HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma

Hypoxia promotes progression of hepatocellular carcinoma (HCC), not only affecting tumor cell proliferation and invasion, but also angiogenesis and thus, increasing the risk of metastasis. Hypoxia inducible factors (HIF)-1 alpha and -2 alpha cause adaptation of tumors to hypoxia, still with uncertainties towards the angiogenic switch. We created a stable knockdown of HIF-1 alpha and HIF-2 alpha in HepG2 cells and generated cocultures of HepG2 spheroids with embryonic bodies as an in vitro tumor model mimicking the cancer microenvironment. The naturally occuring oxygen and nutrient gradients within the cocultures allow us to question the role of distinct HIF isoforms in regulating HCC angiogenesis. In cocultures with a HIF-2 alpha knockdown, angiogenesis was attenuated, while the knockdown of HIF-1 alpha was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1) as a HIF-2 alpha target gene in HepG2 cells. The knockdown of PAI-1 in HepG2 cells also lowered angiogenesis. Blocking plasmin, the downstream target of PAI-1, with aprotinin in HIF-2 alpha knockdown (k/d) cells proved a cause-effect relation and restored angiogenesis, with no effect on control cocultures. Suggestively, HIF-2 alpha increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. We conclude that the HIF-2 alpha target gene PAI-1 favors the angiogenic switch in HCC. (C) 2014 Elsevier Inc. All rights reserved.