Ligand-independent EphB1 signaling mediates TGF-β-activated CDH2 and promotes lung cancer cell invasion and migration

Purpose: The initial step of cancer metastasis is that cancer cells acquire the capability to migrate and invade. Eph receptors comprise the largest family of receptor tyrosine and display dual role in tumor progression due to unique ephrin cis- or trans- signaling. The roles of EphB1 and its phosphorylation signaling in lung cancer remain to be elucidated. Patients and Methods: We analyzed the expression of EphB1 in both publicly available database and 60 cases of NSCLC patients with or without metastasis. The migration and invasion of lung cancer cells were assessed by a transwell assay. The activation of EphB1 signaling was assessed by western blot and real-time PCR. The EphB1 mutant was used to evaluate the effect of phosphorylation of EphB1. Results: Here, we showed that increased expression of EphB1 was detected in Non-Small-Cell Lung Cancer (NSCLC) biopies compared to non-cancer controls. Significant higher expression of EphB1 in lung biopsies were found in patients with metastasis compared to non-metastatic NSCLC patients. Higher EphB1 expression was correlated with poor patient survival in lung cancer. Overexpression of EphB1 promoted the migration and invasion of lung cancer cells. On the contrast, Ephrin-B2, a transmembrane ligand for EphB1 forward signaling, inhibited migration and invasion of lung cancer cells. TGF-β-activated Smad2 transcriptionally upregulated the endogenous expression of EphB1. Ligand-independent EphB1 promoted Epithelial-mesenchymal transition (EMT) through upregulating CDH2. Conclusion: Our results showed that the effect of EphB1 on the migration and invasion was context-specific and was dependent on EphB1 phosphorylation.


Introduction
Non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer cases. Metastasis is the major reason for the mortality of lung cancer patients. Cancer cell migration and invasion are initial steps in metastasis 1,2 . Epithelial-mesenchymal transition (EMT) of cancer cells is believed to be crucial for cancer cell invasion 3 .
The process that epithelial cancer cells lose their polarity and displays mesenchymal phenotype is called EMT. Eph receptors, which comprise the largest family of receptor tyrosine kinase, have been found to play a role in EMT 4 . The Eph receptors are divided into 2 subclasses: nine EphA receptors and five EphB receptors. Ephs and their ligands ephrins trigger Ivyspring International Publisher bidirectional signal pathway upon cell-to-cell contact 5 . Eph/ephrin interactions have been implicated in various pathologic processes, including inflammation, neural development, and angiogenesis [6][7][8] . Eph forward signaling that depends on Eph kinase activity has been involved in cell migration and evasion 5 . However, bidirectional signals can also mediate cell repulsion 5 .
Eph receptors display dual role in tumor progression and tumor suppression 9 . EphB subgroup and the Ephrin-B subgroup are coexpressed in SCLC cell lines and tumors, modulating the behavior of SCLC through autocrine or juxtacrine activation 10 . EphA/B mutation or amplification can be found in 16% of lung adenocarcinoma patients 11 . EphB3 promotes cancer cell survival and migration by enhancing DNA synthesis and inhibiting apoptosis in NSCLC cells 12 . Eph receptors that are activated by ephrins can inhibit oncogenic signaling pathways, such as the HRAS-Erk, PI3K-Akt and Abl-Crk pathways 5 . The paradox may be because of the cis and trans signaling or ligand-dependent or ligandindependent Eph signaling.
In this study, we found that ligand-independent EphB1 promoted lung cancer cell mobility and invasion. TGF-β-activated Smad2 transcriptionally interacted with a Smad2-binding element in EphB1. Ligand-independent EphB1 promotes EMT through upregulating CDH2. However, the ligand induced EphB1 phosphorylation inhibited lung cancer mobility and invasion. A better understanding of context of EphB1 signaling can help to explain the paradox roles in cancer progression.
Cell culture, plasmid construction, siRNA and patients NSCLC cell lines A549 and H460 were cultured in RPMI-1640 medium supplemented with penicillin G (100 U/mL), streptomycin (100 mg/mL) and 10% fetal calf serum. HEK293 cells were cultured in Dulbecco's modified Eagle medium (Gibco) with 1 g/L glucose and 10% FBS. All cell lines were obtained from ATCC. Cells were grown at 37°C in a humidified atmosphere of 5% CO2 and were routinely sub-cultured using 0.25% (w/v) trypsin-EDTA solution.
Patients diagnosed with NSCLC (n=60) were included in this study, 13 of which were diagnosed with lymph node-positive lung cancer and 47 of which were diagnosed with lymph node-negative lung cancer. All cases enrolled in this study were diagnosed at the second Xiangya hospital, Central South University, China. The clinical characteristics of the cases are summarized in Table 1. The patients were informed of the sample collection and signed informed consent forms. The collection and use of samples were approved by the ethical review committees of the second Xiangya Hospital, Central South University. Clinicopathological characteristics of these patients are presented in Table 1.

Western blotting
The protein lysate used for western blotting was extracted using RIPA buffer (Biotime, Hangzhou, China) containing protease inhibitors (Roche, Basel, Switzerland). Proteins were quantified using the BCA TM Protein Assay Kit (Pierce, USA). A western blot system was set up using a Bio-Rad Bis-Tris Gel system, according to the manufacturer's instructions (Bio-Rad, CA, USA). The cell protein lysates were separated on 10% SDS-polyacrylamidegels and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Danvers, MA, USA). The primary antibody solution was prepared in 5% blocking buffer. Primary antibodies against EphB1 (Abcam, USA), p-EphB1 (Abcam, USA) were incubated with the membrane at 4 ºC overnight, followed by a brief wash and incubation with secondary antibody for 1 h at room temperature. An anti-GAPDH antibody control was purchased from Proteintech (Chicago, USA) and was used as a loading control. Finally, a 40:1 solution of peroxide and luminol was added to cover the blot surface for five minutes at room temperature. The chemiluminescent signals were captured, and the intensity of the bands was quantified using a Bio-Rad ChemiDoc XRS system (Bio-Rad, CA,USA).

Cell migration and invasion assays
Cell migration and invasion assays were both performed using a transwell insert that contains polycarbonate filters with 8-μm pores (cat. no. 3422; Corning). Cells (5x10 4 ) were suspended in 200 µl of serum-free medium and added to the transwell membrane in the upper chamber. Migrated cells were fixed in 4% paraformaldehyde and stained with crystal violet. Migrated cell images were observed and imaged under microscope (CKX41, Olympus, Japan). Cell migration was quantitated by counting in 10 random fields on the lower membrane surface. Invasion capacity of cells was measured by Matrigel matrix gel invasion assay. The surface of the filter (8-μm pore size) of the upper chamber was coated with 1 mg/ml Matrigel matrix. Cells (5x10 4 ) were suspended in 200 µl of serum-free medium and added to the transwell membrane in the upper chamber. Invaded cells were fixed in 4% paraformaldehyde and stained with crystal violet. Cell invasion was observed and imaged under microscope (CKX41, Olympus, Japan). Cell invasion was quantitated by counting in 10 random fields on the lower membrane surface.

Immunohistochemistry
Lung biopsies were fixed and embedded in paraffin wax. Four-to six-μm thick paraffin sections were defaced followed by hydration. Tissue sections were incubated with primary antibody at 4°C overnight in a humidified chamber. After extensive washing with PBS, sections were incubated with biotin-linked goat anti-rabbit IgG antibodies (UltraSensitive S-P Kit, Maixin Biotechnology Company, Fuzhou, China). The sections were then washed and followed by developing in 3'-diaminobenzidine hydrochloride (DAB) as chromogen, and sections were counterstained with haematoxylin. Finally, after dehydration and mounting, the sections were observed and imaged under microscope (OLYMPUS BX-51, Japan). Goat serum and PBS were used instead of the first antibody as a negative control and blank control respectively. A semi-quantitative scoring criterion for IHC was used in which both the staining intensity and positive areas were recorded.

Quantitative real-time PCR
Total RNA for RNA-seq experiment was used for real-time PCR to confirm the expression of genes. cDNA was synthesized from total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). GAPDH was used as the endogenous control. Quantitation PCR was performed according to the indications. Real-time PCR was performed using the Bio-Rad IQ TM5 Multicolor real-time PCR detection System (Bio-Rad, Berkeley, CA, USA). Relative mRNA expression levels were calculated by the 2 -ΔΔCt method. The siRNA sequences for knockdown of target genes are shown in Table 2. Table 2. Primer sequence for real-time PCR

Bioinformatics analysis
Six independent cohorts of lung cancer data and their correlated clinic data, GSE10072, GSE19188, GSE7670, GSE68465 13 , GSE50081 14  Overall survival was measured using the Kaplan-Meier method, and the log-rank test was used for comparison between low EphB1 expression group and high EphB1 expression group.

In vitro cell proliferation assessment
The proliferation of lung cancer cells was measured using the CCK-8 assay (Bimake, China). The cell suspension was inoculated in a 96-well plate. After treatment, 10 μl of CCK-8 solution was added to each well and the plate was incubated for an additional 4 hrs. Next, the absorbance measured at 450 nm using a microplate reader. The experiment was repeated three times, and six parallel samples were measured each time.

Chromatin immunoprecipitation (ChIP)
ChIP assays were performed as described 20 . Briefly, A549 cells were crosslinked in 1% formaldehyde for 10 min at 37 °C to generate DNA-protein complex. Cell lysates were then sonicated and immunoprecipitated with anti-Smad2 or with IgG (control). The precipitated DNA fragments were purified and analyzed by PCR and agarose gel electrophoresis. PCR was performed using promoter-specific primers for EphB1 with amplification of the Smad2-binding regions. Primers were synthesized as follows: Forward: CCTTCCCA CCCACACTGAAG; Reverse: GGTTGCCTTTGGTGT TCACTT.

Statistical Analysis
Data are presented as the mean ± S.D. from at least three separate experiments. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., CA, USA). Multiple group comparisons were performed using ANOVA with a post hoc test for the subsequent individual group comparisons. A p value of less than 0.05 was considered to be significant. The survival of tumour-bearing mice was analysed by Kaplan-Meier. A p value of less than 0.05 was considered to be significant.

EphB1 expression is correlated with poor patient survival in lung cancer
To investigate the relationship between EphB1 and lung cancer, we analyzed EphB1 expression in lung samples from cancer patients. Publicly accessible gene expression data of EphB1 was obtained from Gene Expression Ominibus (GEO) database (GSE10072, GSE19188, GSE7670, GSE68465, GSE30219, GSE50081) and The Cancer Genome Atlas (TCGA) database. EphB1 expression was significantly higher in NSCLC samples compared to non-cancer controls ( Figure 1A, Figure 1B). Significant higher expression of EphB1 in cancer biopsies were found in patients with metastasis compared to non-metastatic patients with NSCLC ( Figure 1B). Gene expression data for NSCLC patients was used to analysis the correlation of EphB1 and overall survival (OS). Patients with higher levels of EphB1 expression showed shorter OS compared with the patients with lower levels of EphB1 (p<0.001) ( Figure 1C). EphB1 expression in lung biopsies was correlated with poor patient survival in lung cancer ( Figure 1B). We verified EphB1 expression in patients by recruiting 60 NSCLC patients with or without metastasis. Clinicopathological characteristics of these patients are presented in Table 1. Consistent with results obtained from public database, the higher EphB1 expression was detected in metastatic lung cancer samples than in non-metastatic lung cancer samples ( Figure 1D).

Ligand-independent EphB1 signaling promotes cancer cell migration and invasion
To investigate the roles of EphB1 in the migration and mobility of lung cancer cell, we transfected EphB1 expression vector into H460 cells or EphB1 siRNA into A549 cells. The transwell assay revealed that EphB1 promoted the migration and invasion of lung cancer cells and knockdown of EphB1 resulted in reduced migration and invasion in A549 cells (Figure 2A, 2B and 2C). However, the ligand EphrinB2-Fc treatment on the contrary reduced migration and invasion of lung cancer cells ( Figure  2D). The overexpression of EphB1 did not affect the lung cancer cell growth ( Figure 2E, 2F).

Ligand-dependent EphB1 signaling inhibits cancer cell migration and invasion through inducing the phosphorylation of EphB1
As the phosphorylation of EphB1 mediated by its Tyr-594 is crucial to cell migration, we then examined the effect of EphB1 forced expression and ligand EphrinB2-Fc treatment on the phosphorylation of Tyr-594. We found that transfection of EphB1 inhibited EphB1 Tyr-594 phosphorylation, while treatment of EphrinB2-Fc promoted EphB1 Tyr-594 phosphorylation ( Figure 3A). It suggests the forced overexpression of EphB1 inhibits EphB1 forward signaling and exogenous EphrinB2-Fc promotes EphB1 forward signaling.
In order to investigate if the activation of EphB1 forward signaling affect the cell mobility, we transfected wild type EphB1 or EphB1 Y594 mutant into A549 cells. The exogeneous treatment of EphrinB2-Fc obviously inhibited the migration and invasion of EphB1 wt transfected cells, but significantly improved the migration and invasion of EphB1 Y594 mutant transfected cells ( Figure 3B). It demonstrated that phosphorylation of EphB1 reduces migration and invasion of lung cancer cells, whereas the ligand-independent EphB1 promotes migration and invasion of lung cancer cells.

Ligand-independent EphB1 mediates TGF-β-activated CDH2
To investigate the mechanism of how EphB1 overexpression promotes the migration and invasion of lung cancer cells, we compared the expression of EMT related molecules between cells with or without forced expression of EphB1. We found that the transfection of EphB1 promoted the expressions of mesenchymal molecules such as Snail, Slug, CDH2, Zeb1, whereas knockdown the expression of EphB1 inhibited the expressions of mesenchymal molecules ( Figure 4A). Western blot was performed to confirm the upregulation of CDH2 induced by EphB1 overexpression ( Figure 4B).
TGF-β signaling is the main regulator in cell migration and invasion. We then examined if TGF-β regulates EphB1 expression. We found that TGF-β enhanced the expression of EphB1 ( Figure 4C). Transfection of Smad2 enhanced the expression of EphB1 ( Figure 4D, 4E). We then performed a ChIP assay to elucidate if TGF-β-activated Smad2 can be recruited to EphB1 promoter. The putative Smad2/3 binding sites were shown in Figure 4F. ChIP assay performed on Smad2-transfected HEK293 cells revealed that Smad2 bound to EphB1 promoters ( Figure 4G).

Discussion
Here, we studied the ligand mediated trans-forward EphB1 signaling and ligandindependent cis-attenuation signaling contribute to the migration and invasion of lung cancer cells.
Eph family of receptor tyrosine kinases and their ligands mediate many physiologic and pathologic effects by a multiple signaling mode 21 . Ephrins and Eph tyrosine kinases initiate a unique bidirectional signal (forward and reverse signals) on both the receptor-expressing and ligand-expressing cells 22,23 . Furthermore, Eph receptors and ligand ephrins make their cis-interaction when the ligand and receptor are expressed in the same cells, while they interact in trans when ligand and receptor are expressed in different cells 21 . Cis interactions may be one of the strategies that adopted by cancer cells to escape the tumor suppressive effects of Eph receptor signaling induced by ephrins binding in trans [24][25][26] . Eph receptors and ephrins coexpressed in the same cells can attenuate receptor activation in trans by hindering the binding of ephrins and Eph receptors in trans 27 . The coexpression of Ephrin-A3 can block the ability of EphA2 and EphA3 to link ephrins in trans and become activated, while Ephrin-B2 can deter not only EphB4 but also EphA3 in the cancer cells 27 . Eph receptors are often upregulated in many types of cancer, although decreased Eph receptor levels were also been observed in certain types of cancer 28 . In contrast to the overexpression of Eph receptors in cancer, Eph receptor forward signaling that triggered by tyrosine phosphorylation inhibits tumor cell growth 29,30 . Higher expression of Ephrin-B2 is correlated with poor overall survival and disease-free survival in head and neck squamous cell carcinoma, pancreatic adenocarcinoma and bladder urothelial carcinoma 31 . The paradoxical functions of Eph are influenced by the cis-or trans-signaling. In this study, EphrinB2-Fc provided in trans mode can elicit EphB1 forward signaling, leading to reduced mobility and invasion. The overexpression of EphB1 without exogenous stimulation of Ephrin-B2, however, promotes mobility and invasion through upregulating EMT molecules.
TGF-β signaling is thought to drive EMT and trigger apoptosis. TGF-β-activated Smad3/4 directly binds to CDH2 promoter and transcriptionally regulates CDH2 in NSCLC 32 . Our study found that Smad2 binds to EphB1 promoter and transcriptionally regulates EphB1 expression. The endogenous expression of EphB1 promotes migration and invasion of NSCLC through upregulating CDH2. It demonstrated that TGF-β regulates CDH2 by directly binding to CDH2 promoter or indirectly through transcriptionally regulating EphB1.

Conclusion
In conclusion, the roles of EphB1 in cancer cell invasion and migration are context-dependent and involve the cis-or trans-interactions between receptor and ligands. EphB1 is transcriptionally regulated by Smad2 and mediates TGF-β signaling in a ligand-independent manner.