HMQ‐T‐B10 induces human liver cell apoptosis by competitively targeting EphrinB2 and regulating its pathway

Abstract Hepatocellular carcinoma (HCC) is a highly prevalent cancer worldwide and it is necessary to discover and develop novel preventive strategies and therapeutic approaches for HCC. Herein, we report that EphrinB2 expression is correlated with liver cancer progression. Moreover, by using phosphorylated proteomics array, we reveal a pro‐apoptosis protein whose phosphorylation and activation levels are up‐regulated upon EphrinB2 knockdown. These results suggest that EphrinB2 may act as an anti‐apoptotic protein in liver cancer cells. We also explored the therapeutic potential of HMQ‐T‐B10 (B10), which was designed and synthesized in our laboratory, for HCC and its underlying mechanisms in vitro and in vivo. Our data demonstrate that B10 could bind EphrinB2 and show inhibitory activity on human liver cancer cells. Moreover, induction of human liver cancer cell apoptosis by B10 could be augmented upon EphrinB2 knockdown. B10 inhibited HCC cell growth and induced HCC cell apoptosis by repressing the EphrinB2 and VEGFR2 signalling pathway. Growth of xenograft tumours derived from Hep3B in nude mice was also significantly inhibited by B10. Collectively, these findings highlight the potential molecular mechanisms of B10 and its potential as an effective antitumour agent for HCC.


| INTRODUCTION
EphrinB2 is a cell surface transmembrane protein encoded by the EFNB2 gene in humans. 1 It is widely expressed in tumour cells and mediates tumour cell proliferation, invasion and migration. 2,3 EphrinB2 can activate several Eph receptors (termed "forward" signalling), but can also serve as a receptor ("reverse signalling"). The reverse signal activity promotes tumourigenesis and epithelial-mesenchymal transition through its signalling molecules for tyrosine phosphorylation sites and a PDZ binding motif in the EphrinB2 cytoplasmic domain. 4 EphrinB2 has been shown to undergo internalization and is involved in VEGFR2-and VEGFR3-mediated angiogenesis in cultured cells.
EphrinB2 promotes VEGFR endocytosis in endothelial cells and enhances VEGF-mediated angiogenesis. 5,6 Regulation of VEGFR signalling in cancer cells further results in the activation of PI3K/AKT and MAPK/ERK pathways which regulate cell proliferation, migration, and angiogenesis. EphrinB2 has been verified as a poor prognostic indicator in several solid tumours including pancreatic adenocarcinoma, bladder urothelial carcinoma, and thyroid carcinoma. [7][8][9] Hepatocellular carcinoma (HCC) is a major type of primary liver cancer with an annual incidence of more than half a million new cases worldwide, 10,11 ranking as the fifth most frequently diagnosed malignant tumour and the third leading cause of cancer-related death globally. 12 Attributed to its dismal prognosis, high mortality, and rapid progression and metastasis, liver cancer is still associated with severe disease-and treatment-related morbidity. 13 New drugs are being developed that work differently from standard chemotherapeutic drugs. These targeted drugs act on specific parts of cancer cells or their surrounding environment. Sorafenib, which acts as a multiple tyrosine kinase inhibitor, is a mainstream molecular targeted drug approved for HCC treatment. [14][15][16] Previous studies have identified multiple mechanisms underlying reduced sensitivity to sorafenib in HCC, 17 including various molecular and signalling pathway alterations such as activation of the EGFR pathway, 18 epithelial mesenchymal transition 19 and induction of autophagy. 20,21 Demonstrations of the efficacy of targeted molecular therapies have triggered the search for additional molecules with therapeutic potential in HCC.
In this study, we explored EphrinB2 as a promising marker for HCC prognosis and therapy. Its association with HCC clinical characteristics and the potential underlying mechanisms were explored.
Moreover, we aimed to reveal the functions and mechanisms of B10 ( Figure 1A), which was synthesized in our laboratory, in modulating the in vitro and in vivo growth of HCC. We found that B10 inhibited HCC cell proliferation, induced HCC cell apoptosis, and suppressed xenograft growth in nude mice by targeting EphrinB2 and its pathway.

| Phospho-antibody microarray analysis
The expression profile of 12 signalling pathway phosphor-related proteins was detected and analysed using a human CSP100 Antibody The obtained data were analysed using the Modfit LT software. For Hoechst staining, cells were fixed with 4% paraformaldehyde and stained with Hoechst 33258. Images were photographed under the inverted fluorescence microscope.

| Western blot analyses
Following treatment with B10 and sorafenib for 48 hours, Hep3B, HepG2, and SMMC-7721 cells were collected and lysed. The insoluble protein lysates were denatured and analysed for western blot analysis with primary antibodies, followed by use of the ECL kit. The Image-Pro Plus software (Image-Pro Plus 5.1, Media Cybernetics, Inc., Rockville, MD, USA) was used to quantify the protein.

| RNA interference studies
Silencing RNA oligonucleotides targeting EphrinB2 were obtained from Shanghai GenePharma Co., Ltd (Shanghai, China). The EphrinB2 siRNA was designed to target the following sequence: EphrinB2,

| In vivo tumour suppression assay
BALB/c-nude mice aged 4-6 weeks were injected subcutaneously with 4 9 10 6 Hep3B cells into the right flanks. Tumour growth was recorded by measurement of two perpendicular diameters of the tumours every other day and calculated by using the formula: volume = (length 9 width 2 )/2. When the tumour volume reached 80-100 cm 3 , mice were randomly assigned to 4 groups (6 mice/group) and treated with the vehicle (0.5% CMC-Na), B10 (20, 80 mg/kg in 0.5% CMC-Na) or sorafenib (40 mg/kg in 0.5% CMC-Na) every day by intragastric administration. The mouse weight and tumour volume were monitored every other day.

| Statistical analysis
Quantitative data were expressed as means AE SEM from three separate experiments for each condition. Statistical analysis was performed using the one-way ANOVA and further Tukey's multiple comparison test was used to analyse statistical differences between groups under different conditions. For all other data, an independent-samples t test was used. A *P-value < .05 was considered statistically significant.

| Effect of B10 on VEGFR2
B10 was synthesized by using computer-aided drug design, which was a tool for designing novel compounds for protein targets. In order to confirm whether B10 could inhibit the activity of VEGFR kinase in vitro, the phosphorylation of a peptide substrate by VEGFR kinase was evaluated in a microtitre plate format using LANCE. As shown in Figure S1a, B10 treatment attenuated VEGFR2 activity in a dose-dependent manner and the IC50 of B10 to VEGFR2 kinase was 2.150 lmol/L (IC50 of sorafenib to VEGFR2 kinase was 1.329 lmol/ L), suggesting that B10 altered VEGFR2 kinase activity effectively.
Cell membrane chromatography (CMC) method is an effective technique to study the characteristics of drug-membrane receptor affinity. 25 Elution profiles of B10 and sorafenib on VEGFR2/HEK293 CMC columns are shown in Figure 1B. The retention behaviour indicated that both B10 and sorafenib could bind to VEGFR2. Furthermore, the retention time of B10 was 38.01 minutes and that of sorafenib was 11.42 minutes ( Figure 1C).

| EphrinB2 expression correlates with human liver cancer cell lines
To investigate the clinical significance of EFNB2 in liver cancer, we firstly analysed the EFNB2 gene in the TCGA database to examine the correlation of EFNB2 with normal and clinical liver tissues. EFNB2 was significantly highly expressed in liver cancer tissues than in normal tissues. As shown in Figure 1D, there was a significant difference in EFNB2 expression between normal and clinical liver tissues (P < .0001). Next, we conducted a study to detect EFNB2-related protein EphrinB2 expression levels in liver cancer cells by western blotting. Results showed that EphrinB2 was highly expressed in liver cancer cell lines ( Figure 1E). Taken together, these results indicate that EphrinB2 expression is closely related to liver cancer development.
We then used the CMC method to evaluate B10 and EphrinB2 receptor affinity. Elution profiles of B10 and sorafenib for EphrinB2/ HEK293 CMC columns were shown in Figure 1F. B10 in the effluent could combine with the immobilized receptor EphrinB2, which was present at the stationary phase surface. Furthermore, the retention time of B10 (56.39 minutes) was longer than that of sorafenib (11.22 minutes) ( Figure 1G). Therefore, B10 could bind to EphrinB2, and their combined strength is higher than that of sorafenib.

| B10 inhibits liver cancer cell growth and colony formation
Based on the high expression of EphrinB2 in liver cancer cell lines, we used MTT and colony formation assays to examine the effect of  Figure 2K). These findings indicate that B10 exhibits potential anti-tumour properties in liver cancer cells.

| Effect of B10 on EphrinB2
Furthermore, we used the fluorescent competitive study to explore whether B10 could competitively bind to the site on EphrinB2 occupied by EphB4. Results showed that EphB4 expression was decreased ( Figure 3A). This indicated that B10 could compete with EphB4 for binding to EphrinB2.
We also detected the effect of B10 on EphrinB2 expression in liver cancer cells, including Hep3B, SMMC-7721 and HepG2 cells.

| Differentially expressed proteins induced by B10 and siRNA EphrinB2
To comprehensively determine the mechanism of B10 functioning, we performed a phosphor-proteomics-based study using a phosphoantibody microarray (Full Moon BioSystems Inc.), which provides a high-throughput platform for efficient protein phosphorylation status profiling, with detection and analysis of phosphorylation events at specific sites to identity pathways that are regulated by B10 and EphrinB2 knockdown ( Figure 4A). In total, 42 differentially phosphorylated proteins were identified between control and B10 treated Hep3B cells when the fold-change was ≥2; 52 differentially phosphorylated proteins were identified between control and siRNA of EphrinB2 and Hep3B cells when the fold-change was ≥1.5. Proteins obtained from the array were further analysed by KEGG and pathway mapping analysis. Results indicated that these proteins were associated with several cancer biological processes such as cell growth, cell apoptosis and cell migration ( Figure 4B,C). Interestingly, both B10 treatment and siRNA EphrinB2 induced up-regulation of pro-apoptosis protein phosphorylation ( Figure 4D). Therefore, EphrinB2 might be an anti-apoptotic protein, and B10 could induce cell apoptosis.

| B10-induced liver cell apoptosis
To confirm the phosphor-antibody array, we used a combination of flow cytometry analysis and Hoechst 33258 staining to monitor human liver cell apoptosis both with and without treatment of B10 and sorafenib. As shown in Figure

| EphrinB2 modulates cell apoptosis in liver cancer cells
We further established liver cancer cell lines in which EphrinB2 was inhibited via siRNA expression. These cell lines were used to determine whether EphrinB2 could affect cell apoptosis. As shown in Figure 6A-C, the protein level of EphrinB2 was down-regulated in EphrinB2-silencing cells. Cell proliferation was determined at 48 hours after seeding was done using an MTT assay. As shown in Figure 6D-F, Hep3B, SMMC7721 and HepG2 cell proliferation remained unchanged upon a decrease in EphrinB2 level, after silencing of endogenous EphrinB2.
However, knockdown of EphrinB2 by siRNA significantly enhanced the apoptotic induction of B10 ( Figure 6G-I EphrinB2 was a key factor in apoptosis induction by B10.

| Effect of B10 on molecular of EphrinB2 signalling pathway
EphrinB2 signalling has been reported to regulate the internalization of VEGFR2 and VEGFR3 activity. VEGFR2 downstream signalling

| B10 has a potent antitumour effect in vivo
To test the efficacy of B10 on tumour growth in vivo, a xenograft tumour model was established in nude mice. The anticancer

| DISCUSSION
Hepatocellular carcinoma is the third leading cause of deaths occurring because of cancer worldwide. 26 Currently, most HCC patients cannot be diagnosed in the early stage, and therefore, they lose the best opportunities for treatment. Despite tremendous advances in early diagnosis and surgery, HCC incidence continues to increase worldwide. Therefore, novel preventive strategies and therapeutic approaches for HCC urgently need to be discovered and developed.
B10 was synthesized to target VEGFR2 by computer-aided drug design, a tool for designing novel compounds for protein targets. We found that B10 could inhibit the VEGFR2 kinase activity in vitro by evaluating the phosphorylation of a peptide substrate by VEGFR2 kinase in a microtitre plate format using LANCE. In addition, the elution profiles of B10 and sorafenib on the HEK293/VEGFR2 CMC column indicated that B10 and sorafenib could bind to VEGFR2. It is well-known that VEGFR2 plays a vital role in tumour angiogenesis and metastasis. 27 EphrinB2, which is expressed in cancer cells, was proven to be involved in VEGF/VEGFR mediated angiogenesis. 28 We address this issue by examining the correlation between the expression of EphrinB2 and the progression of liver cancer. Here, we examined the expression of EFNB2 in liver cell carcinoma specimens revealed that EFNB2 overexpression was higher in cancerous liver tissue than normal tissue. It indicated that EFNB2 was overexpressed in human liver cancer, and the overexpression was correlated with tumour progression and poor patient outcome.
Additionally, western blot results showed that EphrinB2 was predominantly expressed in a spectrum of human liver cancer cell lines,  To gain insights into the mechanisms by which B10 bound to HCC is inhibited, a phosphor-antibody array was used to screen the potential targets and signalling pathways. The major findings from array analysis were that differentially phosphorylated proteins associated with apoptosis and growth were obtained after B10 treatment. Such proteins are associated with the PI3K-Akt, MAPK, VEGF and mTOR signalling pathways. Importantly, we found that the phosphorylation of pro-apoptotic proteins were up-regulated after B10 This also suggests that EphrinB2 might be a target forB10. EphrinB2 promotes VEGFR endocytosis in endothelial cells, thereby enhancing VEGF-mediated angiogenesis, 32 which is essential in normal and pathological situations. 33 It has been implicated in mediating angiogenesis signalling in human cancer cells by promoting the internalization of VEGFR2 and VEGFR3, which in turn are linked to downstream effectors involved in PI3K/AKT/mTOR and ERK/MAPK pathways. 34,35 The proteins selected by phosphor-antibody array were confirmed by western blot. As expected, our results showed that B10 inhibited the phosphorylation of VEGFR2 and the expression of VEGFR3. Meanwhile, B10 decreased the activation of VEGFR2 downstream signal molecules, including the activation of AKT, mTOR, and ERK1/2 in Hep3B, SMMC-7721, and HepG2 cells.
Most importantly, B10 caused an effective inhibition of tumour cell growth in the xenografts in athymic mice.
In summary, this study reports that EphrinB2, which is overexpressed in liver cancer, could serve as an apoptosis promoter, and hence, it plays a functional role in liver cancer. B10 has an inhibitory effect on HCC cells, whose underlying mechanism is via the targeting of the EphrinB2 signalling pathway and apoptosis induction. It represents a promising anticancer agent for conducting further clinical trials, for the use of B10 in liver cancer treatment.

ACKNOWLEDG EMENTS
We express our gratitude to Prof. Peng Hou, Chen Huang and Qingyong Ma for technical assistance. This work was supported by