Background
Malignant pleural mesothelioma (MPM) is a uniformly fatal disease. It originates from normal mesothelial cells lining the pleural or peritoneal cavity long after exposure to asbestos [
1,
2]. There are three main histological types of malignant mesothelioma (epitheloid, sarcomatoid, and mixed or biphasic), with longer survival in epitheloid and shorter survival in sarcomatoid types [
3]. Nearly 3,000 new cases are diagnosed each year in the United States [
2]. The median overall survival of MPM patients ranges from 12 to 24 months [
3].
The most effective treatment regimen (cisplatin and pemetrexed) induces partial response in half of patients and improves survival from 9 to 12 months [
4]. Novel targeted therapies have been investigated in MPM with limited success, including vascular endothelial growth factor (VEGF) inhibitors [
5,
6]. Discovery of additional targets and rational combinations of targeted therapies may lead to effective novel therapies. The type 1 receptor tyrosine kinase EphB4 and its cognate ligand Ephrin-B2 are a pair of potential novel targets.
EphB4 and Ephrin-B2 are normally expressed on endothelial cells of venous and arterial lineage, respectively, and their interaction is critically required for new vessel formation, fusion between vessel compartments, and blood flow [
7,
8]. In addition, Ephrin-B2 is also expressed on pericytes and vascular smooth muscle cells, where it plays critical role in vessel maturation [
9,
10]. In tumor angiogenesis, loss of Ephrin-B2 leads both to significantly reduced tumor vessel density and to tumor growth [
11‐
14]. EphB4, on the other hand, is overexpressed in a variety of epithelial cancers, including breast, prostate, ovarian, esophageal, colon, and head and neck cancers [
15‐
23]. Importantly, we have also shown that EphB4 is expressed in mesothelioma and provides a survival advantage to tumor cells [
24].
Both EphB4 and Ephrin-B2 are transmembrane proteins and direct cell-cell contact leads to bidirectional signaling. EphB4 activation leads to downstream activation of the phosphoinositide kinase-3 (PI3K) pathway in tumor cells [
20], while Ephrin-B2 activation leads to activation of Src [
25,
26]. Inhibition of EphB4-Ephrin-B2 signaling blocks tumor angiogenesis, which in turn leads to hypoxia and induces VEGF expression [
14]. Targeting VEGF and EphB4-Ephrin-B2 simultaneously is thus a potentially effective therapy.
In this study, we investigated the aberrant expression of EphB4 in a cohort of primary MPM tissues. We show that a significant proportion of MPM tumors expressed EphB4, which provides survival advantage to tumor cells. We also investigated the efficacy of sEphB4-HSA as an inhibitor of EphB4-Ephrin-B2 in MPM xenograft models. sEphB4-HSA induces cell death in MPM tumor xenografts in vivo and down-regulates major signaling pathways including PI3K and Src. In addition, we demonstrate that the combination of sEphB4-HSA and VEGF antibody has superior efficacy than either single agent alone, leading to complete tumor regression. Based on these promising preclinical results, future clinical investigation of the efficacy of sEphB4-HSA combined with VEGF inhibitors in MPM is warranted.
Methods
Materials
Soluble EphB4 cDNA fused in-frame with human serum albumin cDNA [
14] was expressed as a seamless fusion protein in CHO cells and purified to homogeneity. EphB4-specific antibody (MAb131) was produced by VasGene Therapeutics Inc. Bevacizumab (Genentech Inc) was purchased. Phosphorylated AKT (Ser473), S6 (Ser235/Ser236) and Src (Tyr416) antibodies were from Cell Signaling, Ki67 antibody was from Abcam, CD31 and NG2 antibodies were from BD Biosciences, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) fluorescent kit was from Promega.
Cell lines
NCI-H2373 and MSTO-211H mesothelioma cell lines were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD) and penicillin/streptomycin (Invitrogen, Carlsbad, CA).
Immunohistochemistry
Formalin-fixed paraffin-embedded malignant mesothelioma tumors were analyzed. Tissue analysis was approved by the institutional review board. 4-μm sections were deparaffinized, rehydrated, and washed with TBS/Tween-20. Antigens were retrieved with exposure to 1 mM EDTA (pH 8.0; DakoCytomation) for 20 minutes. Endogenous peroxidase activity in samples was blocked by exposure to 3% hydrogen peroxide/PBS (Fisher Scientific, Fair Lawn, NJ) and serum-free protein block (DakoCytomation). Tissue sections were incubated with primary antibodies overnight at 4°C. Standard avidin/biotin immunoperoxidase methods with diaminobenzidines as the chromogen were used for detection (DakoCytomation). The intensity of staining was quantified with ImageJ (NIH). EphB4-specific monoclonal mouse anti-human antibody MAb131 was used for MPM tissues. Positive controls included the 293T cell line stably expressing full-length EphB4. Negative controls included co-incubation of tissues with primary antibody and immunizing peptide.
In vivo tumor growth studies
Male BALB/c nu/nu mice (9 weeks old) were injected with 5 × 106 tumor cells in the flank. When tumor sizes reached 150 mm3, mice were grouped (8 tumors per group) and treated with intraperitoneal (i.p.) injection of PBS (control, 3 times per week), sEphB4-HSA (20 mg/kg, 3 times a week), Bevacizumab (20 mg/kg, 3 times a week), or a combination of sEphB4-HSA and Bevacizumab. Tumor volume was measured three times a week and calculated using the following formula: tumor volume = 0.52 × length × width2, where length and width are the longest and shortest dimensions of a palpable tumor. All procedures were approved by Institutional Animal Care and Use Committee and performed in accordance with the Animal Welfare Act regulations.
Immunofluorescence
Xenograft tumors were harvested and immediately snap frozen. 5-μm fresh frozen tissue sections were fixed in phosphate-buffered 4% paraformaldehyde, blocked with goat serum, and incubated with primary antibody overnight at 4°C. Antibody binding was localized with appropriate AlexaFluor-conjugated secondary antibodies (Invitrogen, Carlsbad, CA). Nuclei were counterstained with 6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI). Images were obtained with a Nikon Eclipse 80i fluorescence microscope and Meta Morph imaging series system. The intensity of staining and the positive signal coverage area were quantified with ImageJ (NIH).
Statistics
A student’s t-test (two-tailed, unpaired) was used to calculate P values between groups where indicated.
Conclusions
In this study, we found that EphB4 is highly expressed in MPM, especially in epithelioid subtype, and represents a potential therapeutic target. We also found that the EphB4-Ephrin-B2 inhibitor sEphB4-HSA, alone or combined with the anti-VEGF antibody Bevacizumab was highly active in inhibiting mesothelioma growth in xenograft models. sEphB4-HSA is currently in a clinical Phase 1 trial (ClinicalTrials.gov Identifier: NCT01642342). The data presented here suggest that mesothelioma should be a target disease in clinical investigation of this novel therapy, and the combination of sEphB4-HSA and Bevacizumab should undergo further clinical investigation.
Competing interests
Kranthi Naga and Valery Krasnoperov are employees of VasGene Therapeutics Inc.
Authors’ contributions
RL and YZ carried out the xenograft studies and immunoanalysis of tumor tissues. BF performed immunoanalysis of human MPM tissues. KN and VK produced EphB4 antibody and sEphB4-HSA for this study. RS, PSG, and VK participated in the design of the study. RL, BF, RS, PSG, and VK drafted the manuscript. All authors read and approved the final manuscript.