Introduction
VEGFR2 (Vascular endothelial growth factor receptor 2) is one of the two tyrosine kinase receptors involved in angiogenesis. When activated by its ligand VEGF, VEGFR2 promotes neighbouring vessel formation to facilitate the delivery of growth factors, nutrients and oxygen for cancer proliferation, migration, metastasis and survival [
1]. VEGF and VEGFR2 mediated angiogenesis contributes to the aggressive natures and leads to high mortality rate in gastric cancer, which is the third leading cause of cancer deaths worldwide [
2]. In patients with advanced gastric cancer, pharmacologic agents that specifically targeting VEGF ligand, or receptors by specific kinase inhibitors or antibodies, exhibited efficacy in clinical trials in combination with chemotherapy or not [
3]. Of note, monoclonal antibody ramucirumab and tyrosine kinase inhibitor apatinib, both targeting VEGFR2, showed more favourable benefit compared to direct block of angiogenesis stimuli by bevacizumab [
4], implying the complicated implication of VEGFR2 involved in tumorigenesis and progression of gastric cancer.
Over the past decades, besides of initiation of angiogenesis of VEGFR2 signaling in different malignancies, the lesser known direct impact of which on cancer cells cannot be overlooked [
5]. In addition to its constitutive expression on endothelial cells, presence of VEGFR2 on cancer cells was also confirmed in breast cancer [
6,
7] [
8,
9], lung cancer [
10], glioblastoma [
11], gastrointestinal cancer [
12], hepatocellular carcinoma, renal cell carcinoma [
13], ovarian cancer [
14], bladder cancer [
15], and osteosarcoma [
16]. Besides of the contribution of VEGFR2 that leads to tumor neovascularity in peri-cancer cellular niche, how or whether this proangiogenic factor receptor and its regulatory axis could regulate survival, malignant progression and invasion of gastric cancer, independent of VEGF-induced angiogenesis, is still not clearly understood. In the present study, we investigated the angiogenesis independent pro-malignancy function of VEGFR2 signaling in gastric cancer cells. These results may provide novel insights of VEGFR2 inhibitors in cancer cell level and new anticancer strategies for management of gastric cancer.
Methods and materials
Patients and samples
The experimental protocol was approved by the Human Ethics Committee of the Xiangcheng People’s Hospital and the First Affiliated Hospital of Soochow University. Written informed consent was collected from individual patient. A total of 156 surgical cancer specimens were obtained from patients with gastric cancer.
Immunohistochemistry
All resection specimens in this study were fixed in 10% buffered formalin and paraffin embedded by routinely processing. Sections were cut at a thickness of 4 μm, heated at 60 °C for 30 min, then deparaffinized and hydrated through a series of xylene and alcohol baths before staining. The slides were microwaved with antigen retrieval solution (citrate buffer, pH 6.0, containing 0.3% trisodium citrate and 0.04% citric acid) for 5 min. After replenishment of this solution, the slides were microwaved again for an additional 5 min and then allowed to cool for 20 min. The sections were then rinsed in PBS (phosphate-buffered saline), and immersed in 3% H2O2 for 15 min to block the endogenous peroxidase. Thereafter, the sections were incubated with 10% BSA (bull serum albumin) at room temperature for 60 min to block nonspecific antibodies. Immunohistochemical staining was performed with rabbit anti-VEGFR2 antibody (Proteintech Group) or mouse anti-VTN (Vitronectin) antibody (Proteintech Group) respectively at room temperature for 2 h. After incubation with the corresponding secondary antibodies for 20 min, the bound complex was visualized by using the SuperPicTure polymer detection kit (No.87–8963; Invitrogen). To evaluate expressions of target genes, four representative areas were selected and were observed at 400× magnifcation respectively. The percentage of positive cells was assessed by counting the number of positive cells divided by all cancer cells under a selected area. The positive grades were determined as follows: 0, no positive tumor cells; 1, 1–10% positive tumor cells; 2, 11–50%; 3, 51–100%. The stating intensities were interpreted by the presence of yellow- or brown-colored cells in the target antigen sites, and were scored as follows: 0, no detectable signal; 1, mild staining –light yellow color; 2, moderate – yellow color; 3, intense – brown color. The final scores were obtained through multiplying the positive grade by the staining intensity score, leading to seven scores including 0, 1, 2, 3, 4, 6, and 9. Final scores ≤4 were regarded as low expression, while ≥6 were defined as high expression.
Survival analysis
For analysis of survival data of 156 surgical cancer specimens, Kaplan–Meier curves were constructed, and statistical analysis was carried out using the log-rank test. Overall survival (OS) was defined as the time from beginning of surgery to death from any cause or the last date of follow-up. DFS (disease-free survival), or recurrence-free survival, is defined as the time from randomization to the first of either recurrence or relapse, second cancer, or death. Kaplan–Meier plotter (KM plotter,
www.kmplot.com) was capable to assess the effect of 54,675 genes on survival using over 10,000 cancer samples. The 870 patients with gastric cancer were split into two groups according to the expression of a particular gene (high vs. low expression). The overall survival was analyzed using a Kaplan–Meier plot. The hazard ratio (HR) with 95% confidence intervals and log rank
P value were calculated and displayed on the webpage.
Cell culture and reagents
Human gastric cancer cell lines MKN-45, MKN-28, NCI-N87 and SCH and immortalized normal human gastric mucosal epithelial cell line GES-1 were obtained from American Type Culture Collection (ATCC). All cells were cultured in RPMI1640 medium (Invitrogen) with 10% fetal bovine serum (Invitrogen) and 37 °C 5% CO2. Apatinib was purchased from Hengrui Medicine Co. Ltd. (Jiangsu, China).
Real-time PCR
Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. After spectrophotometric quantification, 1 μg total RNA in a final volume of 20 μL was used for reverse transcription with PrimeScript RT Reagent kit (Takara, Otsu, Shiga, Japan) according to the manufacturer’s protocol. Aliquots of cDNA corresponding to equal amounts of RNA were used for quantification of mRNA by real-time PCR using the LightCycler 96 Real-time Quantitative PCR Detection system (Roche, Indianapolis, IN, USA). The reaction system (25 μL) contained the corresponding cDNA, forward and reverse primers, and SYBR Green PCR master mix (Roche). All data were analyzed using GAPDH gene expression as an internal standard. The specific primers are presented as follows: ①. VEGFR2 forward:5’-GGACTCTCTCTGCCTACCTCAC-3′, VEGFR2 reverse:5’-GGCTCTTTCGCTTACTGTTCTG-3′; ②. VTN forward:5’-TCACCAAGAGTCATGCAAGGG-3′, VTN reverse:5’-ACTCAGCCGTATAGTCTGTGC-3′; ③. GAPDH forward:5’-AGAAGGCTGGGGCTCATTTG-3′, GAPDH reverse:5’-AGGGGCCATCCACAGTCTTC-3′.
Western blot
Total protein was extracted using a lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and supplemented with protease inhibitor cocktail kit (Roche). The protein extract was loaded onto an SDS-polyacrylamide gel, size-fractionated by electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, CA, USA). After blocking in 5% non-fat milk for 1 h, the membranes were incubated overnight with primary antibodies at 4 °C. The protein expression was determined using horseradish peroxidase-conjugated antibodies followed by enhanced chemiluminescence (ECL, Millipore, St Charles, MO, USA) detection. The intensity of the bands was captured by JS-1035 image analysis scanning system (Peiqing Science & Technology, Shanghai, China). β-actin was used as the internal control.
RNA interference and generation of stably knockdown cell lines
The sequences of small interfering RNA against human VEGFR2 (①. 5′-GCGGCTACCAGTCCGGATA-3′, ②. 5′-GGAAATCTCTTGCAAGCTA-3′) or VTN (①. 5’-GCAGACACCTGTTCTGAAA-3′, ②. 5’-GGAAGACCTACCTCTTCAA-3′) were cloned into a pGCL-EGFP plasmid (Genechem, Shanghai, China), which encodes an HIV-derived lentiviral vector containing a multiple cloning site for insertion of short hairpin RNA (shRNA) constructs to be driven by an upstream U6 promoter and a downstream CMV promoter–EGFP fluorescent protein. A negative control vector containing the sequence of 5’-TTCTCCGAACGTGTCACGT-3′ was used. Cells were infected with lentivirus produced by Genechem. Forty-eight hours later, EGFP positive cells were sorted by using flow cytometry and expanded for further experiments.
Plasmids construction and generation of stably expressing cell lines
The coding sequences of VEGFR2 and VTN were amplified by PCR from homo cDNA using PrimerSTAR HS DNA polymerase (TAKARA, Otsu, Shiga, Japan), and the resulting PCR products were cloned into pcDNA3.1(+) (Invitrogen). All plasmid constructs were confirmed by sequencing. Cells were transfected with plasmids by Lipofectamine 3000 (Invitrogen) according to manufacturer’s protocol. Forty-eight hours later, the transfected cells were selected and maintained in medium containing 800 μg/ml G418 (Invitrogen) for 14 days. The drug resistant stably clones were isolated, collected and expanded for further experiments.
Cell proliferation assay
Cell proliferation was measured by using Cell Counting Kit-8 (CCK-8) detection kit (Beyotime Biotechnology, China). Cells with a concentration of 5 × 103 per well were seeded in 96-well plates. To evaluate the cytotoxicity of apatinib on cancer cells, different doses of apatinib were added to the culture. 24, 48, 72, 96 and 120 h later, 10 μL CCK-8 solution was added to each well and followed an incubation at 37 °C for 4 h. The absorbance in each well was measured at 450 nm using a microplate ELISA reader (Bio-Rad Laboratories, CA, USA). The relative cell viability was calculated as follows: relative absorbance = (mean absorbance at each time point/mean respective absorbance at 24 h).
Invasion assay
A total of 100 μL of Matrigel (1:30 dilution in serum-free RPMI1640 medium) was added to each Transwell polycarbonate filter (8-μm pore size, Corning, NY, USA) and incubated with the filters at 37 °C for 6 h. Cells were trypsinized and washed three times with DMEM medium containing 1% FBS, followed by resuspention in RPMI1640 containing 1% FBS at a density of 2 × 106 cells/ml. The cell suspensions (100 μL) were seeded into the upper chambers, and 600 mL of RPMI1640 medium containing 10% FBS were was added to the lower chambers. Cells (2 × 105/well) were allowed to invade for 12 h and membranes were then stained with 1% methylrosanilinium chloride. Cells that had migrated to the underside of the filter were counted using a light microscope in five randomly selected fields.
Nude mouse tumor xenograft model and treatment
Four-week-old female BALB/c athymic nude mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and received humane care according to the Soochow University Institutional Animal Care and Treatment Committee. Cells were injected into the left flanks of the mice in a total volume of 100 μL (0.5 × 107 cells). At the end of this experiment, the tumor tissues were dissected out, imaged, and weighed up. Apatinib was dissolved in DMSO and administered daily by intragastric administration at a dose of 10 mg/kg for 3 weeks.
Microarray assay
Total RNA was extracted using TRIzol Reagent (Life technologies, Carlsbad, CA, US) according to the manufacturer’s instructions. RNA integrity was checked by an Agilent Bioanalyzer 2100. Gene expression was performed by Affymetrix GeneChip Human Genome U133 Plus 2.0 array platform. The hybridization occurred in a Hybridization Oven 645 (Affymetrix Inc.). The chips were then washed and stained in a Fluidics Station 450 (Affymetrix Inc.) and the arrays were scanned with a Gene Chip Scanner 3000 and Command Console Software 3.1 with default settings. The selection criterion was > 1.3-fold difference in expression (difference in upregulated expression > 1.3-fold; difference in downregulated expression < 0.77-fold). Hierarchical clustering of samples was performed using an average linkage algorithm using TIGR Multiexperiment Viewer (The Institute for Genomic Research, Rockville, MD, USA).
Gene ontology (GO) is a common method for annotating genes, gene products and sequences to underlying biological phenomena; the Kyoto Encyclopedia of Genes and Genomes (KEGG) is an integrated database resource for biological interpretation of genome sequences and other high-throughput data. Both GO and KEGG analyses were performed using the DAVID database (
https://david.ncifcrf.gov/), which is a bioinformatics data resource composed of an integrated biology knowledge base and analysis tools to extract meaningful biological information from large quantities of genes and protein collections. Genetic networks and functional classification of differentially expressed genes were investigated with the ingenuity pathway analysis (IPA, Ingenuity Systems, Mountain View, CA, USA,
http://www.ingenuity.com), a web delivered tool that enables the discovery, visualization, and exploration of molecular interaction networks in gene expression data.
Statistical analysis
Data was represented as the mean ± SD and the difference among groups was evaluated by one- way ANONA, two-tailed Student’s T test. Repeated ANONA analysis was performed in analysing the difference in growth of implanted tumors between groups. All statistical analyses were performed using the SPSS 16.0 (Chicago, USA). A p value of < 0.05 was considered statistically significant.
Discussion
The regulation of VEGFR2 has been a topic of interest for numerous investigators in tumor pathophysiology, given its importance in the tumorigenesis and development of multiple cancers, including gastric cancer. It has been well-established that the expression of VRGFR2, and its ligands, was correlated with a poor prognosis, and the application of VEGFR2 targeted therapies, inhibiting the tumor related angiogenesis, has improved the outcomes of gastric cancer patients at advanced stages [
3]. In breast cancer, VEGFR2 was only detected in the most aggressive subtype, triple negative breast cancer, leading to enhanced EMT (epithelial-mesenchymal transition) process and activation of NF-κB and β-catenin signaling pathways [
8]. In breast cancer-initiating stem cells, when stimulated by VEGF, VEGFR2 binds to JAK2 and activates STAT3, and maintains cell self-renewal by promoting MYC and SOX2 expression and induces sphere formation [
9]. Besides, in ovarian caner, VEGFR2 also mediates stem cell ability by activation of Src, which increased DNMT3A for miR-128-2 methylation and upregulated Bmi for stem-like cell proliferation [
14]. Interestingly, VEGFR2 could also act as the downstream driver gene of circRNA-MYLK when binding to miR-29a and relieving its suppression for VEGFA, and promote Ras/ERK signaling pathway in bladder cancer [
15]. Loss of PTEN and activated PI3K/AKT/mTOR are required for up-regulation of VEGFR2, and then activating NF-κB-dependent transcriptional activity to induce the loss of sensitivity of chemotherapy in glioma [
17].
With respect to targeted therapy to VEGFR2 by targeting the cancer cells directly, VEGFR2 and its company STAT3 are inhibited by Apatinib and BCL-2 was consequently supressed, inducing autophagy and apoptosis eventually, implying the potential benefits when combing Apatinib and autophagy inhibitors in treatment of osteosarcoma [
16]. However, the involvement of VEGFR2, the target of Apatinib, on the tumorigenesis and metastasis and the molecular mechanisms underlying the regulatory effects in gastric cancer cells remains elucidated.
In this study, we characterized the specific roles and functions of VEGFR2 in gastric cancer pathogenesis, not on the vascular compartment but on the whole cancer cell system. We first characterized VEGFR2 expression in primary gastric cancer tissues and found that level of VEGFR2 expression in gastric cancer were associated with poor survival in subjects from our institution and online database population. VEGFR2 promoted gastric cancer cell proliferation and invasion in vitro, and accelerated tumor growth in vivo. Furthermore, by high-throughput analysis, we predicted its regulatory pathway and delineated the downstream molecules and their relevance for survival. The target signaling pathway, such as MAPK and focal adhesion pathways, as well as target molecules, including VTN, were validated. These findings supported the angiogenesis independent manner of VEGFR2 functioning as a carcinogenic factor in gastric cancer.
VTN (vitronectin), as a cell-adhesion glycoprotein, is primarily localized in the ECM (extracellular matrix) and provides the facility to help tumor cells to breach through to the basement membrane in the process of cancer cell invasion [
18]. Besides of ability to affect cancer cell adhesion, motility and invasion, VTN also assists cancer cells to resist the cell death induced by apoptotic induction [
19]. In drug resistant multiple myeloma cells, the adhesion signaling molecules, such as VTN, was upregulated by Notch signaling pathway and conferred cell protection from drug induced apoptosis [
20]. In nasopharyngeal carcinoma, VTN was identified as once of BPIFB1-interacting proteins and could be reduced by BPIFB1, leading to less formation of VTN-integrin αV complex, suppressing the EMT process, and inhibition of the activation of downstream FAK/Src/ERK signaling pathway [
21]. However, in angiogenic cascade, the engagement of VTN and its receptor integrin αVβ3 needs the activation of Src, serving as a upstream factor, to activate the VEGFR2 signaling for endothelial cell adhesion and migration [
22]. In the cancer development from the early stages to the advanced stages, VTN can be detected in up-regulated levels in a large scale clinical proteomics study in metastatic colorectal cancer, indicating its pro-invasion potential [
23]. Intriguingly, VTN can also be detected to be secreted to blood serum in prostate cancer patients and act as circulating biomarker when combined with PSA (prostate-specific antigen) for early diagnosis of prostate cancer [
24]. With respect of association of gastric cancer and VTN, only one document revealed that VTN acted as the downstream molecule of CEP65, which may provide gastric cancer cells with capacity to detach from the ECM, decreasing cell adhesion [
25]. To our knowledge, for the first time, our results indicate that VTN also acts as the downstream of VEGFR2 pathway in gastric cancer tissues and as a poor prognostic factor in gastric cancer patients. Hence, VEGFR2 and VTN expression in cancer tissues may also serve as biomarkers for tumorigenesis and metastasis of gastric cancer and as direct therapeutic targets in cancer cell level for traditional anti-angiogenesis treatment.
In summary, our data indicated that higher levels of VEGFR2 and its target VTN in cancer specimens were associated with aggressiveness and poorer prognosis of gastric cancer. VEGFR2 promoted the proliferation and invasion, and provided the capacities of tumor formation in xenograft models in gastric cancer, which may be associated with downstream VTN. In addition, our data also suggests that multiple predicted target genes of VEGFR2 in co-expression network may act as prognostic factors in bioinformatic analysis with a larger sample of gastric cancer. VEGFR2/VTN axis in gastric cancer cells may be involved in tumorigenesis and metastasis in a pro-angiogenic-independent way although the precise mechanisms need to be further elucidated. Nevertheless, our study may provide a new and valuable target for design of therapies for intervention and a new cognitive perspective for the anti-angiogenesis therapies.