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

Abstract

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.

An Eight-Gene Hypoxia Signature Predicts Survival in Pancreatic Cancer and Is Associated With an Immunosuppressed Tumor Microenvironment

“…The presence of hypoxia as reflected by our signature was associated with worse OS in two independent datasets of PDA. This provides for a more robust signature than what has been previously published in this cancer (9,38). Indeed, while a recent thirty-gene hypoxia signature was reported to be associated with worse OS and PFS in the TCGA dataset (38), this was not tested in an independent patient cohort.…”

“…This provides for a more robust signature than what has been previously published in this cancer (9,38). Indeed, while a recent thirty-gene hypoxia signature was reported to be associated with worse OS and PFS in the TCGA dataset (38), this was not tested in an independent patient cohort. Moreover, when we compared survival parameters in tumors scored based on that signature, to those scored with our eightgene signature, only ours maintained a significant association with OS and DSS.…”

“…Molecular prognostication based on transcriptomes, genetic variations, and cellular components of pancreatic tumors have classified PDA into different subtypes (18)(19)(20). In this setting, hypoxia was found to dominate in the subtypes with worse prognosis (9,20,38). Recently, five transcriptomic subtypes that integrated elements from existing clusters, but additionally touching on the immune and inflammatory components, were put forth by a large multicentric study using gene expression profiles from FFPE samples of resectable pancreatic tumors (50).…”

“…Common detection methods for hypoxia include the application of immunohistochemistry for determining hypoxiainduced proteins, imaging techniques for visualizing hypoxia sensitive agents, and more recently, there has been the novel approach of incorporating hypoxia gene signatures (32)(33)(34)(35)(36)(37). Given the broad impact of hypoxia on gene expression profiles of both tumor cells and the surrounding stroma, multigene prognostic signatures have been published that act as a proxy to this condition in distinct tumor types (33)(34)(35)(36)(37)(38). With respect to PDA, hypoxia signatures were found to be enriched in patients exhibiting worse prognosis (11).…”

“…In an independent study a hypoxia signature was associated with worse overall survival (OS) and progression free survival (PFS) in patients with primary tumor; however, associations were lost in the presence of other clinical covariates (9). Recently, a thirty gene hypoxia signature was similarly found to be associated with worse OS and PFS in PDA (38). Despite these findings, no hypoxia signature to date has been tested in pancreatic cancer cell lines and validated in multiple patient datasets.…”