Die Rolle von CUX1 bei Hypoxie im hepatozellulären Karzinom

Hepatocellular carcinoma is characterised by a hypoxic environment. Adaption of tumor cells to hypoxia is mediated by the central transcription factor HIF-1α that leads to angiogenesis as well as adaptation of metabolism and cellular proliferation.The transcription factor CUX1 (CUTL1) is part of the homeodomain gene family. It is responsible for mechanisms of cellular proliferation and differentiation. In tumor cells, it has been linked to increased invasion, migration and resistance to apoptosis. Its exact role in tumorgenesis is not completely clarified yet.The aim of this study is the demonstration of a link between CUX1 and the adaptation mechanisms to hypoxia in hepatocellular carcinoma cell lines. It was attempted to transiently knock down CUX1 by siRNA in HepG2 and Hep3B cells. A Knock down of CUX1 transcript was repeatedly observerved by RNA analysis although not statistically confirmed and not supported by a clear Westen Blot. Hypoxia was induced by treatment with 150 µM CoCl2 or incubation with 0.5% oxygen atmosphere. CUX1 as well as markers of hypoxia and cellular stress were detected by q-RT-PCR, western blot and antibody array. The influence of CUX1 on the transcriptional activity of HIF-1α was analyzed by luciferase assay using co-transfected luciferase plasmid and CUX1-siRNA. Here, the measured light intensity of the HIF-α binding sequence HRE correlated with the activity of HIF1-α, which was compared in cells with and without CUX1 knock-down. Whilst no influence of CUX1 on HIF-1α expression was detected, a decreased expression of HIF-1α target genes GLUT1 and VEGFA after knock-down of CUX1 in RT-qPCR was observed. The antagonist to HIF-1α, FIH-1, showed up-regulation after knock-down of CUX1 in HepG2 cells. CUX1 suppression impaired the transcriptional activity of HIF-1α on its ectopic promoter sequence transfected in HCC cells. Moreover, after knock-down of CUX1, a heightened expression of cyclin-dependent kinase inhibitors p21 (CDKNA1) and p27 (CDKNA2) was observed. Generally, CUX1 knock�down seamed to decrease the cell stress reaction in both cell lines and thus seamed to lead to increased sensitivity to hypoxic cytotoxicity, especially in the p53-null Hep3B cell line. It can be speculated that CUX1 plays an important role during hypoxia response in hepatocellular carcinoma. This is probably achieved by sustaining the transcriptional activity of HIF-1α, most likely by regulation of its inhibitor FIH-1. At the same time, CUX1 seemed to counteract cell cycle inhibition and could then promote cell proliferation despite the toxicity caused by hypoxia. These results support the function of CUX1 as an oncogene, as discussed in literature. However further experimental reproduction and a statistical confirmation of the observed effects is needed.