HIF-1 steuert die Angioinvasion und Metastasierung durch Regulation der uPAR — Genexpression beim Pankreaskarzinom

Abstract

Angioinvasion is the rate limiting step in metastatic tumor progression. Among the very few proteins ultimately necessary for angioinvasion is urokinase plasminogen activator receptor (uPAR). Hypoxia-Inducible-Factor-1 (HIF-1) is found to be activated during progression of human pancreatic cancer, a tumor which grows in a microenvironment of low oxygen. However, it is not know whether a relationship between HIF-1 and uPAR exists. Transcriptional control of uPAR expression by HIF-1 has never been reported. AimThe aims of this study were to analyze whether HIF-1 transcriptionally regulates uPAR and to analyze the role of hypoxia in angioinvasion and metastasis formation. MethodsFour human pancreatic cancer cell lines Capan-2 (C2), HPAF-2 (HP2), MIA PaCa-2 (MP2) and PANC-1 were used. Northern blot analysis was used to quantify mRNA expression and a nuclear run of assay to determine newly transcribed mRNA. DNA binding was studied with gelshift assays. PCR generated deletion mutants of the uPAR promotor were used in Luciferase assays to determine reporter gene activity. To study angioinvasion of PaCa cell lines Matrigel invasion assays and fertilized chicken eggs were used for chorioallantoic membrane (CAM) assays. ResultsuPAR but not uPA mRNA expression is upregulated by low oxygen levels. Cell lines not expressing uPAR under normoxic (C2) conditions reconstitute uPAR production under hypoxia. Actual hypoxia induced uPAR mRNA transcription. Transcriptional activation of HIF-1 by binding to the uPAR promotor was detectable within the first hour after onset of hypoxia. Promotor deletion mutants containing the putative HIF-1 binding site showed a significant increase in reporter gene activity. In vitro invasion assays revealed that hypoxia increased the relative number of invading tumor cells. Moreover, hypoxia was a strong uPAR dependent stimulus in angioinvasion assays in vivo in all cell lines tested. ConclusionIn this study we present for the first time, that uPAR is under transcriptional control of HIF-1 and further show that hypoxic upregulation of uPAR is caused by an actual increase in transcription of uPAR mRNA synthesis. We further show that this mechanism also increased the potential of metastatic growth of pancreatic cancer, both in vitro and in vivo in an uPAR dependent manner.