ABSTRACT
The tyrosine kinase receptor EphB4 and its ligand ephrinB2 interact through celltocell contacts. Upon interaction, EphB4 transmits bidirectional signals. A forward signal inside EphB4expressing cells is believed to suppress tumor growth, while inside the ephrinexpressing 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 ephrinB2. Therefore, we hypothesized that by reintroducing the ligand in EphB4positive 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 wildtype EFNB2 or with a mutant EFNB25F, unable to transmit reverse signaling. Furthermore, we investigated ephrinB and EphB4 protein expression in 216 paraffinembedded tumors using immunohistochemistry. The in vitro results indicated that ephrinB2 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 (B25F). In clinical material, ephrinB protein expression was associated with a positive estrogen receptor (ER) status, a low HER2 expression and was negatively associated with Nottingham histologic grade (NHG) III. EphrinB expression indicated a good prognosis, whereas EphB4 expression was associated with a shorter metastasisfree 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 ephrinB2 expression is associated with less proliferation and lower motility of breast cancer cells and with a longer patient survival in breast cancer.
Subject(s)
Breast Neoplasms/mortality , Breast/pathology , Ephrin-B2/metabolism , Neoplasm Recurrence, Local/diagnosis , Receptor, EphB4/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Cell Movement , Cell Proliferation , Datasets as Topic , Disease-Free Survival , Ephrin-B2/analysis , Ephrin-B2/genetics , Female , Humans , Kaplan-Meier Estimate , MCF-7 Cells , Neoplasm Recurrence, Local/pathology , Prognosis , RNA, Small Interfering/metabolism , Randomized Controlled Trials as Topic , Receptor, EphB4/analysis , Receptor, EphB4/genetics , Time FactorsABSTRACT
The EPH and ephrins function as both receptor and ligands and the output on their complex signaling is currently investigated in cancer. Previous work shows that some EPH family members have clinical value in breast cancer, suggesting that this family could be a source of novel clinical targets. Here we quantified the mRNA expression levels of EPH receptors and their ligands, ephrins, in 65 node positive breast cancer samples by RT-PCR with TaqMan® Micro Fluidics Cards Microarray. Upon hierarchical clustering of the mRNA expression levels, we identified a subgroup of patients with high expression, and poor clinical outcome. EPHA2, EPHA4, EFNB1, EFNB2, EPHB2 and EPHB6 were significantly correlated with the cluster groups and particularly EPHB2 was an independent prognostic factor in multivariate analysis and in four public databases. The EPHB2 protein expression was also analyzed by immunohistochemistry in paraffin embedded material (cohort 2). EPHB2 was detected in the membrane and cytoplasmic cell compartments and there was an inverse correlation between membranous and cytoplasmic EPHB2. Membranous EPHB2 predicted longer breast cancer survival in both univariate and multivariate analysis while cytoplasmic EPHB2 indicated shorter breast cancer survival in univariate analysis. Concluding: the EPH/EFN cluster analysis revealed that high EPH/EFN mRNA expression is an independent prognostic factor for poor survival. Especially EPHB2 predicted poor breast cancer survival in several materials and EPHB2 protein expression has also prognostic value depending on cell localization.