NADPH Oxidase 4 Promotes Hypoxia-induced Epithelial-to-mesenchymal Transition via Histone Modification in Pancreatic cancer

Background: Hypoxia is a characteristic of the tumor microenvironments within Pancreatic cancer (PC) which has been linked to its malignancy. Oxidative stress, characterized by NADPH oxidase (NOX) activation, and epithelial-to-mesenchymal transition (EMT) could be induced by hypoxia which involved in tumor progression and metastasis. However, the relationship between hypoxia-induced oxidative stress and EMT has not been clarified, and the regulatory mechanism of NADPH oxidase is still unknown. Methods: A hypoxic-related gene signature and its associated pathways in PC were identified by bioinformatics method. Candidate downstream gene (NOX4), responding to hypoxia was validated by RT-PCR and western blot. In vitro and in vivo assays as well as tumor samples from our centre were preformed to explore the phenotype of NOX4 in PC. Immunofluorescence, western blot and chromatin immunoprecipitation assays were further applied to search for detailed mechanism. Results: We established a hypoxia-related gene signature within PC which was prognostic and linked with up-regulated EMT pathway. Then we found that hypoxia could induce stable up-regulation of NOX4, which is essential for EMT activation. Elevated expression of NOX4 was observed in PC samples and positively associated with advanced tumor grade and unfavorable prognosis. In vivo and in vitro experiments demonstrated NOX4 overexpress or inhibition in pancreatic cancer cells caused changes of proliferation and invasion ability. Then we found NOX4 could increase the methylation modification of histone H3 and regulated the transcription of EMT-associated gene_ snail family transcriptional repressor 1 (SNAIL1). Conclusions: This study highlights the prognostic role of hypoxia-related genes in PC and strong correlation with EMT pathway. Our results also creatively discovered that NOX4 was an essential mediator for hypoxia-induced histone methylation modification and EMT in PC cells.