br Author Contributions br Acknowledgments This work was sup
Acknowledgments This work was supported, in part, by a grant from the National Cancer Institute of the National Institutes of Health under Grant U01CA202241 and National Institutes of Health under Grant R01NS083856.
Introduction The Eph (erythropoietin-producing human hepatoma) family is the largest among transmembrane receptor tyrosine kinases (RTKs). Fifteen Eph RTKs are classified into two subclasses based on their structural features and binding affinities to various ephrin ligands (Lemke, 1997). The Eph receptors and their membrane-anchored ephrin ligands regulate tumorigenesis and angiogenesis (Heroult et al., 2006). Among the EphB receptors, increased Chlorothiazide of EphB4 has been reported in various tumour types, including colon (Kumar et al., 2009), prostate (Li et al., 2015), breast (Brantley-Sieders et al., 2011), oesophageal (Hu et al., 2014), pancreatic (Bai et al., 2014), lung (Ferguson et al., 2013), and mesothelioma (Liu et al., 2013), but with low or no expression in most normal tissues. As for CRC tissue, EphB4 was shown to be expressed in all 102 CRC specimens analysed, but was absent in normal colon mucosae, and its expression level was correlated with higher tumour grade and stage. Additionally, EphB4 inhibition decreased tumour growth and metastasis (Kumar et al., 2009). Another study analysing expression of all Eph and ephrin genes in 153 clinical specimens and CRC cell lines showed that EphA1, EphA2, EphB2, and EphB4 were all significantly overexpressed in CRC (Herath et al., 2012). Other studies showed that EphB4 inactivation resulted in higher proliferation of both intestinal tumours and normal epithelium (Dopeso et al., 2009). These opposing conclusions illustrate why the functional role of EphB4 in colorectal tumours remains undefined. EphB4 and EphrinB2 play critical roles in the development of the vascular system and contribute to vascular function (Pitulescu and Adams, 2010). Research concerning the angiogenic role of EphB4 mainly focuses on embryonic and retinal vasculogenesis (Katsuta et al., 2013, Pitulescu et al., 2010, Xue et al., 2014). The role of EphB4 signalling in CRC has only recently been elucidated, and there are limited studies focusing on the relationship between microvessel density in human CRC and EphB4 expression. In a preliminary study, we stably transfected EphB4 overexpression and knockdown lentiviral vectors into CRC cell line SW480, and in vitro experiments showed that EphB4 overexpression promoted cell proliferation and stimulated migration and invasion, while EphB4 knockdown resulted in reduced cell viability, migration, and invasion. Here, we evaluated the expression of EphB4 and EphrinB2 using tissue microarrays from 200 colorectal tumours by immunohistochemistry. Furthermore, we performed in vivo experiments using xenograft models to investigate the role of EphB4 in CRC proliferation, invasion, and angiogenesis.
Material and methods
Discussion As a family of RTKs, nine types of EphA receptors (EphA1–8 and EphA10) and six types of EphB receptors (EphB1–B6) have been identified. Corresponding ligands include six types of EphrinA (EphrinA1–6) and three types of EphrinB (EphrinB1–3) (Zhang and Hughes, 2006). They are frequently reported to regulate the proliferation, migration, and attachment of tumour cells, and regulate numerous developmental processes, particularly in the vasculature and nervous system. For EphB receptors, EphB4 is reported to potentially exert an oncogenic effect and is an important regulator of fundamental physiological and pathophysiological processes, such as tissue patterning, during development, angiogenesis, and tumour progression (Lisabeth et al., 2013). Wu Q showed that high levels of EphB2 and EphB4 protein expression were associated with poor prognosis in patients with ovarian cancer (Wu et al., 2006). EphB4 was overexpressed three-fold in lung tumours, and overexpression promoted cellular proliferation, colony formation, and the motility of lung cancer cell line H661 (Ferguson et al., 2013). In oesophageal squamous cell carcinoma (ESCC), EphB4 expression was upregulated in tumour tissues, while EphrinB2 was downregulated in ESCC samples compared with paired normal tissues (Liu et al., 2013). EphB4 expression was also significantly higher in soft tissue sarcomas, and both mRNA and protein expression were markedly increased in synovial sarcoma (Becerikli et al., 2015).