Background
Gastric cancer is the second leading cause of cancer death in China, accounting for about 17.6% of all cancer deaths [
1], although its incidence and mortality rates decreased worldwide [
2]. Despite advances in diagnosis and treatment, the outcome of gastric cancer patients remains poor [
3,
4]. Local invasion and distal metastasis largely account for the poor prognosis in these patients. Therefore, it is imperative to explore the underlying mechanism of gastric cancer metastasis in order to identify novel therapeutic approaches and to improve the patient survival.
Testes-specific protease 50 (TSP50) is a novel member of cancer/testis antigens (CTAs), which is not expressed in normal tissues except testes and cancers, including breast cancer, colorectal cancer,
laryngocarcinoma and cervical cancer [
5‐
10]. Accumulating evidences implicate that TSP50 is involved in proliferation, apoptosis, migration and metastasis in various types of tumors except gastric cancer [
8‐
10]. We recently reported that TSP50 expression was up-regulated in gastric cancer tissues compared with adjacent non-tumor mucosa. TSP50 overexpression was associated with lymph node metastasis and poor prognosis in gastric cancer patients [
11]. However, the biologic role and molecular mechanisms of TSP50 in gastric cancer metastasis remain to be elucidated.
NF-κB is known to be a tumorigenic and prometastatic factor in gastric cancer [
12,
13]. NF-κB signaling has been proved to be a downstream target of TSP50 [
14,
15]. Moreover, deregulation of NF-κB has been reported to induce the epithelial-to–mesechymal transition (EMT) in various cancers [
16‐
18], which is believed to be an essential step for tumor cell invasion and metastasis.
In this study, we investigated whether TSP50 activates EMT in gastric cancer cells by NF-κB signaling pathway thus promoting cancer invasion and metastasis. Genetic manipulation of TSP50 levels showed that TSP50, being highly expressed in gastric cancer cells, promoted the proliferation, migration and invasion in vitro. Mechanistically, TSP50 activates EMT in gastric cancer by up-regulating Vimentin and Twist whereas down-regulating E-Cadherin. The control of TSP50 on EMT activation was also confirmed in human gastric cancer tissues. Statistical analysis showed a significant negative correlation between TSP50 and E-Cadherin expression in human gastric cancer tissues. Combining TSP50 and E-Cadherin provide superior performance in the prediction of prognosis and metastasis as compared to TSP50 or E-Cadherin alone. In addition, we showed that TSP50 activates EMT process in gastric cancer via augmenting NF-κB signaling pathway. Pharmacological inhibition of NF-κB pathway by its specific inhibitor blocks TSP50 induced migration and invasion. Our data for the first time identified the mechanism by which TSP50 may promote tumor cell invasion and metastasis in gastric cancer.
Methods
Patients and specimens
Formalin-fixed, paraffin-embedded tissues from 334 patients with gastric cancer were collected as described in our previous study [
11], 30 corresponding lymph node metastatic lesions were added in the present study.
Cell lines and cell culture
Human gastric adenocarcinoma cell line MKN-45, BGC-823, MGC-803, SGC-7901, AGS and human gastric epithelial cell line GES-1 (Shanghai Institute of Cell Biology, China) were grown in F-12 k (ATCC) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C with humidified 5% CO2. For inhibitor treatment, the cultured cells were incubated with 10 μmol/l BAY-117082 (Selleck, USA) for 48 h. Cells were collected in logarithmic growth phase for all experiments.
RNA extract and quantitative real time PCR
Total RNA was extracted from MKN-45, BGC-823, MGC-803, SGC-7901, AGS and GES-1 using Trizol reagent (Invitrogen, USA) according to protocol. Complementary DNA was prepared using oligodT primers according to the protocol supplied with the Primer Script TM RT Reagent (TaKaRa, Japan). Expression of TSP50 was determined by quantitative real-time PCR using Power SYBR green PCR master mix (Applied Biosystems). Results were normalized to the expression of GAPDH. The primers for TSP50 were: forward: 5’-TCGTGCTCGTTCCAAAGG-3′ and reverse: 5’-GGCAATAGGTGGGTTCGTT-3′.
Establishment of stably transfected cell lines
For TSP50 overexpression, ectopic TSP50 coding sequence was amplified by polymerase chain reaction (PCR). The primer sequences were: forward: 5’-GTAGGATCCGCGAGGGGAAGCCCCGG-3′ and reverse: 5’-CCGAATTCTTATCACTGCCCGTTGAGGCAGTCC-3′. The amplified product was cloned into the pBaBb-puromycin plasmid and confirmed by sequencing. For TSP50 and p65 silencing, sequences of short hairpin RNA targeting TSP50 (shTSP50) and p65 (shp65) werecloned into the pSUPER-retro-puromycin plasmid. The shTSP50 and shp65 sequences were: 5’-GTTCTGCTATGAGCTAACT-3′ and 5’-GCCCTAUCCCTTTACGTCA-3′, respectively. The sequence of scrambled control shRNA was: 5’-GACGCTTACCGATTCAGAA-3′. GC cell lines were transfected with aforementioned constructed plasmids or empty vector. Stably transfected cell lines were selected with 0.5 μg/ml puromycin at 48 h after infection.
Cell proliferation assay
BrdU incorporation and Cell count were used to assess cell proliferation as described previously [
19]. BrdU incorporation was examined using 5-Bromo-2′-deoxy-uridine Labeling and Dectection kit III (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, cells were serum free for 24 h. Then cells were trypsinized and equal number (2 × 10
4) of cells from each group was plated into a 96-well plate and grown in complete culture medium with 10 μM BrdU for 2, 4 or 6 h. BrdU incorporation into cellular DNA was assessed using a microplate reader (Safie II; Tecan, Mannedorf, Switzerland). The experiment was repeated three times independently. For the cell counts, cells were serum free for 24 h. Then cells were trypsinized and equal number (2 × 10
5) of cells from each group was plated into 6-well culture plates in complete culture medium for 1, 2, 3 days. Then the cell number was determined in triplicate using a hemocytometer.
Cell migration and invasion assay
Wound healing assay and transwell assay were employed to evaluate the ability of migration and invasion. For the wound healing assay, cells were serum free for 24 h. Then cells were trypsinized and equal number (3.5 × 105) of cells from each group was plated into 6-well culture plates in complete culture medium for 4 h, then a scratch lesion was created using a 200 μl pipette tip. To avoid dislodged cells, culture medium was removed and wells were washed gently with PBS. Then cells were grown in serum-free culture medium for 24 h until the digital images of cells migrated into the scratch were taken on an inverted microscope. Measurement of wound area was done using the Adobe Photoshop software. Wound closure was quantified as the mean ± standard deviation(SD) of three independent experiments. The control wound closure was set as 100%, and the wound closure of overexpression or knockdown group was represented as the percent of the control. Transwell inserts with 8 μm pores (BD Biosciences, San Jose, CA, USA) for transwell migration assays, 2 × 105 cells in serum free medium were added to each upper compartment of the chamber. After 48 h incubation, noninvasive cells were removed from the upper surface of the transwell membrane, and migrated cells were fixed with methanol, stained with 1% crystal violet and counted using a light microscope in 5 random visual fields at the magnification of 100 × .
Immunohistochemistry analysis
Immunohistochemistry was carried out with the Dako Envision System (Dako, Denmark). Target protein expression level was evaluated by integrating the percentage of positive tumor cells and the intensity of positive staining. Briefly, sections were scored as 0 (negative), 1 (bordering), 2 (weak), 3 (moderate) or 4 (strong), whereas the staining extent was scored according to the area percentages: 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51%–75%) or 4 (76–100%). The products of staining intensity and extent scores were the final staining scores (0–16). The median score was used as cut-off point to divide the patients into high or low expression group.
Western blot analysis
Cells were collected and lysed with the RIPA buffer containing protease inhibitor. Protein concentration was determined by the Bradford method with bovine serum albumin as the control. Equal amounts of tissue lysates (50 μg) were run by SDS-PAGE, and electro-transferred on a polyvinylidene difloride membrane. The membrane was then blocked and incubated with primary antibodies against TSP50 (1:400, Proteintech, USA), E-cadherin (1:500, Abcam, UK), Vimentin(1:2000, Abcam, UK), Twist(1:1000, Abcam, UK), P65(1:500, Santa Cruz, CA), Histone1 (1:500, Dako, Denmark) and β-actin antibody (1:1000, Santa Cruz, CA) respectively, for 2 h at room temperature, and then incubated with appropriate horseradish peroxidaseconjugated secondary antibodies (1:1000, Cell Signaling Technology) for 1 h at room temperature. Final detection was carried out with LumiGLO chemiluminescent reagent (New England Biolabs) as described by the manufacturer. The densities of target bands was accurately determined by the computer-aided 1-D gel analysis system.
Statistical analysis
Statistical analysis was performed using SPSS standard version 19.0 (SPSS Inc) and GraphPad Prism 5 (GraphPad Software). Survival curves calculation and OS curve plotting used the Kaplan-Meier method, and the Log-Rank test was applied to compare the distribution between patient subsets. The association between TSP50 and E-Cadherin was estimated by Phiand Cramers V correlation analysis. ANOVA or Student’s unpaired t-test were used to analyze the cellular proliferation, migration and invasion. Data from all quantitative assays were demonstrated as the mean ± standard and values of P < 0.05 were considered statistically significant.
Discussion
The present study has provided the first evidence concerning the role of TSP50 in gastric cancer. Our data showed that: (1) TSP50 was significantly up-regulated in most of the gastric cancer cell lines, and contributed to their proliferation and invasion; (2) TSP50 was negatively related with E-Cadherin expression in gastric cancer tissues as well as lymph node metastasis, and combination of TSP50 and E-Cadherin improved the prediction for prognosis and lymph node metastasis; (3) overexpression of TSP50 induced EMT through activating NF-κB signaling pathway to promote gastric cancer metastasis.
TSP50 was first identified in human breast cancer. After that TSP50 was shown to be involved in proliferation and metastasis of various cancer cells. In P19 murine embryonal carcinoma stem cells, knockdown of TSP50 inhibited cell proliferation and induced apoptosis [
24]. Similar results were illustrated in laryngocarcinoma and cervical cancer [
9,
10]. Song et al. recently found that TSP50 overexpression facilitated breast cancer cells motility and contribute to the development of metastasis both in vitro and in vivo [
15]. These data suggested that TSP50 may serve as a common mechanism to promote tumorigenesis in different types of cancers. Consistently, we found that TSP50 was elevated in most of the gastric cancer cell lines, and overexpression or knockdown of TSP50 significantly affected cellular proliferation, migration and invasion.
EMT, characterized by loss of epithelial features (e.g. E-Cadherin) and acquired characteristics of mesenchymal cells (e.g. Vimentine, Twist), facilitate cells motility and invasion during the tumor development [
18,
25]. Accumulating evidence has established the role of aberrant EMT activation in gastric tumorigenesis and cancer progression. However, the mechanism by which EMT is activated in the carcinogenesis of gastric cancer remains elusive [
26,
27]. Recent study showed that TSP50 promotes cell invasion and metastasis by augmenting matrix metalloproteinase-9 expression in human breast cancer [
15]. Our results showing modulation of TSP50 altered the phenotypes of gastric cancer cells in vitro prompted us to investigate whether EMT is the primary downstream target of TSP50 regulated effects. Since loss of epithelial marker E-Cadherin is one of the most important molecular events during EMT [
20‐
22], we examined the expression pattern of E-Cadherin and its relation with TSP50 level in tissue microarray of a large number of archived human gastric cancers. First, we found that TSP50 overexpression was significantly correlated with E-Cadherin down-regulation in primary gastric cancer tissues and lymph node metastasis, and combination of them was a more powerful predictor for gastric cancer prognosis. Further, we showed that overexpression of TSP50 up-regulated mesenchymal maker Vimentin, EMT related transcript factor Twist and down-regulated epithelial marker E-Cadherinin in gastric cancer cells. Therefore, these data support the fact that TSP50 acts by enhancing EMT in gastric cancer progression. In fact, several oncogenic CTAs were recently shown to be involved in EMT and cancer metastasis. In this regard, CT45A1 is a potent inducer of the expression of the EMT master gene Twist1 in breast cancer and thereby promotes tumor invasion, and metastasis [
28]. SSX (CTA5) was reported to be interacted with β-catenin leading to alterations of the transcription profile of target genes including Snail-2, E-cadherin, and Vimentin [
29]. In gastric cancer, there were a few studies showing an association between CTAs and poor prognosis [
30,
31]. However, the mechanism underlying its pathogenesis is rather lacking. Our study thus provided the first mechanistic evidence corroborating the important role of TSP50 in promotion of EMT and metastasis in gastric cancer.
Our study also demonstrated that TSP50 activated EMT in a NF-κB dependent manner in gastric cancer cells. NF-κB signaling plays a critical role in promoting and maintaining invasiveness of cancer cells via controlling of EMT process in different tumors including gastric cancer [
16‐
18,
32,
33]. An earlier study also showed that NF-kB is required in TSP50-induced migration and invasion of breast cancer cells [
15]. However, the effect of TSP50 on NF-kB in gastric cancer has not been reported in the literature. Therefore, our data provide a critical link between TSP50 and NF-kB in terms of gastric cancer progression. Nevertheless, given the fact that NF-kB signaling is broadly involved in the regulation of metastasis [
17,
34‐
36], other signaling pathway other than EMT may also account for the TSP50 dependent invasive phenotype in gastric cancer. For instance, NF-κB activation was required for the transcription of a group of adhesion molecules including endothelial-leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1), which facilitate the extravasation of cancer cells [
37,
38]. NF-κB binding sites were identified in the promoters of genes that encode several matrix metalloproteinases (MMPs) including MMP-2, MMP-9, and so forth, which degrade the extracellular matrix (ECM) to facilitate tumor cell invasion in tissues [
39]. Further studies are needed to address these concerns in the pathogenesis of gastric cancer progression. Furthermore, it should be noted that inhibition of NF-κB signaling activity by its specific inhibitor BAY117082 or shp65 did not completely abrogate the TSP50 mediated activation of EMT, migration and invasion of gastric cancer cells, suggesting that other molecular mechanisms might be involved. Various cell signaling pathways are involved in the regulation of EMT, including Wnt/β-catenin, TGF-β, Notch, Hedgehog and others [
40‐
43]. The pathways involved in TSP50-induced EMT besides NF-κB are also needed to be investigated in our future studies.