Background: The EphB4 receptor tyrosine kinase is commonly over-expressed in epithelial cancers including 66% of prostate cancers, promoting tumor angiogenesis, cancer cell survival and facilitating invasion and migration within the tumor microenvironment. The intricacies of EphB4 function in the cancer cell are not completely determined, but mechanisms disrupting the balance between the EphB4 receptor and its sole physiologically relevant ligand, ephrin-B2, allow tumor promotion through ligand-independent EphB4 forward signaling. Such mechanisms may include extracellular proteolytic cleavage, and our in silico data predicts that EphB4 is a substrate of the kallikrein-related peptidase 4 (KLK4), a serine protease also commonly elevated in prostate cancers, with major interactions within the tumor microenvironment. KLK4 has particularly strong expression in tumors that have metastasized to bone, and is reported to have significant functional roles in prostate cancer progression.
Methods: Recombinant proteins were used to confirm a biochemical interaction between KLK4 and EphB4. Cell-based assays were used to determine the biological consequences of protease cleavage of EphB4 expressed by cancer cells.
Results: KLK4 cleaved EphB4 after arginine-508 in the extracellular domain, close to the transmembrane domain. The remaining membrane-tethered intracellular fragment was then further processed by the action of the protease, γ-secretase. When this intracellular C-terminal fragment was over-expressed in prostate cancer cells, the cell morphology was altered and this correlated with nuclear translocation of the C-terminal fragment, via β-importin interaction, and increased expression of LEF1 - a gene that encodes a key transcription factor in the Wnt-β catenin pathway linked to EMT in androgen-independent prostate cancer.
Conclusions: This study has not only identified a new substrate for the prostate cancer-associated protease KLK4, but has also provided, to our knowledge, the first direct evidence for serine protease-mediated cleavage of the EphB4 receptor, with significant implications for the mechanism of action for the known tumor-promoting effects of EphB4.