The EphB4 receptor tyrosine kinase is over-expressed in several epithelial cancers including prostate (PCa), and is involved in cell survival, migration and angiogenesis. We recently reported that immunoreactive EphB4 is present in PCa cell nuclei and that two functional nuclear localisation signals and co-immunoprecipitation with importin-alpha, provides a potential mechanism for nuclear translocation. EphB4-specific antibodies, directed to either the C- or N-terminus, identified specific fragments of EphB4 in PCa cell lysates and nuclear extracts, which we predicted were generated by sequential proteolytic cleavage events releasing both extracellular (ECF – 70 kDa) and intracellular (ICF – 47 kDa) fragments.
In silico proteomic modelling identified the PCa-associated Kallikrein-like peptidase 4 (KLK4) as a candidate protease responsible for the first cleavage event. This was confirmed by treating cells with recombinant KLK4 and a specific KLK4 inhibitor. Involvement of a second protease, γ-secretase, often linked to further processing of cleaved surface proteins, was indicated using the specific inhibitor, Compound E. Subcellular fractionation of the KLK4-positive PCa cell line LNCaP identified the ICF fragment in the nucleus. Treatment with ivermectin, an inhibitor of importin-alpha-mediated nuclear transport, reduced the nuclear presence of the ICF. DU145 and PC3 cells engineered to over-express the ICF were more mesenchymal-like in morphology compared to vector-only control cells, and had reduced E-cadherin, suggesting an epithelial to mesenchymal transition. Chromatin immunoprecipitation (ChIP) implicated nuclear EphB4 in gene regulatory functions and several potential EphB4-binding genomic loci were identified. Lef1, a gene linked to PCa, is a potential target of EphB4-mediated gene regulation. This study provides the first evidence for proteolytic regulation of EphB4 in PCa and for unique mechanisms of action which may provide novel insights into how EphB4 contributes to PCa. Development of strategies for inhibition of proteolysis and/or nuclear translocation may prove to be a potential novel avenue for anti-cancer therapies.