Hypoxia promotes the metastasis of pancreatic cancer through regulating NOX4/KDM5A-mediated histone methylation modification changes in a HIF1A-independent manner

BACKGROUND: Hypoxia is a characteristic of the tumor microenvironments within pancreatic cancer (PC), which has been linked to its malignancy. Recently, hypoxia has been reported to regulate the activity of important carcinogenic pathways by changing the status of histone modification. NOX4, a member of NADPH oxidase (NOX), has been found to be activated by hypoxia and promote cancer progression in several cancers. But whether it is involved in the epigenetic changes of tumor cells induced by hypoxia is still unclear, and its biological roles in PC also need to be explored. METHODS: A hypoxic-related gene signature and its associated pathways in PC were identified by analyzing the pancreatic cancer gene expression data from GEO and TCGA database. Candidate downstream gene (NOX4), responding to hypoxia, was validated by RT-PCR and western blot. Then, we evaluated the relationship between NOX4 expression and clinicopathologic parameters in 56 PC patients from our center. In vitro and in vivo assays were preformed to explore the phenotype of NOX4 in PC. Immunofluorescence, western blot and chromatin immunoprecipitation assays were further applied to search for a detailed mechanism. RESULTS: We quantified hypoxia and developed a hypoxia signature, which was associated with worse prognosis and elevated malignant potential in PC. Furthermore, we found that NADPH oxidase 4 (NOX4), which was induced by hypoxia and upregulated in PC in a HIF1A-independent manner, caused inactivation of lysine demethylase 5A (KDM5A), increased the methylation modification of histone H3 and regulated the transcription of EMT-associated gene_ snail family transcriptional repressor 1 (SNAIL1). This served to promote the invasion and metastasis of PC. NOX4 deficiency repressed hypoxia-induced EMT, reduced expression of H3K4ME3 and impaired the invasion and metastasis of PC cells; however, knockdown of KDM5A reversed the poor expression of H3KEME3 induced by NOX4 deficiency, thereby promoting EMT. 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.