Over-expression of EphB3 enhances cell–cell contacts and suppresses tumor growth in HT-29 human colon cancer cells

Abstract

Receptor tyrosine kinase EphB3 is expressed in cells in the bottom of intestinal crypts near stem cell niches. Loss of Ephb3 has recently been reported to produce invasive colorectal carcinoma in Apc(Min/+) mice and EphB-mediated compartmentalization was demonstrated to be a mechanism suppressing colorectal cancer progression; however, it is unknown whether other factors contribute to EphB-mediated tumor suppression. EphA4-ephrin-A and EphB4-ephrin-B2 signaling have been reported to promote mesenchymal-to-epithelial transition (MET). Here, we examine whether EphB3-ephrin-B interaction has a similar effect and investigate its role in tumor suppression. We found in a clinical cohort that EphB3 expression was significantly reduced in advanced Dukes' stage tumor specimens, so we over-expressed EphB3 in HT-29 cells by stable transfection. EphB3 over-expression inhibited HT-29 growth in monolayer cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice and initiated morphological, behavioral and molecular changes consistent with MET. Specifically, EphB3 over-expression re-organized cytoskeleton (converting spreading cells to a cobble-like epithelial morphology, patterning cortical actin cytoskeleton and polarizing E-cadherin and ZO-1), induced functional changes favoring MET (decreased transwell migration, increased apoptosis and Ca(2+)-dependent cell-cell adhesion), decreased mesenchymal markers (fibronectin and nuclear beta-catenin), increased epithelial markers (ZO-1, E-cadherin and plakoglobin) and inactivated CrkL-Rac1, a known epithelial-to-mesenchymal transition signaling pathway. Additionally, cross talk from Wnt signaling potentiated the restoration of epithelial cell polarity. Noteworthily, the same factors contributing to MET, owing to EphB3 signaling, also facilitated tumor suppression. We conclude that EphB3-ephrin-B interaction promotes MET by re-establishing epithelial cell-cell junctions and such an MET-promoting effect contributes to EphB3-mediated tumor suppression.

EphB3 suppresses non-small-cell lung cancer metastasis via a PP2A/RACK1/Akt signalling complex

“…Most previous studies could not easily distinguish the contributions of Eph receptors specifically to metastasis regulation due to their mitogenic and/or antiapoptotic roles 34,38 . In contrast, our findings here show that ligand-stimulated EphB3 inhibits cell migration without exerting confounding influences on proliferation and apoptosis.…”

Class I and III HDACs and loss of active chromatin features contribute to epigenetic silencing ofCDX1andEPHBtumor suppressor genes in colorectal cancer

“…3 At the adenoma-carcinoma transition, dysplastic cells then breach this barrier and acquire tumor cell invasiveness. [4][5][6] Inactivation of the EphB-ephrin system can occur by mutational crippling or transcriptional silencing of EphB receptor genes. 2 In fact, EPHB2 and EPHB3 are transcriptionally inactive in a sizeable fraction of colorectal cancers.…”

Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer

“…tumor progression | metastasis | Ephrin signaling | EPHB2 I n colorectal tumorigenesis, signaling by the protein tyrosine kinases Ephrin type-B receptor 2 (EPHB2) and 3 (EPHB3) represents a powerful barrier against tumor-cell spreading and the onset of metastasis, the main cause for cancer-related mortality (1,2). Through repulsive interactions between cells expressing EPHB receptors and cells presenting EphrinB ligands, EPHB/ EphrinB signaling compartmentalizes tumors and locally confines their growth (3).…”

“…In addition, EPHB/EphrinB signaling affects the function of the cell-cell adhesion molecule E-cadherin, thereby adding to the stabilization of a noninvasive epithelial-cell phenotype (3). However, colorectal carcinomas frequently overcome the invasion/tumor suppressor function of the EPHB/EphrinB system by transcriptional down-regulation of receptor expression (1,2,4).…”

“…In colorectal tumors, EPHB3 exhibits biphasic expression profiles that are distinguished by a surge in expression in adenomas and secondary down-regulation in carcinomas (1,2,4). EPHB3 is a direct target of Wnt/β-catenin signaling (9), which may explain EPHB3 up-regulation in early tumorigenesis.…”