2048

EPHB2

CAPB|DRT|EK5|EPHT3|ERK|Hek5|PCBC|Tyro5;EPH receptor B2;EPHB2;EPH receptor B2

EPH-like kinase 5|developmentally-regulated Eph-related tyrosine kinase|elk-related tyrosine kinase|eph tyrosine kinase 3|ephrin type-B receptor 2|protein-tyrosine kinase HEK5|renal carcinoma antigen NY-REN-47|tyrosine-protein kinase TYRO5|tyrosine-protei

1p36.1-p35

protein-coding

Count: 3; Pubmed_search,Generif,UniProt

Receptor tyrosine kinase EphB2 and autophagic machinery are known as tumor suppressors; however, the connection remains to be elucidated. Here, we show the link between EphB2 and autophagy. Sesamin, a major lignan in sesame oil, induced autophagy in the human colon cancer cell lines HT29 and LS180, as shown by electron microscopy, as well as Western blotting and immunofluorescence imaging using an anti-LC3 antibody. Receptor tyrosine kinase array analysis revealed that sesamin treatment increased the levels of unphosphorylated -EphA1 and -EphB2 in HT29 cells. Silencing of EphA1 and EphB2 blocked sesamin-induced autophagy as well as sesamin-induced loss of cell viability. These results show that EphA1 and EphB2 play a critical role in this process. The present study reveals a novel function for EphA1 and EphB2 in the induction of autophagy, suggesting a tumor suppressor role for these proteins in colorectal cancer.

Several studies have reported the change of EphB2 in a variety of carcinomas and suggested a functional relation between EphB2 and tumor progression. However, its role in human pancreatic carcinoma has not been described. The aim of this study was to evaluate the significance of EphB2 in human pancreatic carcinoma CFPAC-1 cells. A lentivirus-based RNA interference (RNAi) vector was designed, synthesized and transfected into CFPAC-1 cells to inhibit EphB2 expression. WST-8 based Colorimetric Assay Cell Counting kit 8 (CCK-8) in vitro and xenograft transplantation model in nude mice was used to evaluate cell proliferation and growth respectively. Cell-cycle and apoptosis were analyzed by flow cytometry (FCM). RT-PCR and Western blot were used to assess mRNA expression and protein levels. EphB2 expression was significantly suppressed both in mRNA and protein levels using the lentivirus-based EphB2 RNAi in CFPAC-1 cells (P<0.01, P<0.01). Silencing EphB2 stimulated cell growth in vitro (P<0.05) and proliferation in vivo (P<0.01) versus Control RNAi. EphB2 RNAi significantly increased S phase cells from 18.15 to 27.18% (P<0.05), and significantly decreased G1 phase cells from 72.93 to 57.61% compared with Control RNAi (P<0.05). In addition, decreased apoptosis was observed in CFPAC-1 EphB2 RNAi cells compared with Control RNAi cells (P<0.01). The apoptosis rate was 1.63% and 7.44%, respectively. Silencing EphB2 increased CyclinD1, cyclindependent kinase 6 (CDK6) and Bcl-2 expression in both mRNA and protein levels compared with Control RNAi. A lentivirus-based EphB2 RNAi efficiently inhibited EphB2 gene and its protein expression. Silencing EphB2 stimulated pancreatic carcinoma growth by increasing cell proliferation through G1/S phase breakthrough, which relied on a CyclinD1/CDK6 cell-cycle regulated signal. Similarly, EphB2 inhibition also reduced CFPAC-1 cells apoptosis by up-regulating Bcl-2 expression. Thus, at least in the context of pancreatic carcinoma CFPAC-1 cells, EphB2 plays a tumor suppressor role in cell proliferation and apoptosis.CI - Copyright (c) 2011 Elsevier Masson SAS. All rights reserved.

EphB2 is a tyrosine kinase receptor that has been shown to be a tumor suppressor gene in various cancers. However the mechanisms of this function are unknown. We report that EphB2 induces a form of cell death that does not involve the formation of apoptotic bodies or nuclear fragmentation and is instead accompanied by extensive vacuolization. Transmission electron microscopy demonstrates cytoplasmic vacuoles in EphB2-overexpressing cells that resembled autophagosomes. Using an EYFP-LC3 fusion protein and immunoblotting, we detected LC3 aggregation and conversion from form I to form II, both hallmarks of autophagy, in EphB2-transfected cells. Silencing of the autophagy regulating genes ATG5 or ATG7 using shRNAs, strongly prevented EphB2-induced cell death, further confirming its autophagic nature. EphB2 expression results in mitochondrial depolarization and translocation of cytochrome c from the mitochondria to the cytosol. Mapping of signaling pathways revealed novel information about the mechanisms of action of EphB2. We demonstrated that the MAPK pathway is important in the pro-death action of EphB2, through ERK1/2 phosphorylation and inhibition of this pathway using PD98059 counters EphB2-driven cell death. In addition, we found that inhibition of class III PI3K pathway, using the autophagy inhibitor 3MA, but not class I PI3K inhibition using LY294002, also effectively blocks EphB2- induced cell death. Finally, EphB2 expression inactivates Akt, which is a known inhibitor of autophagy. In conclusion, the EphB2 receptor induces an autophagic cell death that is mediated through the ERK1/2 and PI3K/Akt pathways.

Familial clustering of colorectal cancer occurs in 15-20% of cases, however recognized cancer syndromes explain only a small fraction of this disease. Thus, the genetic basis for the majority of hereditary colorectal cancer remains unknown. EPHB2 has recently been implicated as a candidate tumor suppressor gene in colorectal cancer. The aim of this study was to evaluate the contribution of EPHB2 to hereditary colorectal cancer. We screened for germline EPHB2 sequence variants in 116 population-based familial colorectal cancer cases by DNA sequencing. We then estimated the population frequencies and characterized the biological activities of the EPHB2 variants identified. Three novel nonsynonymous missense alterations were detected. Two of these variants (A438T and G787R) result in significant residue changes, while the third leads to a conservative substitution in the carboxy-terminal SAM domain (V945I). The former two variants were found once in the 116 cases, while the V945I variant was present in 2 cases. Genotyping of additional patients with colorectal cancer and control subjects revealed that A438T and G787R represent rare EPHB2 alleles. In vitro functional studies show that the G787R substitution, located in the kinase domain, causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function and likely represents a neutral polymorphism. Tumor tissue from the G787R variant case manifested loss of heterozygosity, with loss of the wild-type allele, supporting a tumor suppressor role for EPHB2 in rare colorectal cancer cases. Rare germline EPHB2 variants may contribute to a small fraction of hereditary colorectal cancer.