ADAM9 contributes to vascular invasion in pancreatic ductal adenocarcinoma

Abstract

A disintegrin and a metalloprotease (ADAM)-9 is a metzincin cell-surface protease with strongly elevated expression in solid tumors, including pancreatic ductal adenocarcinoma (PDAC). In this study, we performed immunohistochemistry (IHC) of a tissue microarray (TMA) to examine the expression of ADAM9 in a cohort of >100 clinically annotated PDAC cases. We report that ADAM9 is prominently expressed by PDAC tumor cells, and increased ADAM9 expression levels correlate with poor tumor grading (P = 0.027) and the presence of vasculature invasion (P = 0.017). We employed gene expression silencing to generate a loss-of-function system for ADAM9 in two established PDAC cell lines. In vitro analysis showed that loss of ADAM9 does not impede cellular proliferation and invasiveness in basement membrane. However, ADAM9 plays a crucial role in mediating cell migration and adhesion to extracellular matrix substrates such as fibronectin, tenascin, and vitronectin. This effect appears to depend on its catalytic activity. In addition, ADAM9 facilitates anchorage-independent growth. In AsPC1 cells, but not in MiaPaCa-2 cells, we noted a pronounced yet heterogeneous impact of ADAM9 on the abundance of various integrins, a process that we characterized as post-translational regulation. Sprout formation of human umbilical vein endothelial cells (HUVECs) is promoted by ADAM9, as examined by transfer of cancer cell conditioned medium; this finding further supports a pro-angiogenic role of ADAM9 expressed by PDAC cancer cells. Immunoblotting analysis of cancer cell conditioned medium highlighted that ADAM9 regulates the levels of angiogenic factors, including shed heparin-binding EGF-like growth factor (HB-EGF). Finally, we carried out orthotopic seeding of either wild-type AsPC-1 cells or AsPC-1 cells with silenced ADAM9 expression into murine pancreas. In this in vivo setting, ADAM9 was also found to foster angiogenesis without an impact on tumor cell proliferation. In summary, our results characterize ADAM9 as an important regulator in PDAC tumor biology with a strong pro-angiogenic impact.

Keywords: ADAM9; adhesion; angiogenesis; heparin-binding EGF-like growth factor; migration.