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Breast cancer remains a leading cause of cancer mortality worldwide, underscoring the urgent need for novel and effective therapeutic strategies. Eph receptor tyrosine kinases, particularly EphB4, exhibit diverse roles in cancer biology, acting as either tumor promoters or suppressors depending on the cellular environment and ligand engagement. EphB4 is frequently overexpressed in breast cancer and contributes to dysregulated signaling and tumor progression through the abnormal interaction with its ligand Ephrin‐B2. We herein developed an improved anti‐EphB4 monoclonal antibody, Eb 4 Mab‐7‐mG 2a , which can be characterized as a subclass‐switched IgG 2a variant designed to enhance immune effector function, specifically antibody‐dependent cellular cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC). Our findings showed that Eb 4 Mab‐7‐mG 2a effectively blocked Ephrin‐B2‐induced ERK phosphorylation and proliferation in EphB4‐positive MCF‐7 breast cancer cells but had no effect on EphB4‐knockout (KO) MCF‐7 cells. Flow cytometry confirmed high‐affinity binding between Eb 4 Mab‐7‐mG 2a and EphB4‐expressing cells, whereas in vitro assays demonstrated potent and selective ADCC and CDC activities against EphB4‐positive tumor cells. In vivo experiments showed that Eb 4 Mab‐7‐mG 2a significantly suppressed xenograft growth in models bearing EphB4‐overexpressing CHO‐K1 and EphB4‐positive MCF‐7, but showed no therapeutic effect in EphB4‐negative CHO‐K1 and EphB4‐KO MCF‐7 xenografts. Immunohistochemical analysis revealed reduced Ki‐67 proliferation indices in treated tumors, supporting the antiproliferative effects of the developed antibody. Overall, these findings demonstrate that Eb 4 Mab‐7‐mG 2a exerts dual‐action antitumor activity through ligand blockade and immune effector engagement. Further evaluations in other EphB4‐overexpressing cancers and in combination with immune checkpoint inhibitors are warranted. Humanization and tumor‐selective engineering may enhance its clinical potential for precision oncology.

Ephrin-B2/VEGF in angiogenesis control Ephrin-B ligands are well known as axon guidance molecules. Ephrin-B2 is also known to play a role in angiogenic remodelling. Two studies now show that signalling through ephrin-B2 controls vessel sprouting. Mechanistically, ephrin-B2 seems to function in part by regulating VEGFR internalization and signalling. The finding suggests that blocking ephrin-B2 signalling may be an alternative approach to blocking VEGFR function in angiogenesis. The protein ephrin-B2 is known to be upregulated during angiogenesis — the growth of new blood vessels — but its precise function has been unclear. Here it is shown that signalling through ephrin-B2 controls vessel sprouting. Mechanistically, ephrin-B2 seems to function in part by regulating the internalization of vascular endothelial growth factor receptors (VEGFRs). The results indicate that blocking ephrin-B2 signalling might be an alternative to blocking VEGFR function to disrupt angiogenesis in tumours. In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions 1 , 2 , 3 . Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

The EPHB4 receptor is implicated in the development of several epithelial tumors and is a promising therapeutic target, including in prostate tumors in which EPHB4 is overexpressed and promotes tumorigenicity. Here, we show that high expression of EPHB4 correlated with poor survival in prostate cancer patients and EPHB4 inhibition induced cell death in both hormone sensitive and castration-resistant prostate cancer cells. EPHB4 inhibition reduced expression of the glucose transporter, GLUT3, impaired glucose uptake, and reduced cellular ATP levels. This was associated with the activation of endoplasmic reticulum stress and tumor cell death with features of immunogenic cell death (ICD), including phosphorylation of eIF2α, increased cell surface calreticulin levels, and release of HMGB1 and ATP. The changes in tumor cell metabolism after EPHB4 inhibition were associated with MYC downregulation, likely mediated by the SRC/p38 MAPK/4EBP1 signaling cascade, known to impair cap-dependent translation. Together, our study indicates a role for EPHB4 inhibition in the induction of immunogenic cell death with implication for prostate cancer therapy.

Osimertinib is the latest generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor used for patients with EGFR-mutated non-small cell lung cancer (NSCLC). We aimed to explore the novel mechanisms of osimertinib by particularly focusing on EGFR-independent effects, which have not been well characterized. We explored the EGFR-independent effects of osimertinib on cell proliferation using NSCLC cell lines, an antibody array analysis, and the association between the action of osimertinib and the ephrin receptor B4 (EphB4). We also studied the clinicopathological significance of EphB4 in 84 lung adenocarcinoma patients. Osimertinib exerted significant inhibitory effects on cell growth and cell cycle progression by promoting the phosphorylation of p53 and p21 and decreasing cyclin D1 expression independently of EGFR. EphB4 was significantly suppressed by osimertinib and promoted cell growth and sensitivity to osimertinib. The EphB4 status in carcinoma cells was positively correlated with tumor size, T factor, and Ki-67 labeling index in all patients and was associated with poor relapse-free survival in EGFR mutation-positive patients. EphB4 is associated with the EGFR-independent suppressive effects of osimertinib on cell cycle and with a poor clinical outcome. Osimertinib can exert significant growth inhibitory effects in EGFR-mutated NSCLC patients with a high EphB4 status.

Hypoxia during early pregnancy critically regulates trophoblast function, and its dysregulation has been implicated in unexplained recurrent spontaneous abortion (URSA). This study investigated the circ-TBC1D1/miR-520 h/EPHB4 regulatory axis in HTR-8/SVneo trophoblast function under hypoxic conditions (2% O 2 ) and its association with URSA. Clinical analysis of chorionic tissues from 14 URSA patients and 14 controls revealed upregulated EPHB4 expression in URSA samples. EPHB4 suppressed proliferation, migration, and invasion while increasing apoptosis and altering angiogenesis markers (VEGFA, CD31). Mechanistically, circ-TBC1D1 acted as a competitive endogenous RNA by sequestering miR-520 h, thereby elevating EPHB4 expression and modulating HTR-8/SVneo trophoblast function under hypoxia. Additionally, downregulated miR-520 h and a non-significant trend toward elevated circ-TBC1D1 were observed in chorionic tissues from URSA patients. These findings demonstrate that the circ-TBC1D1/miR-520 h/EPHB4 axis may impair extravillous trophoblast cell functionality, and dysregulation of this pathway in URSA patients may correlate with compromised uterine spiral artery remodeling. This study highlights potential molecular targets involved in URSA pathogenesis associated with hypoxic stress.

The tyrosine kinase receptor EphB4 and its ligand ephrin-B2 interact through cell-to-cell contacts. Upon interaction, EphB4 transmits bidirectional signals. A forward signal inside EphB4-expressing cells is believed to suppress tumor growth, while inside the ephrin-expressing cells, an oncogenic reverse signal arises. In breast cancer cells with a high EphB4 receptor expression the forward signal is low, in part due to the low expression of the ligand ephrin-B2. Therefore, we hypothesized that by re-introducing the ligand in EphB4-positive cells, tumor suppression could be induced by the stimulation of the forward signal. This question was addressed in vitro by the stable lentiviral infection of breast cancer cells with either wild-type EFNB2 or with a mutant EFNB2-5F, unable to transmit reverse signaling. Furthermore, we investigated ephrin-B and EphB4 protein expression in 216 paraffin-embedded tumors using immunohistochemistry. The in vitro results indicated that ephrin-B2 expression was associated with a lower cell proliferation, migration and motility compared with the control cells. These effects were more pronounced when the cells lacked the ability to transmit the reverse signal (B2-5F). In clinical material, ephrin-B protein expression was associated with a positive estrogen receptor (ER) status, a low HER-2 expression and was negatively associated with Nottingham histologic grade (NHG) III. Ephrin-B expression indicated a good prognosis, whereas EphB4 expression was associated with a shorter metastasis-free survival in univariate and multivariate analysis. Furthermore, the prognostic value of EFNB2 and EPHB4 was confirmed at the gene expression level in public datasets. Thus, on the whole, the findings of this study suggest that ephrin-B2 expression is associated with less proliferation and lower motility of breast cancer cells and with a longer patient survival in breast cancer.

Oral squamous cell carcinoma (OSCC) represents the most common type of oral cancer, and its prognosis remains poor. In this study, we found that almost OSCC cases showed high Ephrin type‐B receptor 4 (EPHB4) expression that was mainly localized on the membrane of tumor cells. Therefore, EPHB4 represents a potential target of chimeric antigen receptor (CAR) T cell therapy for OSCC treatment. Because the oral cavity can be directly accessed, local administration of CAR‐T cells is feasible for treating OSCC. In this study, we investigated the efficacy of intratumoral injection of EPHB4‐specific CAR‐T cells in OSCC using xenograft models. To evaluate the anti‐tumor effect, the SAS OSCC cell line or an OSCC patient‐derived xenograft (PDX) tumor was subcutaneously implanted into NOD SCID gamma mice, and EPHB4‐CAR‐T cells were intratumorally injected twice. As expected, administration of CAR‐T cells suppressed tumor growth of both SAS cells and PDX tumor. EPHB4 expression in tumor tissues was attenuated by CAR‐T cell treatment, which was accompanied by a reduction in tumor area and accumulation of CAR‐T cells. Our findings suggest that intratumoral injection of EPHB4‐CAR‐T cells represents a potential therapeutic strategy for OSCC. Oral squamous cell carcinoma (OSCC), the most common type of oral cancer, has a poor prognosis. This study found high Ephrin type‐B receptor 4 (EPHB4) expression, mainly localized on the tumor cell membrane, in most OSCC cases, suggesting EPHB4 as a promising target for chimeric antigen receptor (CAR) T cell therapy. Intratumoral injection of EPHB4‐specific CAR‐T cells suppressed tumor growth in SAS and PDX mouse models, accompanied by reduced EPHB4 expression, decreased carcinoma area, and CAR‐T cell accumulation within the tumor tissues.

The important role of the EphrinB2–EphB4 signalling pathway in bone remodelling has been demonstrated, while its effect on inflammatory bone defect regeneration remains poorly understood. This study was to assess the effect of EphB4–EphrinB2 signalling on inflammation‐mediated bone defect repair in murine models. The modelling method of inflammation‐mediated bone defect in mice was established by intraperitoneally injecting different concentrations of TNF‐α. Then, three randomly assigned groups were administered vehicle (PBS, control), EphrinB2 siRNA, and EphB4 siRNA into a 1.5‐mm diameter mandibular bone defect with 5 μg/kg TNF‐α intraperitoneally injected every 2 days. The gene expression of osteogenic differentiation markers Runx2, Osterix, ALP, OCN and BSP in healing tissue of the bone defect was examined by quantitative real‐time polymerase chain reaction (PCR). Runx2 and BSP protein expressions were examined by western blot, and the decalcified tissues were subjected to histological examination. Compared with the control group, the EphB4 siRNA group mice exhibited lower levels of osteogenic differentiation markers and higher levels of the osteoclastogenic marker. H&E staining, TRACP staining and bone histomorphometry showed that the bones were thinner and the number of giant osteoclasts in the EphB4 siRNA group was higher compared with the control group, whereas there were no significant differences in osteoblastic and osteoclastic differentiation between EphrinB2 siRNA mice and control mice. In conclusion, the EphrinB2–EphB4 signalling pathway plays a critical role in the inflammation‐induced bone defect repair process; selective inhibition of EphB4 using siRNA results in decreased bone formation and increased bone resorption under high inflammatory circumstances in vivo.

Bidirectional signalling is regarded as a notable hallmark of the Eph-ephrin signalling system: Eph-dependent forward signalling in Eph-expressing cells and ephrin-dependent reverse signalling in Ephrin-expressing cells. The notion of ephrin-dependent reverse signalling derives from genetic experiments utilizing mice carrying mutations in the intracellular region of ephrinBs. Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling. We develop antibodies that selectively target EphB4 and ephrinB2. We find that mice bearing genetically altered cytoplasmic region of ephrinB2 have significantly altered EphB4-dependent forward signalling. Selective inhibition of EphB4 using a functional blocking antibody results in defective lymphatic valve development. Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development. The bidirectional Eph-ephrin signalling regulates a myriad of developmental programmes. Zhang et al . show that EphB4 forward signalling is crucial for lymphatic valve development, providing new insight into this important developmental process previously thought to be regulated by ephrinB2-dependent reverse signalling.

The significant role of VEGF (vascular endothelial growth factor) as an angiogenesis inducer is well recognized. Besides VEGF, EphrinB2/EphB4 also plays essential roles in vascular development and postnatal angiogenesis. Compared with classical proangiogenic factors, not only does EphrinB2/EphB4 promote sprouting of new vessels, it is also involved in the vessel maturation. Given their involvement in many physiologic and pathological conditions, EphB4 and EphrinB2 are increasingly recognized as attractive therapeutic targets for angiogenesis-related diseases through modulating their expression and function. Previous works mainly focused on the individual role of VEGF and EphrinB2/EphB4 in angiogenesis, respectively, but the correlation between EphrinB2/EphB4 and VEGF in angiogenesis has not been fully disclosed. Here, we summarize the structure and bidirectional signaling of EphrinB2/EphB4, provide an overview on the relationship between EphrinB2/EphB4 signaling and VEGF pathway in angiogenesis and highlight the associated potential usefulness in anti-angiogenetic therapy.