EphrinB2/EphB4 Interaction Promotes Myeloid Leukemia Cell Invasion through RhoA-Mediated Mechanism

Background and Objective: Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrinB ligands in a variety of tumors, suggesting a functional relation between EphB/ephrinB signaling and tumor progression. However, how they regulate the invasiveness of myeloid leukemia cells were still unknown. Our previously study suggested that EphB4 were highly expressed in patients with extramedullary leukemia compared with patients without extramedullary leukemia, which indicated that the expression of EphB4 was related with myeloid leukemia cell invasion. To address the molecular mechanism, we aimed to characterize the role of EphB4 and ephrinB2 ligands in the interaction of myeloid leukemia cells.

Methods: To clarify the question, myeloid leukemia cell lines (K562 cells and THP-1 cells) treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins were cultured in vitro, then migration and invasion were determined by transwell assay according to different time. Pulldown western immunoblot analysis were used to detect the level of GTP-RhoA and total RhoA; the phosphorylation of EphB4 and MMP9 expression were also determined by immunoblot analysis before and after the treatment of different clustered Fc proteins.

Results: The results showed that after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (1.85-fold, P=0.033), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.46 -fold, P=0.025). However, the numbers of K562 cells migrating through transwell chamber after ephrinA1–Fc stimulation didn’t significantly increase compared to Fc proteins stimulation (P=0.411), and the numbers of K562 cells invading the matrigel also didn’t enhanced (P=0.072) after ephrinA1–Fc stimulation. Moreover, after ephrinB2–Fc stimulation, the numbers of THP-1 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (2.25-fold, P<0.01), meanwhile, the numbers of THP-1 cells invading the matrigel also enhanced (1.66 -fold, P<0.01). However, the numbers of THP-1 cells migrating through transwell chamber and the numbers of THP-1 cells invading the matrigel didn’t significantly enhanced (P>0.05, P>0.05) after ephrinA1–Fc stimulation. Furthermore, EphB4 immunoprecipitation followed by immunoblotting with anti-phosphotyrosine antibody revealed that EphB4 is phosphorylated on tyrosine in K562 cells after ephrinB2–Fc stimulation. Additionally, the level of active RhoA (GTP-RhoA) and MMP9 in K562 cells were both significantly increased in response to EphB4 receptor activation with its ligand ephrin-B2-Fc ( P<0.05).

Conclusions: These findings suggested that EphB4/EprinB2 signaling played an important role in myeloid leukemia cells progression by promoting their migratory ability, activating RhoA activity and increasing MMP9 expression. Our findings reveal a novel regulation of this intriguing receptor/ligand family that contributes to the cell invasiveness of myeloid leukemia cells.