ABSTRACT
Members of the Eph family of receptor tyrosine kinases have been implicated in a wide array of human cancers. The EphB4 receptor is ubiquitously expressed in head and neck squamous cell carcinoma (HNSCC) and has been shown to impart tumorigenic and invasive characteristics to these cancers. In this study, we investigated whether EphB4 receptor targeting can enhance the radiosensitization of HNSCC. Our data show that EphB4 is expressed at high to moderate levels in HNSCC cell lines and patient-derived xenograft (PDX) tumors. We observed decreased survival fractions in HNSCC cells following EphB4 knockdown in clonogenic assays. An enhanced G2 cell cycle arrest with activation of DNA damage response pathway and increased apoptosis was evident in HNSCC cells following combined EphB4 downregulation and radiation compared to EphB4 knockdown and radiation alone. Data using HNSCC PDX models showed significant reduction in tumor volume and enhanced delay in tumor regrowth following sEphB4-HSA administration with radiation compared to single agent treatment. sEphB4-HSA is a protein known to block the interaction between the EphB4 receptor and its ephrin-B2 ligand. Overall, our findings emphasize the therapeutic relevance of EphB4 targeting as a radiosensitizer that can be exploited for the treatment of human head and neck carcinomas.
Subject(s)
Carcinoma, Squamous Cell/enzymology , Head and Neck Neoplasms/enzymology , Neoplasm Proteins/physiology , Receptor, EphB4/physiology , Animals , Apoptosis , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/radiotherapy , Cell Line, Tumor/radiation effects , DNA Repair , G2 Phase Cell Cycle Checkpoints , Gene Knockdown Techniques , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/radiotherapy , Humans , Keratinocytes/enzymology , Mice , Molecular Targeted Therapy , Neoplasm Proteins/deficiency , RNA Interference , RNA, Small Interfering/genetics , Radiation Tolerance , Receptor, EphB4/deficiency , Tumor Burden , Tumor Stem Cell Assay , Xenograft Model Antitumor AssaysABSTRACT
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. 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.
Subject(s)
Ephrin-B2/metabolism , Lymphangiogenesis , Neovascularization, Physiologic , Vascular Endothelial Growth Factor C/metabolism , Animals , Cells, Cultured , Embryo Loss , Embryo, Mammalian/blood supply , Embryo, Mammalian/metabolism , Endocytosis , Endothelial Cells/cytology , Endothelial Cells/metabolism , Ephrin-B2/deficiency , Ephrin-B2/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Lymphangiogenesis/genetics , Lymphatic Vessels , Mice , Mice, Transgenic , Neovascularization, Physiologic/genetics , Neuropeptides/metabolism , Pregnancy , Proto-Oncogene Proteins c-akt/metabolism , Receptor, EphB4/deficiency , Receptor, EphB4/genetics , Receptor, EphB4/metabolism , Signal Transduction , Vascular Endothelial Growth Factor Receptor-3/metabolism , Zebrafish , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding ProteinABSTRACT
Differentiation of pluripotent embryonic stem (ES) cells is associated with expression of fate-specifying gene products. Coordinated development, however, must involve modifying factors that enable differentiation and growth to adjust in response to local microenvironmental determinants. We report here that the ephrin receptor, EphB4, known to be spatially restricted in expression and critical for organized vessel formation, modifies the rate and magnitude of ES cells acquiring genotypic and phenotypic characteristics of mesodermal tissues. Hemangioblast, blood cell, cardiomyocyte, and vascular differentiation was impaired in EphB4-/- ES cells in conjunction with decreased expression of mesoderm-associated, but not neuroectoderm-associated, genes. Therefore, EphB4 modulates the response to mesoderm induction signals. These data add differentiation kinetics to the known effects of ephrin receptors on mammalian cell migration and adhesion. We propose that modifying sensitivity to differentiation cues is a further means for ephrin receptors to contribute to tissue patterning and organization.