4Department of Clinical Oncology, the Cancer Hospital, Zhengzhou
University, Zhengzhou, PR China.
Authors’ contributions
FX and HL performed the experimental procedures with support from YZ,
YQ, YD, TZ, LC, CN, TH and YL. FX, LF and XYG were responsible for
experimental design, interpretation of the results and writing the
manuscript. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 26 April 2010 Accepted: 25 February 2011
Published: 25 February 2011
References
1.Enzinger PC, Mayer RJ: Esophageal cancer. The New England journal of
medicine 2003, 349(23):2241-2252.
2. Chen BS, Wang MR, Xu X, Cai Y, Xu ZX, Han YL, Wu M: Transglutaminase-3,
an esophageal cancer-related gene. International journal of cancer 2000,
88(6):862-865.
3.Metzger R, Schneider PM, Warnecke-Eberz U, Brabender J, Holscher AH:
Molecular biology of esophageal cancer. Onkologie 2004, 27(2):200-206.
4.Montesano R, Hollstein M, Hainaut P: Genetic alterations in esophageal
cancer and their relevance to etiology and pathogenesis: a review.
International journal of cancer 1996, 69(3):225-235.
5.Qin YR, Wang LD, Fan ZM, Kwong D, Guan XY: Comparative genomic
hybridization analysis of genetic aberrations associated with
development of esophageal squamous cell carcinoma in Henan, China.
World J Gastroenterol 2008, 14(12):1828-1835.
6. Fu L, Qin YR, Xie D, Hu L, Kwong DL, Srivastava G, Tsao SW, Guan XY:
Characterization of a novel tumor-suppressor gene PLC delta 1 at 3p22
in esophageal squamous cell carcinoma. Cancer research 2007,
67(22):10720-10726.
7.Zhu C, Qin YR, Xie D, Chua DT, Fung JM, Chen L, Fu L, Hu L, Guan XY:
Characterization of tumor suppressive function of P300/CBP-associated
factor at frequently deleted region 3p24 in esophageal squamous cell
carcinoma. Oncogene 2009, 28(31):2821-2828.
Moechars D, Depoortere I, Moreaux B, de Smet B, Goris I, Hoskens L,
Daneels G, Kass S, Ver Donck L, Peeters T, Coulie B: Altered gastrointestinal
and metabolic function in the GPR39-obestatin receptor-knockout
mouse. Gastroenterology 2006, 131(4):1131-1141.
Popovics P, Stewart AJ: GPR39: a Zn(2+)-activated G protein-coupled
receptor that regulates pancreatic, gastrointestinal and neuronal
functions. Cell Mol Life Sci 2010.
Holst B, Holliday ND, Bach A, Elling CE, Cox HM, Schwartz TW: Common
structural basis for constitutive activity of the ghrelin receptor family.
The Journal of biological chemistry 2004, 279(51):53806-53817.
Holst B, Egerod KL, Schild E, Vickers SP, Cheetham S, Gerlach LO,
Storjohann L, Stidsen CE, Jones R, Beck-Sickinger AG, Schwartz TW: GPR39
signaling is stimulated by zinc ions but not by obestatin. Endocrinology
2007, 148(1):13-20.
Dittmer S, Sahin M, Pantlen A, Saxena A, Toutzaris D, Pina AL, Geerts A,
Golz S, Methner A: The constitutively active orphan G-protein-coupled
receptor GPR39 protects from cell death by increasing secretion of
pigment epithelium-derived growth factor. The Journal of biological
chemistry 2008, 283(11):7074-7081.
Cotton M, Claing A: G protein-coupled receptors stimulation and the
control of cell migration. Cellular signalling 2009, 21(7):1045-1053.
Dorsam RT, Gutkind JS: G-protein-coupled receptors and cancer. Nature
reviews 2007, 7(2):79-94.
Shimada Y, Imamura M, Wagata T, Yamaguchi N, Tobe T: Characterization
of 21 newly established esophageal cancer cell lines. Cancer 1992,
69(2):277-284.
Xie D, Sham JS, Zeng WF, Lin HL, Che LH, Wu HX, Wen JM, Fang Y, Hu L,
Guan XY: Heterogeneous expression and association of beta-catenin,
p16 and c-myc in multistage colorectal tumorigenesis and progression
detected by tissue microarray. International journal of cancer 2003,
107(6):896-902.
Cao ZA, Daniel D, Hanahan D: Sub-lethal radiation enhances anti-tumor
immunotherapy in a transgenic mouse model of pancreatic cancer. BMC
cancer 2002, 2:11.
Burger M, Hartmann T, Burger JA, Schraufstatter I: KSHV-GPCR and CXCR2
transforming capacity and angiogenic responses are mediated through
a JAK2-STAT3-dependent pathway. Oncogene 2005, 24(12):2067-2075.
Marin YE, Chen S: Involvement of metabotropic glutamate receptor 1, a
G protein coupled receptor, in melanoma development. Journal of
molecular medicine (Berlin, Germany) 2004, 82(11):735-749.
Spiegelberg BD, Hamm HE: Roles of G-protein-coupled receptor signaling
in cancer biology and gene transcription. Current opinion in genetics &
development 2007, 17(1):40-44.
Thomas SM, Bhola NE, Zhang Q, Contrucci SC, Wentzel AL, Freilino ML,
Gooding WE, Siegfried JM, Chan DC, Grandis JR: Cross-talk between G
protein-coupled receptor and epidermal growth factor receptor
signaling pathways contributes to growth and invasion of head and
neck squamous cell carcinoma. Cancer research 2006,
66(24):11831-11839.
Even-Ram S, Uziely B, Cohen P, Grisaru-Granovsky S, Maoz M, Ginzburg Y,
Reich R, Vlodavsky I, Bar-Shavit R: Thrombin receptor overexpression in
malignant and physiological invasion processes. Nature medicine 1998,
4(8):909-914.
Uchida D, Begum NM, Tomizuka Y, Bando T, Almofti A, Yoshida H, Sato M:
Acquisition of lymph node, but not distant metastatic potentials, by the
overexpression of CXCR4 in human oral squamous cell carcinoma.
Laboratory investigation; a journal of technical methods and pathology 2004,
84(12):1538-1546.
Fong LY, Sivak A, Newberne PM: Zinc deficiency and
methylbenzylnitrosamine-induced esophageal cancer in rats. J NatI
Cancer Inst 1978, 61(1):145-150.
Fong LY, Li JX, Farber JL, Magee PN: Cell proliferation and esophageal
carcinogenesis in the zinc-deficient rat. Carcinogenesis 1996,
17(9):1841-1848.
Gutierrez C, Ramirez-Parra E, Castellano MM, del Pozo JC: G(1) to S
transition: more than a cell cycle engine switch. Current opinion in plant
biology 2002, 5(6):480-486.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
Page 11 of 12

Page 12

27.Vermeulen K, Van Bockstaele DR, Berneman ZN: The cell cycle: a review of
regulation, deregulation and therapeutic targets in cancer. Cell
proliferation 2003, 36(3):131-149.
Guarino M, Rubino B, Ballabio G: The role of epithelial-mesenchymal
transition in cancer pathology. Pathology 2007, 39(3):305-318.
Li S, Huang S, Peng SB: Overexpression of G protein-coupled receptors in
cancer cells: involvement in tumor progression. International journal of
oncology 2005, 27(5):1329-1339.
Liebmann C: G protein-coupled receptors and their signaling pathways:
classical therapeutical targets susceptible to novel therapeutic concepts.
Current pharmaceutical design 2004, 10(16):1937-1958.
28.
29.
30.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2407/11/86/prepub
doi:10.1186/1471-2407-11-86
Cite this article as: Xie et al.: Overexpression of GPR39 contributes to
malignant development of human esophageal squamous cell
carcinoma. BMC Cancer 2011 11:86.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Xie et al. BMC Cancer 2011, 11:86
http://www.biomedcentral.com/1471-2407/11/86
Page 12 of 12