Cyclin-dependent kinase 5 stabilizes hypoxia-inducible factor-1α: a novel approach for inhibiting angiogenesis in hepatocellular carcinoma

// Julia Herzog 1 , Sandra M. Ehrlich 1 , Lisa Pfitzer 1 , Johanna Liebl 1 , Thomas Frohlich 2 , Georg J. Arnold 2 , Wolfgang Mikulits 3 , Christine Haider 3 , Angelika M. Vollmar 1 and Stefan Zahler 1 1 Department of Pharmacy, Pharmaceutical Biology, University of Munich, Munich, Germany 2 Laboratory for Functional Genome Analysis (LAFUGA), Gene Center Munich, University of Munich, Munich, Germany 3 Department of Medicine I, Institute of Cancer Research, Comprehensive Cancer Center Vienna, Medical University of Vienna, Vienna, Austria Correspondence to: Stefan Zahler, email: // Keywords : angiogenesis, HIF-1α, CDK5, HCC Received : September 17, 2015 Accepted : March 16, 2016 Published : March 24, 2016 Abstract We recently introduced CDK5 as target in HCC, regulating DNA damage response. Based on this and on our previous knowledge about vascular effects of CDK5, we investigated the role of CDK5 in angiogenesis in HCC, one of the most vascularized tumors. We put a special focus on the transcription factor HIF-1α, a master regulator of tumor angiogenesis. The interaction of CDK5 with HIF-1α was tested by Western blot, PCR, reporter gene assay, immunohistochemistry, kinase assay, co-immunoprecipitation, mass spectrometry, and mutation studies. In vivo , different murine HCC models, were either induced by diethylnitrosamine or subcutaneous injection of HUH7 or HepG2 cells. The correlation of vascular density and CDK5 was assessed by immunostaining of a microarray of liver tissues from HCC patients. Inhibition of CDK5 in endothelial or HCC cells reduced HIF-1α levels in vitro and in vivo , and transcription of HIF-1α target genes ( VEGFA , VEGFR1 , EphrinA1 ). Mass spectrometry and site directed mutagenesis revealed a stabilizing phosphorylation of HIF-1α at Ser687 by CDK5. Vascular density was decreased in murine HCC models by CDK5 inhibition. In conclusion, inhibiting CDK5 is a multi-modal systemic approach to treat HCC, hitting angiogenesis, as well as the tumor cells themselves.