Hypoxia Accelerates Aggressiveness of Hepatocellular Carcinoma Cells Involving Oxidative Stress, Epithelial-Mesenchymal Transition and Non-Canonical Hedgehog Signaling.

BACKGROUND/AIMS: Hypoxic microenvironment, a common feature of hepatocellular carcinoma (HCC), can induce HIF-1α expression and promote the epithelial-mesenchymal transition (EMT) and invasion of cancer cells. However, the underlying molecular mechanisms have not fully elucidated.

METHODS

HCC cells were cultured under controlled hypoxia conditions or normoxic conditions. Transwell assays were used to examine the migration and invasion capacity. HIF-1α siRNA, cyclopamine (a SMO antagonist) and GLI1 siRNA were used to inhibit HIF-1α transcription or Hh signaling activation.

RESULTS

In present study, we first observed a strongly positive correlation between HIF-1α and GLI1 expression in HCC tissues. Then, we showed that hypoxia significantly promoted EMT process and invasion of HCC cells, associated with activating the non-canonical Hh pathway without affecting SHH and PTCH1 expression. HIF-1α knockdown mitigated hypoxia-induced SMO and GLI1 expression, EMT invasion of HCC cells. Moreover, the SMO inhibitor or GLI1 siRNA also reversed the hypoxia-driven EMT and invasion of HCC cells under hypoxia condition. Here, we show that non-canonical Hh signaling is required as an important role to switch on hypoxia-induced EMT and invasion in HCC cells. In addition, we found that hypoxia increased ROS production and that ROS inhibitors (NAC) blocked GLI1-dependent EMT process and invasion under hypoxic conditions. To determine a major route of ROS production, we tested whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) is involved in hypoxia-induced ROS production. NOX4 expression was found to be increased at both mRNA and protein levels in hypoxic HCC cells. Furthermore, siRNA-mediated knockdown of NOX4 expression abolished hypoxia induced ROS generation and GLI1-dependent activation and invasion of HCC cells.

CONCLUSION

Our findings indicate that hypoxia triggers ROS-mediated GLI1-dependent EMT progress and invasion of HCC cells through induction of NOX4 expression. Thus, hypoxia-driven ROS mediated non-canonical Hh signaling may play an important role in the initiation of EMT and provides a potential marker for cancer prevention and treatment.