Med Oncol (2015) 32:83 DOI 10.1007/s12032-015-0539-5
ORIGINAL PAPER
VGLL4 inhibits EMT in part through suppressing Wnt/b-catenin signaling pathway in gastric cancer Hui Li • Ziwei Wang • Wei Zhang • Kun Qian • Gang Liao • Wei Xu • Shouru Zhang
Received: 7 January 2015 / Accepted: 13 February 2015 Ó Springer Science+Business Media New York 2015
Abstract VGLL4 is a member of the Vestigial-like proteins that functions as a tumor suppressor, which directly competes with YAP for binding TEADs in several cancer types. Recently, an increasing number of studies have reported that VGLL4 acts as a crucial role in regulating cell mobility, migration, and invasion. However, little is known about the signaling mechanisms in regulating epithelial– mesenchymal transition (EMT) of gastric cancer. In our study, we confirmed that the expression level of VGLL4 was down-regulated in gastric cancer tissues, and reduced VGLL4 expression levels inhibited apoptosis and promoted proliferation, migration, and invasion. Additionally, we found a phenomenon that VGLL4 was associated with the change in nuclear location of b-catenin, which suggested that b-catenin was a significant downstream factor of VGLL4. These results suggest that VGLL4 suppressed EMT in part via negative regulation of Wnt/b-catenin signaling pathway. Taken together, our study demonstrated that VGLL4 is important in the process of suppressing tumor progression of gastric cancer and provided a potential therapeutic strategy for gastric cancer. Keywords VGLL4 Gastric cancer Wnt/b-catenin Tumor progression EMT
H. Li Z. Wang (&) W. Zhang K. Qian G. Liao W. Xu S. Zhang Department of Gastrointestinal Surgery, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China e-mail: [email protected]
Introduction Gastric cancer is the fourth most frequent malignant neoplasm and the second leading cause of cancer death worldwide [1]. Invasiveness and metastasis are considered as a main reason for gastric cancer death. In the last few years, an increasing number of studies reported that epithelial–mesenchymal transition (EMT) plays an important role in cancer progression and metastasis [2–4]. EMT is a process that is characterized by the loss of epithelial characteristics and acquisition of a mesenchymal phenotype to produce enhanced motility, resistance to apoptosis, and the capacity to invade the surrounding tissues [5–8]. Many reports about EMT had been observed in various gastric cell lines such as MGC-803, MKN-45, SGC-7901, BGC-823 [9, 10]. It is well known that the occurrence of EMT is associated with several distinct pathways, including Notch, TGF-b, and several receptor tyrosine kinase [11–13]. Furthermore, dysregulation of Wnt/b-catenin signaling has been proved to play a significant role in the progression of both the EMT and cancer metastasis [14– 17]. In addition, accumulation of nuclear b-catenin is a significant marker of enhanced Wnt signaling [18]. VGLL4 is a member of Vestigial-like (VGLL) proteins that has four subtypes in mammals, and these VGLL proteins contain the TEAD-interacting domain (TDU) [19– 22]. Some studies have found that VGLL1 and VGLL3 were up-regulated in basal-like breast cancer samples and soft tissue sarcoma, respectively [23–25]. However, VGLL4 may have different functions because it contains two TDU domains, unlike other members in VGLL family. As the literatures reported, VGLL4 is a tumor suppressor in pancreatic adenocarcinoma and promotes cell apoptosis through down-regulating the inhibitor of apoptosis proteins [26, 27]. In addition, a recent study showed that VGLL4
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directly competed with YAP for binding TEADs to suppress tumor growth [28]. However, the mechanisms involved in the anti-EMT effects of VGLL4 in gastric cancer are unknown. In this study, we used in vivo and in vitro approaches to demonstrate that whether VGLL4 could inhibit EMT and whether the regulatory effects of VGLL4 on the EMT are partially attributed to the Wnt/b-catenin signaling pathway in gastric cancer.
Materials and methods Cell culture and transfection The human immortalized gastric epithelial cell line GES-1 and human gastric cancer cell lines AZ-521, SGC-7901, BGC-823, and MGC-803 were obtained from Shanghai Center for Life Science Research Institute of Chinese Academy of Sciences cell resources. AZ-521 and BGC-823 cells were both maintained in RPMI 1640 medium (RPMI 1640, Gibco, USA). These media contained 10 % FBS (Gibco, USA) and 2 % penicillin/streptomycin (Hyclone, Shanghai, China). All the cells were cultured in an incubator containing humidified 95 % air and 5 % CO2 at 37 °C. Lentivirus constructs expression VGLL4-shRNA (LVVGLL4-shRNA-Puromycin), VGLL4 (LV-VGLL4-Puromycin), and the nontargeted negative control virus were purchased from Shanghai Genechen Co., Ltd. (China). The human VGLL4-shRNA sequence was 50 -TCACCTGTG CCTCGG CTG GCG-30 , 50 -TGAACAAGACTGCCAA TGGAG-30 . The AZ-521 and BGC-823 cells were digested by 0.125 % trypsase and then seeded on 6-well plate, respectively, at a concentration of 1.5 9 105 and 1.0 9 105. The cells were transfected in the following day at a MOI of 60(AZ-521) and 80(BGC-823) using polybrene (10 lg/ml) and enhanced infection solution (Genechen, China) for 12 h, and the lentiviral empty vector was used with the control cells by a same method. After incubation for 48 h, the cells were selected with corresponding concentration of puromycin. The cells would be collected for mRNA and protein analysis and other assays in the reasonable time. Tissue samples and immunohistochemistry One hundred and fifty gastric cancer samples, paired peritumoral samples ([2 cm distance from the margin of the resection), and 20 normal samples were obtained from the First Affiliated Hospital of Chongqing Medical University, China, between 2011 and 2013. None of the patients underwent any other treatment before operation. Tumor tissues, paired peritumoral tissues, and normal tissues were immediately stored in paraformaldehyde after
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collected. Tissue sections were deparaffinized, and antigens were retrieved by heating in a buffer (0.01 M sodium citrate) in a microwave oven at 95 °C for 15 min. The slides were incubated in 3 % hydrogen peroxide for 15 min and subsequently treated with 0.5 % Triton X-100 (Beyontime, Zhenjiang, Jiangsu, China) for 15 min. Then, we blocked the slides with 5 % BSA for 30 min and treated the slides with anti-VGLL4 primary antibody (1:200 dilution, Sigma, USA) overnight at 4 °C. The next day, the slides were incubated with proper secondary antibodies for 30 min after washing in PBS for 30 min. Then, the slides were exposed to streptavidin-HRP label (Zhongshanjinqiao, Beijing, China) for 30 min. The percentage of positive cells was graded according to the following criteria: 0, \10 %; 1, 10–30 %; 2, 30–50 %; 3, [50 % [29]. This study was supported by the approval and supervision of the Medical Ethics Committee of First Affiliated Hospital of Chongqing Medical University. Meanwhile, we asked for consents from patients and healthy subjects about collecting the tissues specimens. Transwell migration and invasion assay Transwell migration assay was performed as previously described [30]. 1 9 105 cells of AZ-521 and BGC-823 were, respectively, added to the top chamber of transwell (Millicell, Millipore, USA), and the bottom chamber of transwell was placed with corresponding medium supplemented with 10 % FBS. The cells were fixed with 4 % paraformaldehyde for 30 min after 24 h of culture with 37 °C in 5 % CO2. Thereafter, the cells were stained in 0.1 % crystal violet for 10 min and counted under a light microscope at 1009 magnification. Transwell invasion assay was similarly performed through a modified Boyden chamber with polyester membrane inserts that were coated with Matrigel (BD Bioscience, USA). Cell proliferation and apoptosis detection AZ-521 and BGC-823 were seeded with a concentration of 1 9 103 resuspended cells on 96-well plates in triplicate in 100 ll of RPMI 1640 medium. Then, we performed the Cell Counting Kit-8 (CCK8) assay (Dojindo, Japan) to test the cell proliferation. The cells were treated with 10 ll of CCK8 in each well and were incubated for 1 h at 37 °C in 5 % CO2 every day. Finally, we measured the absorbance of the cells by a spectrophotometer at 450 nm. Flow cytometry was used to investigate apoptosis. All the cells were cultured in 6-well plates. Then, each cell of three groups (521-VGLL4-shRNA, 521-NC, 521-BC, 823-VGLL4, 823-NC, 823-BC) was resuspended in a concentration of 1 9 106. Thereafter, we added 5 ll Annexin V-FITC into each well, and the cells were incubated in the dark for 15 min. We subsequently treated the cells
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Fig. 1 Immunohistochemical (IHC) staining of VGLL4 (a–c) in normal, peritumoral, and gastric cancer tissues, respectively. Positive expression is visualized by brown staining. a Nuclear expression of VGLL4 in the normal tissues. b Nuclear expression of VGLL4 in
peritumoral tissues. c Staining of VGLL4 in gastric cancer tissues. d– f Corresponding negative controls of cancer tissues. Original magnification, 9400
with 10 ll propidium iodide which were incubated in the dark for 5 min. Finally, the cell apoptosis was detected by flow cytometry.
fed at a constant temperature and humidity. All the animal protocols were conducted in accordance with Institutional Animal Ethics Care and Use Committee of First Affiliated Hospital of Chongqing Medical University. The AZ-521 and BGC-823 cells transduced with the VGLL4-shRNA and VGLL4 were inoculated subcutaneously with a concentration of 4 9 106 into the right flank of each nude mouse, and the tumor size was measured per week. Eight weeks later, the mice were euthanatized, and tumor samples were taken.
Western blot Total protein and nucleoprotein were extracted via a previous procedure described in [31]. Equal amounts of protein were resolved on SDS–PAGE and transferred to PVDF membranes. We blocked the membranes with 5 % nonfat dry milk in PBST for 1 h, and then, the membranes were incubated with primary antibody overnight at 4 °C against VGLL4 (1:200 dilution; Sigma, USA), E-cadherin (1:1000 dilution; CST, USA), N-cadherin (1:1000 dilution; Epitomics, USA), Vimentin (1:1000 dilution; CST, USA), b-catenin (1:5000 dilution, Epitomics, USA), and b-actin (1:2000, Santa Cruze, USA). The next day, the membranes were washed by PBST for 30 min and treated with corresponding secondary antibodies for 1 h. Then, the membranes were detected by ECL after washing again with PBST for 30 min. In vivo experiments Four-week-old male BALB/c nude mice were purchased from the National Biological Industry Base, Laboratory Animal Center of Chongqing Medical University. These mice were
Statistical analysis All the experimental data are presented as mean ± SEM, and single comparisons between two groups were evaluated by Student’s t test using SPSS 13.0. Survival curves were obtained by the Kaplan–Meier method, and differences were assessed using the log-rank test.
Results The relationship between expression of VGLL4 and clinicopathologic features in gastric cancer To determine the expression level of VGLL4 in gastric cancer, we performed immunohistochemistry in tissue
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Fig. 2 Expression of VGLL4 and its clinical significance in gastric cancer cases. a Representative Western blot analysis of VGLL4 expression in normal, peritumoral, and gastric cancer tissues, respectively. Quantification of the data revealed that VGLL4 protein expression in gastric cancer tissues was significantly lower than that in the normal tissues and normal tumor-adjacent tissues (**P \ 0.01). b Comparison of VGLL4 expression levels in various gastric epithelial cell lines. Quantification of the data revealed that VGLL4
protein expression levels were significantly lower in AZ-521, SGC7901, MGC-803, and BGC-823 cell lines compared to GES-1 cells (**P \ 0.01). c Kaplan–Meier overall 5-year and disease-free survival curves for gastric cancer patients according to their VGLL4 protein expression status. Statistically significant differences in overall survival rate (**P \ 0.01) and disease-free survival rate (*P \ 0.05)
specimens from 150 gastric cancer patients. The results showed that VGLL4 was mainly located in the nuclear and a few was located in the cytoplasm (Fig. 1). Furthermore, the positive rate of VGLL4 in normal tissues and peritumoral tissues was, respectively, 75.3 % (113/150) and 68.7 % (103/150), whereas only 38.7 % (58/150) of the gastric cancer tissues performed a positive expression of VGLL4. The results showed that the expression of VGLL4 in gastric cancer tissues was much lower than that in normal tissues and peritumoral tissues (Fig. 1). There was a significant difference between the VGLL4 expression in gastric cancer tissues and that in normal tissues and peritumoral tissues (P \ 0.01). Additionally, we tested the protein expression of VGLL4 in 20 pair tissue samples via Western blot. As we expected, the protein expression level of VGLL4 in gastric cancer tissues was significantly lower than that in normal tissues and peritumoral tissues (P \ 0.01; Fig. 2a). The association between VGLL4 expression and the various patient clinicopathologic features
is shown in Table 1. We observed that the reduced VGLL4 expression was significantly correlated with tumor size, TNM stage, serosal invasion, vascular invasion, and lymph node metastasis (P \ 0.01). Meanwhile, there were no remarkable differences between reduced expression of VGLL4 and other clinicopathologic factors, including patient age, gender, and histological types (P [ 0.05) (Table 1). Thus, we concluded that VGLL4 may play a role as a tumor suppressor in gastric cancer.
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Positive VGLL4 expression predicts a better 5-year survival for gastric cancer We performed a Kaplan–Meier survival analysis using the overall 5-year survival data on 150 gastric cancer patients to determine whether VGLL4 could play as a predictor on prognosis of gastric cancer. As shown in the Fig. 2c, the overall survival rate in VGLL4-negative expression group (26.08 %) was much lower than that in VGLL4-positive
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0.2381
used Western blot to test the protein expression of VGLL4 in five cell lines, including GES-1, AZ-521, SGC-7901, BGC-823, and MGC-803. As we expected, the protein expression of VGLL4 in four gastric cancer cell lines was significantly decreased (P \ 0.01; Fig. 2b). Therefore, we confirmed that VGLL4 may play an important role in the development and progression of gastric cancer.
0.6709
VGLL4 induces apoptosis and inhibits proliferation in gastric cancer
Table 1 Clinical correlation of VGLL4 expression in gastric cancer Clinicopathologic features
Total no. of patients (n = 150)
No. of patients VGLL4 positive
P
VGLL4 negative
Age (years) \60
58
19
39
C60 Sex
92
39
53
Male
86
32
54
Female
64
26
38
Size (cm) B3 cm
89
26
63
[3 cm
61
32
29
Poorly
77
33
44
Moderately
41
15
26
Well
32
10
22
0.0041*
Histologic type 0.4998
Stage I ? II III ? IV
48
26
22
102
32
70
0.0024*
69 81
35 23
34 58
0.0051*
0.0021*
Serosal invasion Absent Present
Lymph node metastasis Yes
88
25
63
No
62
33
29
Absent
76
38
38
Present
74
20
54
Absent
101
36
65
Present
49
22
27
83
Vascular invasion 0.0039*
Liver metastasis 0.2750
* P \ 0.01
expression group (43.10 %). There was a poorer prognosis in VGLL4-negative expression group (n = 92) than in VGLL4-positive expression group (n = 58) (P \ 0.01; Fig. 2c). The median disease-free survival times were 36.0 and 27.0 months, respectively, in the VGLL4-positive and VGLL4-negative groups of gastric cancer patients. These results also revealed that the down-regulation of VGLL4 was associated with a short disease-free survival time (P \ 0.05; Fig. 2c). These data indicated that down-regulation of VGLL4 predicts an unfavorable prognosis in gastric cancer. VGLL4 protein expression level in gastric cancer cell lines To further investigate whether there is a similar trend in VGLL4 expression levels in gastric cancer cell lines, we
It had been reported previously that VGLL4 could play a crucial role in the process regulating apoptosis and proliferation in various cancers. Thus, we supposed that whether VGLL4 could play a significant role as a tumor suppressor to regulate cell apoptosis and proliferation in gastric cancer. We performed Western blot to test the protein expression of VGLL4 in AZ-521 and BGC-823 cells transduced with VGLL4-shRNA or VGLL4 expression vector or corresponding negative control vector, respectively. We found that the expression level of VGLL4 could be raised in BGC-823 cells transfected with VGLL4 expression (LV-VGLL4-Puromycin) or reduced via using VGLL4 shRNA (LV-VGLL4-shRNAPuromycin) in AZ-521 cells (P \ 0.05 or P \ 0.01 vs. controls; Fig. 6a, b). Detecting the cell apoptosis by flow cytometry, we found that VGLL4 overexpression promoted apoptosis in BGC-823 cells, whereas VGLL4 knockdown prevented apoptosis in AZ-521 cells (P \ 0.01 vs. controls, respectively, Fig. 3b). Next, we tested the cell proliferation by CCK8 assays. As we expected, VGLL4 overexpression prevented cell proliferation in BGC-823 cells and VGLL4 knockdown increased cell proliferation in AZ-521 cells (P \ 0.05 or P \ 0.01 vs. controls, Fig. 3a). Thus, we confirmed that VGLL4 plays a significant role in regulating apoptosis and proliferation in gastric cancer cell lines. Effects of VGLL4 on tumor growth in vivo We established cancer xenograft model in nude mice by inoculating AZ-521 and BGC-823 cells (3 9 106) transfected with VGLL4-shRNA or VGLL4 expression vector or corresponding negative control vector, respectively, to determine whether there was a similar function on tumor growth in vivo. After 8 weeks, we found that tumor xenograft in VGLL4 overexpression groups was smaller than that in control cells, whereas VGLL4 knockdown groups were bigger than that in control cells (Fig. 4a). Additionally, the average volume had a similar trend in upregulated groups of VGLL4 and down-regulated groups of VGLL4 (P \ 0.05 or P \ 0.01 vs. controls; Fig. 4b). These data were consistent with our previous studies
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Fig. 3 Effects of VGLL4 knockdown and overexpression on cell proliferation and apoptosis in vitro. a In AZ-521 cells in which VGLL4 was silenced, the cell proliferation rate was higher than that in the blank control (BC) and negative control (NC) cells. However, the cell proliferation rate was decreased in the BGC-823-V4 cells compared to the BC and NC cells based on the CCK8 assays (*P \ 0.05 vs. controls;
Fig. 4 Effects of VGLL4 on tumor growth in nude mice. a Representative xenograft tumors are shown. b The tumor volume was monitored once per week (*P \ 0.05 vs. controls; **P \ 0.05 vs. controls)
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**P \ 0.01 vs. controls). b In the AZ-521 and BGC-823 cells in which VGLL4 was silenced and overexpression respectively, the apoptosis rate was decreased and increased respectively compared with the blank control (BC) and negative control (NC) based on FACS analysis (**P \ 0.01 vs. controls)
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Fig. 5 Effects of VGLL4 knockdown and overexpression on cell invasion and migration in vitro. a In AZ-521 cells, following VGLL4 silencing, the migratory and invasion capacities were increased compared to the blank control (BC) and negative control (NC) cells
(**P \ 0.01 vs. controls). b The migratory and invasion capacities were decreased in the BGC-823-V4 cells compared to BC and NC cells (**P \ 0.01 vs. controls)
in vitro. Therefore, we concluded that VGLL4 has a close relationship with gastric cancer progression.
important role in regulating migration and invasion in gastric cancer cells.
VGLL4 regulates migration and invasion in gastric cancer
VGLL4 suppresses EMT in part through downregulation of Wnt/b-catenin signal pathway in gastric cancer
Migration and invasion played a key role in initial procedure of tumor metastasis in gastric cancer [29, 30]. Little is known about the relationship between VGLL4 and migration and invasion. Thus, we performed the transwell migration assay and Boyden chamber assay to assess whether VGLL4 participated in the process of regulating migration and invasion in gastric cancer cell lines. As shown in the figure, stable VGLL4 overexpression groups in BGC-823 cells significantly inhibited the migration and invasion compared to control groups (P \ 0.01 vs. controls; Fig. 5a), whereas stable VGLL4 knockdown groups in AZ-521 cells were provided with a higher migration and invasion compared to negative control groups (P \ 0.01 vs. controls; Fig. 5b). In conclusion, VGLL4 plays an
To confirm whether VGLL4 could prevent the EMT to suppress migration and invasion in gastric cancer, we used Western blot to test the protein expression levels of VGLL4, E-cadherin, N-cadherin, and vimentin in AZ-521 and BGC-823 cells that were transduced with VGLL4shRNA or VGLL4 expression vector or corresponding negative control vector, respectively. The results showed that VGLL4 knockdown in AZ-521 cells led to downregulation of E-cadherin and up-regulation of N-cadherin and vimentin (P \ 0.05 or P \ 0.01 vs. controls; Fig. 6a), whereas VGLL4 overexpression in BGC-823 cells resulted in increased expression of E-cadherin and decreased expression of N-cadherin and vimentin (P \ 0.05 or
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Fig. 6 VGLL4 plays a crucial role in the EMT in gastric cancer, and EMT could be stimulated by reduced VGLL4 in part via Wnt/bcatenin signal pathway in gastric cancer. To determine whether VGLL4 suppresses the EMT to inhibit cell migration and invasion, the expression levels of VGLL4, E-cadherin, N-cadherin, and vimentin were detected in all transduced and untransduced cells via Western blot. The results revealed that VGLL4 could suppress the EMT, which increased E-cadherin expression and inhibited
N-cadherin and vimentin expression (*P \ 0.05 vs. controls; **P \ 0.01 vs. controls). Next, we performed Western blot to test the expression levels of N-b-catenin and T-b-catenin. The results showed that the expression levels of N-b-catenin and T-b-catenin were enhanced following knockdown of VGLL4 and were suppressed following VGLL4 overexpression (*P \ 0.05 vs. controls; **P \ 0.01 vs. controls)
P \ 0.01 vs. controls; Fig. 6b). Next, we further investigated the expression levels of N-b-catenin and T-b-catenin. The results showed that VGLL4 knockdown in AZ-521 cells increased the expression levels of N-b-catenin and T-b-catenin compared to control cells (P \ 0.05 or P \ 0.01 vs. controls; Fig. 6a). Meanwhile, the expression levels of N-b-catenin and T-b-catenin in BGC-823-V4 cells were much lower than that in control cells (P \ 0.01 vs. controls; Fig. 6b). These data indicated that b-catenin is a downstream factor of VGLL4, and EMT could be stimulated by reduced VGLL4 in part via Wnt/b-catenin signal pathway in gastric cancer.
potential contribution of VGLL4 to inhibiting the EMT in gastric cancer remained to be elucidated. In this study, we performed a study using a variety of methods, including the clinical samples, cell cultures, and animal model to evaluate the function and the mechanisms of VGLL4 which suppress the EMT in gastric cancer. We firstly tested the expression of VGLL4 in 150 gastric cancer specimens and corresponding normal tumor-adjacent tissues via immunohistochemistry and Western blot. We found that the expression of VGLL4 in tumor tissues was significantly lower than that in normal tissues and peritumoral tissues, and VGLL4 expression was markedly associated with tumor size, TNM stage, serosal invasion, vascular invasion, and lymph node metastasis. In addition, our data demonstrated that down-regulation of VGLL4 results in a worse 5-year survival for gastric cancer patients. These results were all consistent with previous research [28]. Thus, we confirmed that reduced VGLL4 is closely related with clinicopathological features and predicts an unfavorable prognosis in gastric cancer.
Discussion VGLL4 is a member of the VGLL proteins. Recent studies have been reported that VGLL4 could function as a tumor suppressor, which directly competes with YAP for binding TEADs in several cancer types [28, 32]. However, the
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We used flow cytometry and CCK8 assay to test the role of VGLL4 in the apoptosis and proliferation of gastric cancer cells. Our data suggested that VGLL4 overexpression could increase apoptosis and reduce the proliferation, whereas knockdown of VGLL4 has an opposite trend. Furthermore, we established cancer xenograft model in nude mice to explain whether VGLL4 plays a similar role in vivo. The results showed that the tumor xenograft in VGLL4 overexpression groups was smaller than that in control groups, whereas VGLL4 knockdown groups have a bigger tumor xenograft than control groups. Meanwhile, the average tumor volume had a similar tendency. To determine whether VGLL4 regulate the migration and invasion of gastric cancer cells, we performed transwell migration and invasion assay. The results showed that reduced VGLL4 expression could increase migration and invasion in gastric cancer cells, and VGLL4 overexpression could suppress migration and invasion in gastric cancer cells. Thereafter, we used Western blot to test the expression of E-cadherin, N-cadherin, and vimentin. The results indicated that VGLL4 overexpression increased the expression level of E-cadherin and suppressed the expression level of N-cadherin and vimentin, whereas the reverse results were obtained following knockdown of VGLL4. Thus, we confirmed that VGLL4 plays an important role in inhibiting migration and invasion by suppressing EMT in gastric cancer. To further investigate whether VGLL4 regulates EMT via Wnt/b-catenin signal pathway, we performed Western blot to test the expression levels of N-b-catenin and T-bcatenin. The results showed that VGLL4 overexpression suppresses the expression levels of N-b-catenin and T-bcatenin, whereas the expression of N-b-catenin and T-bcatenin was increased following knockdown of VGLL4. Therefore, we confirmed that VGLL4 could induce a change in nuclear location of b-catenin, which may be a downstream factor of VGLL4. We finally concluded that EMT could be suppressed by VGLL4 in part via Wnt/bcatenin signal pathway. In conclusion, we found that overexpression of VGLL4 can suppress oncogenic properties and inhibit migration and invasion of gastric cancer cell lines both in vitro and in vivo. Even more important, we also found that VGLL4 down-regulate Wnt/b-catenin signal pathway to suppressing EMT. We hope that this study will provide the basis for recognizing VGLL4 as a potential therapeutic target in gastric cancer. Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (No. 81272753). Conflict of interest of interest.
The authors declare that they have no conflict
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counteracts apoptosis-inhibitory function of IAPs by nuclear sequestration. Biochem Biophys Res Commun. 2012;412(3):454–9. Jiao S, Wang H, Shi Z, et al. A peptide mimicking VGLL4 function acts as a YAP antagonist therapy against gastric cancer. Cancer Cell. 2014;25(2):166–80. Xu MZ, Yao TJ, Lee NP, et al. Yes-associated protein is an independent prognostic marker in Hepatocellular carcinoma. Cancer. 2009;115:4576–85. Xu W, Wang Z, Zhang W et al. Mutated K-ras activates CDK8 to stimulate the epithelial-to-mesenchymal transition in pancreatic cancer in part via the Wnt/b-catenin signaling pathway. Cancer Lett. 2015;356(2 Pt B):613–27. Zha L, Zhang J, Tang W, et al. HMGA2 elicits EMT by activating the Wnt/b-catenin pathway in gastric cancer. Dig Dis Sci. 2013;58:724–33. Zhang W, Gao Y, Li P, et al. VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAPTEAD transcriptional complex. Cell Res. 2014;24(3):331–43.
ORIGINAL PAPER
VGLL4 inhibits EMT in part through suppressing Wnt/b-catenin signaling pathway in gastric cancer Hui Li • Ziwei Wang • Wei Zhang • Kun Qian • Gang Liao • Wei Xu • Shouru Zhang
Received: 7 January 2015 / Accepted: 13 February 2015 Ó Springer Science+Business Media New York 2015
Abstract VGLL4 is a member of the Vestigial-like proteins that functions as a tumor suppressor, which directly competes with YAP for binding TEADs in several cancer types. Recently, an increasing number of studies have reported that VGLL4 acts as a crucial role in regulating cell mobility, migration, and invasion. However, little is known about the signaling mechanisms in regulating epithelial– mesenchymal transition (EMT) of gastric cancer. In our study, we confirmed that the expression level of VGLL4 was down-regulated in gastric cancer tissues, and reduced VGLL4 expression levels inhibited apoptosis and promoted proliferation, migration, and invasion. Additionally, we found a phenomenon that VGLL4 was associated with the change in nuclear location of b-catenin, which suggested that b-catenin was a significant downstream factor of VGLL4. These results suggest that VGLL4 suppressed EMT in part via negative regulation of Wnt/b-catenin signaling pathway. Taken together, our study demonstrated that VGLL4 is important in the process of suppressing tumor progression of gastric cancer and provided a potential therapeutic strategy for gastric cancer. Keywords VGLL4 Gastric cancer Wnt/b-catenin Tumor progression EMT
H. Li Z. Wang (&) W. Zhang K. Qian G. Liao W. Xu S. Zhang Department of Gastrointestinal Surgery, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China e-mail: [email protected]
Introduction Gastric cancer is the fourth most frequent malignant neoplasm and the second leading cause of cancer death worldwide [1]. Invasiveness and metastasis are considered as a main reason for gastric cancer death. In the last few years, an increasing number of studies reported that epithelial–mesenchymal transition (EMT) plays an important role in cancer progression and metastasis [2–4]. EMT is a process that is characterized by the loss of epithelial characteristics and acquisition of a mesenchymal phenotype to produce enhanced motility, resistance to apoptosis, and the capacity to invade the surrounding tissues [5–8]. Many reports about EMT had been observed in various gastric cell lines such as MGC-803, MKN-45, SGC-7901, BGC-823 [9, 10]. It is well known that the occurrence of EMT is associated with several distinct pathways, including Notch, TGF-b, and several receptor tyrosine kinase [11–13]. Furthermore, dysregulation of Wnt/b-catenin signaling has been proved to play a significant role in the progression of both the EMT and cancer metastasis [14– 17]. In addition, accumulation of nuclear b-catenin is a significant marker of enhanced Wnt signaling [18]. VGLL4 is a member of Vestigial-like (VGLL) proteins that has four subtypes in mammals, and these VGLL proteins contain the TEAD-interacting domain (TDU) [19– 22]. Some studies have found that VGLL1 and VGLL3 were up-regulated in basal-like breast cancer samples and soft tissue sarcoma, respectively [23–25]. However, VGLL4 may have different functions because it contains two TDU domains, unlike other members in VGLL family. As the literatures reported, VGLL4 is a tumor suppressor in pancreatic adenocarcinoma and promotes cell apoptosis through down-regulating the inhibitor of apoptosis proteins [26, 27]. In addition, a recent study showed that VGLL4
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directly competed with YAP for binding TEADs to suppress tumor growth [28]. However, the mechanisms involved in the anti-EMT effects of VGLL4 in gastric cancer are unknown. In this study, we used in vivo and in vitro approaches to demonstrate that whether VGLL4 could inhibit EMT and whether the regulatory effects of VGLL4 on the EMT are partially attributed to the Wnt/b-catenin signaling pathway in gastric cancer.
Materials and methods Cell culture and transfection The human immortalized gastric epithelial cell line GES-1 and human gastric cancer cell lines AZ-521, SGC-7901, BGC-823, and MGC-803 were obtained from Shanghai Center for Life Science Research Institute of Chinese Academy of Sciences cell resources. AZ-521 and BGC-823 cells were both maintained in RPMI 1640 medium (RPMI 1640, Gibco, USA). These media contained 10 % FBS (Gibco, USA) and 2 % penicillin/streptomycin (Hyclone, Shanghai, China). All the cells were cultured in an incubator containing humidified 95 % air and 5 % CO2 at 37 °C. Lentivirus constructs expression VGLL4-shRNA (LVVGLL4-shRNA-Puromycin), VGLL4 (LV-VGLL4-Puromycin), and the nontargeted negative control virus were purchased from Shanghai Genechen Co., Ltd. (China). The human VGLL4-shRNA sequence was 50 -TCACCTGTG CCTCGG CTG GCG-30 , 50 -TGAACAAGACTGCCAA TGGAG-30 . The AZ-521 and BGC-823 cells were digested by 0.125 % trypsase and then seeded on 6-well plate, respectively, at a concentration of 1.5 9 105 and 1.0 9 105. The cells were transfected in the following day at a MOI of 60(AZ-521) and 80(BGC-823) using polybrene (10 lg/ml) and enhanced infection solution (Genechen, China) for 12 h, and the lentiviral empty vector was used with the control cells by a same method. After incubation for 48 h, the cells were selected with corresponding concentration of puromycin. The cells would be collected for mRNA and protein analysis and other assays in the reasonable time. Tissue samples and immunohistochemistry One hundred and fifty gastric cancer samples, paired peritumoral samples ([2 cm distance from the margin of the resection), and 20 normal samples were obtained from the First Affiliated Hospital of Chongqing Medical University, China, between 2011 and 2013. None of the patients underwent any other treatment before operation. Tumor tissues, paired peritumoral tissues, and normal tissues were immediately stored in paraformaldehyde after
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collected. Tissue sections were deparaffinized, and antigens were retrieved by heating in a buffer (0.01 M sodium citrate) in a microwave oven at 95 °C for 15 min. The slides were incubated in 3 % hydrogen peroxide for 15 min and subsequently treated with 0.5 % Triton X-100 (Beyontime, Zhenjiang, Jiangsu, China) for 15 min. Then, we blocked the slides with 5 % BSA for 30 min and treated the slides with anti-VGLL4 primary antibody (1:200 dilution, Sigma, USA) overnight at 4 °C. The next day, the slides were incubated with proper secondary antibodies for 30 min after washing in PBS for 30 min. Then, the slides were exposed to streptavidin-HRP label (Zhongshanjinqiao, Beijing, China) for 30 min. The percentage of positive cells was graded according to the following criteria: 0, \10 %; 1, 10–30 %; 2, 30–50 %; 3, [50 % [29]. This study was supported by the approval and supervision of the Medical Ethics Committee of First Affiliated Hospital of Chongqing Medical University. Meanwhile, we asked for consents from patients and healthy subjects about collecting the tissues specimens. Transwell migration and invasion assay Transwell migration assay was performed as previously described [30]. 1 9 105 cells of AZ-521 and BGC-823 were, respectively, added to the top chamber of transwell (Millicell, Millipore, USA), and the bottom chamber of transwell was placed with corresponding medium supplemented with 10 % FBS. The cells were fixed with 4 % paraformaldehyde for 30 min after 24 h of culture with 37 °C in 5 % CO2. Thereafter, the cells were stained in 0.1 % crystal violet for 10 min and counted under a light microscope at 1009 magnification. Transwell invasion assay was similarly performed through a modified Boyden chamber with polyester membrane inserts that were coated with Matrigel (BD Bioscience, USA). Cell proliferation and apoptosis detection AZ-521 and BGC-823 were seeded with a concentration of 1 9 103 resuspended cells on 96-well plates in triplicate in 100 ll of RPMI 1640 medium. Then, we performed the Cell Counting Kit-8 (CCK8) assay (Dojindo, Japan) to test the cell proliferation. The cells were treated with 10 ll of CCK8 in each well and were incubated for 1 h at 37 °C in 5 % CO2 every day. Finally, we measured the absorbance of the cells by a spectrophotometer at 450 nm. Flow cytometry was used to investigate apoptosis. All the cells were cultured in 6-well plates. Then, each cell of three groups (521-VGLL4-shRNA, 521-NC, 521-BC, 823-VGLL4, 823-NC, 823-BC) was resuspended in a concentration of 1 9 106. Thereafter, we added 5 ll Annexin V-FITC into each well, and the cells were incubated in the dark for 15 min. We subsequently treated the cells
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Fig. 1 Immunohistochemical (IHC) staining of VGLL4 (a–c) in normal, peritumoral, and gastric cancer tissues, respectively. Positive expression is visualized by brown staining. a Nuclear expression of VGLL4 in the normal tissues. b Nuclear expression of VGLL4 in
peritumoral tissues. c Staining of VGLL4 in gastric cancer tissues. d– f Corresponding negative controls of cancer tissues. Original magnification, 9400
with 10 ll propidium iodide which were incubated in the dark for 5 min. Finally, the cell apoptosis was detected by flow cytometry.
fed at a constant temperature and humidity. All the animal protocols were conducted in accordance with Institutional Animal Ethics Care and Use Committee of First Affiliated Hospital of Chongqing Medical University. The AZ-521 and BGC-823 cells transduced with the VGLL4-shRNA and VGLL4 were inoculated subcutaneously with a concentration of 4 9 106 into the right flank of each nude mouse, and the tumor size was measured per week. Eight weeks later, the mice were euthanatized, and tumor samples were taken.
Western blot Total protein and nucleoprotein were extracted via a previous procedure described in [31]. Equal amounts of protein were resolved on SDS–PAGE and transferred to PVDF membranes. We blocked the membranes with 5 % nonfat dry milk in PBST for 1 h, and then, the membranes were incubated with primary antibody overnight at 4 °C against VGLL4 (1:200 dilution; Sigma, USA), E-cadherin (1:1000 dilution; CST, USA), N-cadherin (1:1000 dilution; Epitomics, USA), Vimentin (1:1000 dilution; CST, USA), b-catenin (1:5000 dilution, Epitomics, USA), and b-actin (1:2000, Santa Cruze, USA). The next day, the membranes were washed by PBST for 30 min and treated with corresponding secondary antibodies for 1 h. Then, the membranes were detected by ECL after washing again with PBST for 30 min. In vivo experiments Four-week-old male BALB/c nude mice were purchased from the National Biological Industry Base, Laboratory Animal Center of Chongqing Medical University. These mice were
Statistical analysis All the experimental data are presented as mean ± SEM, and single comparisons between two groups were evaluated by Student’s t test using SPSS 13.0. Survival curves were obtained by the Kaplan–Meier method, and differences were assessed using the log-rank test.
Results The relationship between expression of VGLL4 and clinicopathologic features in gastric cancer To determine the expression level of VGLL4 in gastric cancer, we performed immunohistochemistry in tissue
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Fig. 2 Expression of VGLL4 and its clinical significance in gastric cancer cases. a Representative Western blot analysis of VGLL4 expression in normal, peritumoral, and gastric cancer tissues, respectively. Quantification of the data revealed that VGLL4 protein expression in gastric cancer tissues was significantly lower than that in the normal tissues and normal tumor-adjacent tissues (**P \ 0.01). b Comparison of VGLL4 expression levels in various gastric epithelial cell lines. Quantification of the data revealed that VGLL4
protein expression levels were significantly lower in AZ-521, SGC7901, MGC-803, and BGC-823 cell lines compared to GES-1 cells (**P \ 0.01). c Kaplan–Meier overall 5-year and disease-free survival curves for gastric cancer patients according to their VGLL4 protein expression status. Statistically significant differences in overall survival rate (**P \ 0.01) and disease-free survival rate (*P \ 0.05)
specimens from 150 gastric cancer patients. The results showed that VGLL4 was mainly located in the nuclear and a few was located in the cytoplasm (Fig. 1). Furthermore, the positive rate of VGLL4 in normal tissues and peritumoral tissues was, respectively, 75.3 % (113/150) and 68.7 % (103/150), whereas only 38.7 % (58/150) of the gastric cancer tissues performed a positive expression of VGLL4. The results showed that the expression of VGLL4 in gastric cancer tissues was much lower than that in normal tissues and peritumoral tissues (Fig. 1). There was a significant difference between the VGLL4 expression in gastric cancer tissues and that in normal tissues and peritumoral tissues (P \ 0.01). Additionally, we tested the protein expression of VGLL4 in 20 pair tissue samples via Western blot. As we expected, the protein expression level of VGLL4 in gastric cancer tissues was significantly lower than that in normal tissues and peritumoral tissues (P \ 0.01; Fig. 2a). The association between VGLL4 expression and the various patient clinicopathologic features
is shown in Table 1. We observed that the reduced VGLL4 expression was significantly correlated with tumor size, TNM stage, serosal invasion, vascular invasion, and lymph node metastasis (P \ 0.01). Meanwhile, there were no remarkable differences between reduced expression of VGLL4 and other clinicopathologic factors, including patient age, gender, and histological types (P [ 0.05) (Table 1). Thus, we concluded that VGLL4 may play a role as a tumor suppressor in gastric cancer.
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Positive VGLL4 expression predicts a better 5-year survival for gastric cancer We performed a Kaplan–Meier survival analysis using the overall 5-year survival data on 150 gastric cancer patients to determine whether VGLL4 could play as a predictor on prognosis of gastric cancer. As shown in the Fig. 2c, the overall survival rate in VGLL4-negative expression group (26.08 %) was much lower than that in VGLL4-positive
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0.2381
used Western blot to test the protein expression of VGLL4 in five cell lines, including GES-1, AZ-521, SGC-7901, BGC-823, and MGC-803. As we expected, the protein expression of VGLL4 in four gastric cancer cell lines was significantly decreased (P \ 0.01; Fig. 2b). Therefore, we confirmed that VGLL4 may play an important role in the development and progression of gastric cancer.
0.6709
VGLL4 induces apoptosis and inhibits proliferation in gastric cancer
Table 1 Clinical correlation of VGLL4 expression in gastric cancer Clinicopathologic features
Total no. of patients (n = 150)
No. of patients VGLL4 positive
P
VGLL4 negative
Age (years) \60
58
19
39
C60 Sex
92
39
53
Male
86
32
54
Female
64
26
38
Size (cm) B3 cm
89
26
63
[3 cm
61
32
29
Poorly
77
33
44
Moderately
41
15
26
Well
32
10
22
0.0041*
Histologic type 0.4998
Stage I ? II III ? IV
48
26
22
102
32
70
0.0024*
69 81
35 23
34 58
0.0051*
0.0021*
Serosal invasion Absent Present
Lymph node metastasis Yes
88
25
63
No
62
33
29
Absent
76
38
38
Present
74
20
54
Absent
101
36
65
Present
49
22
27
83
Vascular invasion 0.0039*
Liver metastasis 0.2750
* P \ 0.01
expression group (43.10 %). There was a poorer prognosis in VGLL4-negative expression group (n = 92) than in VGLL4-positive expression group (n = 58) (P \ 0.01; Fig. 2c). The median disease-free survival times were 36.0 and 27.0 months, respectively, in the VGLL4-positive and VGLL4-negative groups of gastric cancer patients. These results also revealed that the down-regulation of VGLL4 was associated with a short disease-free survival time (P \ 0.05; Fig. 2c). These data indicated that down-regulation of VGLL4 predicts an unfavorable prognosis in gastric cancer. VGLL4 protein expression level in gastric cancer cell lines To further investigate whether there is a similar trend in VGLL4 expression levels in gastric cancer cell lines, we
It had been reported previously that VGLL4 could play a crucial role in the process regulating apoptosis and proliferation in various cancers. Thus, we supposed that whether VGLL4 could play a significant role as a tumor suppressor to regulate cell apoptosis and proliferation in gastric cancer. We performed Western blot to test the protein expression of VGLL4 in AZ-521 and BGC-823 cells transduced with VGLL4-shRNA or VGLL4 expression vector or corresponding negative control vector, respectively. We found that the expression level of VGLL4 could be raised in BGC-823 cells transfected with VGLL4 expression (LV-VGLL4-Puromycin) or reduced via using VGLL4 shRNA (LV-VGLL4-shRNAPuromycin) in AZ-521 cells (P \ 0.05 or P \ 0.01 vs. controls; Fig. 6a, b). Detecting the cell apoptosis by flow cytometry, we found that VGLL4 overexpression promoted apoptosis in BGC-823 cells, whereas VGLL4 knockdown prevented apoptosis in AZ-521 cells (P \ 0.01 vs. controls, respectively, Fig. 3b). Next, we tested the cell proliferation by CCK8 assays. As we expected, VGLL4 overexpression prevented cell proliferation in BGC-823 cells and VGLL4 knockdown increased cell proliferation in AZ-521 cells (P \ 0.05 or P \ 0.01 vs. controls, Fig. 3a). Thus, we confirmed that VGLL4 plays a significant role in regulating apoptosis and proliferation in gastric cancer cell lines. Effects of VGLL4 on tumor growth in vivo We established cancer xenograft model in nude mice by inoculating AZ-521 and BGC-823 cells (3 9 106) transfected with VGLL4-shRNA or VGLL4 expression vector or corresponding negative control vector, respectively, to determine whether there was a similar function on tumor growth in vivo. After 8 weeks, we found that tumor xenograft in VGLL4 overexpression groups was smaller than that in control cells, whereas VGLL4 knockdown groups were bigger than that in control cells (Fig. 4a). Additionally, the average volume had a similar trend in upregulated groups of VGLL4 and down-regulated groups of VGLL4 (P \ 0.05 or P \ 0.01 vs. controls; Fig. 4b). These data were consistent with our previous studies
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Fig. 3 Effects of VGLL4 knockdown and overexpression on cell proliferation and apoptosis in vitro. a In AZ-521 cells in which VGLL4 was silenced, the cell proliferation rate was higher than that in the blank control (BC) and negative control (NC) cells. However, the cell proliferation rate was decreased in the BGC-823-V4 cells compared to the BC and NC cells based on the CCK8 assays (*P \ 0.05 vs. controls;
Fig. 4 Effects of VGLL4 on tumor growth in nude mice. a Representative xenograft tumors are shown. b The tumor volume was monitored once per week (*P \ 0.05 vs. controls; **P \ 0.05 vs. controls)
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**P \ 0.01 vs. controls). b In the AZ-521 and BGC-823 cells in which VGLL4 was silenced and overexpression respectively, the apoptosis rate was decreased and increased respectively compared with the blank control (BC) and negative control (NC) based on FACS analysis (**P \ 0.01 vs. controls)
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Fig. 5 Effects of VGLL4 knockdown and overexpression on cell invasion and migration in vitro. a In AZ-521 cells, following VGLL4 silencing, the migratory and invasion capacities were increased compared to the blank control (BC) and negative control (NC) cells
(**P \ 0.01 vs. controls). b The migratory and invasion capacities were decreased in the BGC-823-V4 cells compared to BC and NC cells (**P \ 0.01 vs. controls)
in vitro. Therefore, we concluded that VGLL4 has a close relationship with gastric cancer progression.
important role in regulating migration and invasion in gastric cancer cells.
VGLL4 regulates migration and invasion in gastric cancer
VGLL4 suppresses EMT in part through downregulation of Wnt/b-catenin signal pathway in gastric cancer
Migration and invasion played a key role in initial procedure of tumor metastasis in gastric cancer [29, 30]. Little is known about the relationship between VGLL4 and migration and invasion. Thus, we performed the transwell migration assay and Boyden chamber assay to assess whether VGLL4 participated in the process of regulating migration and invasion in gastric cancer cell lines. As shown in the figure, stable VGLL4 overexpression groups in BGC-823 cells significantly inhibited the migration and invasion compared to control groups (P \ 0.01 vs. controls; Fig. 5a), whereas stable VGLL4 knockdown groups in AZ-521 cells were provided with a higher migration and invasion compared to negative control groups (P \ 0.01 vs. controls; Fig. 5b). In conclusion, VGLL4 plays an
To confirm whether VGLL4 could prevent the EMT to suppress migration and invasion in gastric cancer, we used Western blot to test the protein expression levels of VGLL4, E-cadherin, N-cadherin, and vimentin in AZ-521 and BGC-823 cells that were transduced with VGLL4shRNA or VGLL4 expression vector or corresponding negative control vector, respectively. The results showed that VGLL4 knockdown in AZ-521 cells led to downregulation of E-cadherin and up-regulation of N-cadherin and vimentin (P \ 0.05 or P \ 0.01 vs. controls; Fig. 6a), whereas VGLL4 overexpression in BGC-823 cells resulted in increased expression of E-cadherin and decreased expression of N-cadherin and vimentin (P \ 0.05 or
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Fig. 6 VGLL4 plays a crucial role in the EMT in gastric cancer, and EMT could be stimulated by reduced VGLL4 in part via Wnt/bcatenin signal pathway in gastric cancer. To determine whether VGLL4 suppresses the EMT to inhibit cell migration and invasion, the expression levels of VGLL4, E-cadherin, N-cadherin, and vimentin were detected in all transduced and untransduced cells via Western blot. The results revealed that VGLL4 could suppress the EMT, which increased E-cadherin expression and inhibited
N-cadherin and vimentin expression (*P \ 0.05 vs. controls; **P \ 0.01 vs. controls). Next, we performed Western blot to test the expression levels of N-b-catenin and T-b-catenin. The results showed that the expression levels of N-b-catenin and T-b-catenin were enhanced following knockdown of VGLL4 and were suppressed following VGLL4 overexpression (*P \ 0.05 vs. controls; **P \ 0.01 vs. controls)
P \ 0.01 vs. controls; Fig. 6b). Next, we further investigated the expression levels of N-b-catenin and T-b-catenin. The results showed that VGLL4 knockdown in AZ-521 cells increased the expression levels of N-b-catenin and T-b-catenin compared to control cells (P \ 0.05 or P \ 0.01 vs. controls; Fig. 6a). Meanwhile, the expression levels of N-b-catenin and T-b-catenin in BGC-823-V4 cells were much lower than that in control cells (P \ 0.01 vs. controls; Fig. 6b). These data indicated that b-catenin is a downstream factor of VGLL4, and EMT could be stimulated by reduced VGLL4 in part via Wnt/b-catenin signal pathway in gastric cancer.
potential contribution of VGLL4 to inhibiting the EMT in gastric cancer remained to be elucidated. In this study, we performed a study using a variety of methods, including the clinical samples, cell cultures, and animal model to evaluate the function and the mechanisms of VGLL4 which suppress the EMT in gastric cancer. We firstly tested the expression of VGLL4 in 150 gastric cancer specimens and corresponding normal tumor-adjacent tissues via immunohistochemistry and Western blot. We found that the expression of VGLL4 in tumor tissues was significantly lower than that in normal tissues and peritumoral tissues, and VGLL4 expression was markedly associated with tumor size, TNM stage, serosal invasion, vascular invasion, and lymph node metastasis. In addition, our data demonstrated that down-regulation of VGLL4 results in a worse 5-year survival for gastric cancer patients. These results were all consistent with previous research [28]. Thus, we confirmed that reduced VGLL4 is closely related with clinicopathological features and predicts an unfavorable prognosis in gastric cancer.
Discussion VGLL4 is a member of the VGLL proteins. Recent studies have been reported that VGLL4 could function as a tumor suppressor, which directly competes with YAP for binding TEADs in several cancer types [28, 32]. However, the
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We used flow cytometry and CCK8 assay to test the role of VGLL4 in the apoptosis and proliferation of gastric cancer cells. Our data suggested that VGLL4 overexpression could increase apoptosis and reduce the proliferation, whereas knockdown of VGLL4 has an opposite trend. Furthermore, we established cancer xenograft model in nude mice to explain whether VGLL4 plays a similar role in vivo. The results showed that the tumor xenograft in VGLL4 overexpression groups was smaller than that in control groups, whereas VGLL4 knockdown groups have a bigger tumor xenograft than control groups. Meanwhile, the average tumor volume had a similar tendency. To determine whether VGLL4 regulate the migration and invasion of gastric cancer cells, we performed transwell migration and invasion assay. The results showed that reduced VGLL4 expression could increase migration and invasion in gastric cancer cells, and VGLL4 overexpression could suppress migration and invasion in gastric cancer cells. Thereafter, we used Western blot to test the expression of E-cadherin, N-cadherin, and vimentin. The results indicated that VGLL4 overexpression increased the expression level of E-cadherin and suppressed the expression level of N-cadherin and vimentin, whereas the reverse results were obtained following knockdown of VGLL4. Thus, we confirmed that VGLL4 plays an important role in inhibiting migration and invasion by suppressing EMT in gastric cancer. To further investigate whether VGLL4 regulates EMT via Wnt/b-catenin signal pathway, we performed Western blot to test the expression levels of N-b-catenin and T-bcatenin. The results showed that VGLL4 overexpression suppresses the expression levels of N-b-catenin and T-bcatenin, whereas the expression of N-b-catenin and T-bcatenin was increased following knockdown of VGLL4. Therefore, we confirmed that VGLL4 could induce a change in nuclear location of b-catenin, which may be a downstream factor of VGLL4. We finally concluded that EMT could be suppressed by VGLL4 in part via Wnt/bcatenin signal pathway. In conclusion, we found that overexpression of VGLL4 can suppress oncogenic properties and inhibit migration and invasion of gastric cancer cell lines both in vitro and in vivo. Even more important, we also found that VGLL4 down-regulate Wnt/b-catenin signal pathway to suppressing EMT. We hope that this study will provide the basis for recognizing VGLL4 as a potential therapeutic target in gastric cancer. Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (No. 81272753). Conflict of interest of interest.
The authors declare that they have no conflict
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