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 development 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 characterized 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 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 gastrointestinal and metabolic function [
8]. GPR39 receptor is now thought to be activated by Zn
2+ signals and may have other, as yet unidentified, cognitive ligands [
9]. Moreover, GPR39 receptor also displays a strong ligand-independent signaling activity through Gα
12/13 as well as Gα
q [
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 evidence indicates that G protein-coupled receptors are crucial players in cancer progression and metastasis [
13,
14], however, the role of GPR39 in cancer development 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 Ethical 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, Carlsbad, 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'-GCCACCGGGGTCTCACTTGC-3' and GPR39Rv: 5'-GGCCGCAGCCATGATCCTCC-3'. GAPDH (Fw: 5'-CATGAGAAGTATGACAACAGCCT; Rv: 5'-AGTCCTTCCACGATACCAAAGT) 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 Linzhou 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 percentage 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 transfected 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 × 103 GPR39-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 formation 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 perform cell growth assay, GPR39-expressing cells and control Vec-30 cells were seeded in 96-well plate at a density of 800 cells per well. The cell growth rate was 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).
For
in vivo experiment, stable GPR39-expressing KYSE30 cells or control Vec-30 cells (1 × 10
6) 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 × W
2 [
17]. All experiments were done in accordance with institutional standard guidelines of Sun Yat-Sen University for animal 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 medium 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 permeabilized 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 additional 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 (Invitrogen) 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 standard version 16.0 (SPSS Inc., Chicago, IL). The relationship between the expression of GPR39 protein and clinicopathologic characteristics was assessed by χ2 test. Results expressed as mean ± SD were analyzed using the Student t test. Differences were considered significant when P < 0.05.
Discussion
Many G protein-coupled receptors (GPCRs) have been found to play critical roles in the development and progression 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 frequently overexpressed in human ESCC. To our knowledge, 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. Functional studies showed that GPR39 could effectively promote 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 activation 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 promoting 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 overexpression in human ESCC samples and its association with advanced clinical stage and lymph node metastasis of ESCC. Conversely, when we knocked down the endogenous GPR39 by RNAi in ESCC cells, the mobility of ESCC cells was significantly reduced, suggesting that GPR39 is closely involved in ESCC invasion and metastasis. Moreover, the observation of overexpression of GPR39 resulting in cell morphological alteration promoted 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' regulator 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 cytoskeletal 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 significant alterations on cytoskeleton by inducing the lamellipodia formation in
GPR39-transfected ESCC cells. This finding was consistent to previous studies that some G protein-coupled receptors (GPCRs) were able to promote 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 targeting 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, clinical trials, and cancer treatment [
30]. Therefore, consideration should be given to the development of novel therapeutics targeting GPR39 for use in GPR39-expressing ESCC tumors.
Conclusions
In summary, our findings demonstrate that GPR39 plays an important role in ESCC development and progression 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 progression would provide novel therapeutic strategies to ESCC cancer patients.
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).
Competing interests
The authors declare that they have no competing interests.
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.