Nadph Oxidase 4 Regulates Anoikis Resistance Of Gastric Cancer Through Reactive Oxygen Species Generation And Subsequent Induction Of EGFR

Objective: Anoikis is a type of programed cell death induced by detachment from the extracellular matrix.In cancer cells,anoikis resistance is essential for cancer cell survival in blood circulation and distant metastasis.However,the mechanisms behind anoikis resistance of gastric cancer remain largely unknown.Regulation of redox homeostasis is essential for the cellular metabolism,survival and growth and is likely to be involved in the anoikis of gastric cancer cells.As one of the main sources of reactive oxygen species,NADPH oxidase 4(NOX4)plays a crucial role in the occurrence and development of tumors.However,there is currently little evidence demonstrating that NOX4 is associated with gastric cancer,especially in terms of anoikis.Methods: We cultured gastric cancer cells in the attachment or suspension condition.Cell anoikis rate was measured by flow cytometry using the FITC Annexin V Apoptosis Detection.ROS generation was measured by flow cytometry.Effects of NOX4 depletion or NOX4 overexpression on ROS generation,EGFR regulation and anoikis resistance were explored in vitro.Moreover,the effects of NOX4 on anoikis resistance and distant metastasis of gastric cancer cells were assessed in vivo.We performed immunohistochemistry on gastric cancer tissues and paired adjacent normal tissues from 90 gastric cancer patients to detect and compare NOX4 and EGFR expression.Next,we analyzed the association between NOX4 expression and clinicopathological characteristics.Survival analysis was performed to explore the association between NOX4 expression and the prognosis of gastric cancer patients based on biological database.Results: NADPH oxidase 4(NOX4)expression and reactive oxygen species(ROS)generation are upregulated in suspension gastric cell cultures compared with adherent cultures.Silencing of NOX4 decreases ROS generation and downregulates EGFR,sensitizing cells to anoikis.NOX4 overexpression upregulates ROS and subsequent EGFR levels and promotes anoikis resistance.NOX4 depletion inhibits gastric cancer survival in blood circulation and attenuates distant metastasis.NOX4 expression in gastric cancer tissues is higher than in paired adjacent normal tissues(p = 0.0009).NOX4 expression is significantly correlated with tumor size(p = 0.0321),lymphatic metastasis(p = 0.0125)and vascular invasion(p = 0.0017)and a poor prognosis(p = 0.0000)in gastric cancer patients.NOX4 expression is correlated with EGFR expression in patients.Conclusion: Induction of NOX4 expression by detachment promotes anoikis resistance of gastric cancer through ROS generation and downstream upregulation of EGFR,which is critical for the metastatic progression of gastric cancer.NOX4 is related to gastric cancer development and predicts a poor prognosis.Hence,targeting NOX4 could be a promising therapeutic strategy to prevent metastatic development in GC.