EphB4 is a member of the largest family of receptor
tyrosine kinases and is commonly over-expressed in most epithelial cancers,
including 66% of prostate cancers where it has been shown to promote tumour
angiogenesis, increase cancer cell survival and facilitate cell invasion and
migration. Recent studies have suggested that this is via a ligand-independent process, but how the EphB4 protein is
regulated at the surface of the prostate cancer cell is unclear. We have
identified prostate cancer-associated EphB4 cleavage events using an
EphB4-over-expression model of the prostate cancer cell line 22Rv1 and have
identified the kallikrein-related peptidase 4 (KLK4) as a possible mediator of this cleavage. KLK4 is a serine protease that is commonly
elevated in prostate cancers, with strong expression seen in tumours that have
metastasised to the bone. The ability of
KLK4 to cleave EphB4 was confirmed using recombinant proteins. Recombinant KLK4 was also used to demonstrate
cleavage of EphB4 present on the surface of prostate cancer cells. The primary cleavage site was determined by
N-terminal sequencing to be after R507, in the juxtamembrane extracellular
domain, consistent with the identified fragments of 70 and 50 kDa. A second
C-terminal fragment of 47 kDa was also generated, possibly as a consequence of
ectodomain shedding, with preliminary evidence suggesting this is via the
action of the intracellular protease, γ-secretase. This study has not
only revealed a new substrate for the KLK4 protease, whose strong link to prostate
cancer has been long known, but whose mechanistic contribution still remains
poorly understood, but has also identified a possible mechanism
for the regulation of EphB4
signaling in prostate cancer and therefore the regulation of the
ligand-independent tumour progressive actions of EphB4.