Asparaginyl endopeptidase induces endothelial permeability and tumor metastasis via downregulating zonula occludens protein ZO-1

Zona occludens-1 (ZO-1) is a key component of tight junctions that govern the function of the endothelial barrier against tumor metastasis. Factors secreted by tumor cells contribute to the maintenance of tumor vascular networks. How tumor cell-derived protein signals regulate ZO-1 expression is unclear. Here, we explored the effect of tumor cell -secreted asparaginyl endopeptidase (AEP) on the permeability of endothelial cells in the tumor microenvironment. First, we confirmed the existence of AEP in conditioned medium (CM) from AEP-overexpressing MDA-MB-231 and 4T1 cells. Treatment with CM from AEP-overexpressing tumor cells increased the permeability and tumor cell transversal of an endothelial monolayer. Furthermore, CM from AEP-overexpressing tumor cells suppressed endothelial ZO-1 expression, as well as ZO-1-associated nucleic acid binding protein ZONAB. In addition, the level of phosphorylated STAT3 was increased by treatment with AEP-containing CM. A mutation of RGD or blocking integrin alpha v beta 3 with antibody recovered the ZO-1 downregulation induced by AEP. In vivo, a lung metastatic mouse model showed increased endothelial permeability in the AEP-overexpressing group compared with the control group. An orthotopic tumor transplantation model was established using AEP-overexpression and compared with mice receiving control 4T1 cells. Compared with controls, over expression of AEP increased lung metastatic foci and area, as well as vascular instability in primary tumors or lung metastatic sites. Moreover, endothelial ZO-1 was decreased in the AEP-overexpressing group. Taken together, our data show that tumor cell-derived AEP increases the permeability of endothelial barriers. Interactions between RGD and endothelial integrin alpha v beta 3 mediate this effect by downregulating ZO-1.