title: HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma.
abstract: 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)-1α and -2α cause adaptation of tumors to hypoxia, still with uncertainties towards the angiogenic switch. We created a stable knockdown of HIF-1α and HIF-2α 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-2α knockdown, angiogenesis was attenuated, while the knockdown of HIF-1α was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1) as a HIF-2α 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-2α knockdown (k/d) cells proved a cause-effect relation and restored angiogenesis, with no effect on control cocultures. Suggestively, HIF-2α increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. We conclude that the HIF-2α target gene PAI-1 favors the angiogenic switch in HCC.
authors.affiliation: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. Electronic address: geis@biochem.uni-frankfurt.de.
authors.forename: Theresa
authors.initials: T
authors.lastname: Geis
authors.affiliation: Dr. Senckenberg Institute of Pathology, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. Electronic address: C.Doering@em.uni-frankfurt.de.
authors.forename: Claudia
authors.initials: C
authors.lastname: Döring
authors.affiliation: Institute for Vascular Signalling, Centre for Molecular Medicine, Faculty of Medicine Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany. Electronic address: popp@vrc.uni-frankfurt.de.
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Popp
authors.affiliation: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. Electronic address: grossmann@biochem.uni-frankfurt.de.
authors.forename: Nina
authors.initials: N
authors.lastname: Grossmann
authors.affiliation: Institute for Vascular Signalling, Centre for Molecular Medicine, Faculty of Medicine Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany. Electronic address: fleming@vrc.uni-frankfurt.de.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Fleming
authors.affiliation: Dr. Senckenberg Institute of Pathology, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. Electronic address: m.l.hansmann@em.uni-frankfurt.de.
authors.forename: Martin-Leo
authors.initials: ML
authors.lastname: Hansmann
authors.affiliation: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. Electronic address: dehne@biochem.uni-frankfurt.de.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Dehne
authors.affiliation: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. Electronic address: b.bruene@biochem.uni-frankfurt.de.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Brüne
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007124:Immunoenzyme Techniques; D008113:Liver Neoplasms / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D009389:Neovascularization, Pathologic; D020411:Oligonucleotide Array Sequence Analysis; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D014408:Biomarkers, Tumor; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D000099162:Endothelial PAS Domain-Containing Protein 1; C051321:SERPINE1 protein, human
keywords: Aprotinin; Plasmin; Spheroid-embryonic body-coculture; Tumor hypoxia