Effect And Mechanism Of GPX4 In Proliferation,Invasion And Metastasis Of Lung Adenocarcinoma

Background and ObjectiveLung cancer is the leading cause of cancer-related deaths in the United States and worldwide.Non-small cell lung cancer(NSCLC)accounts for 85% of new lung cancer cases,and the 5-year survival rate ranges from 92% of early disease cases to 13% of locally advanced cases.Radiotherapy,chemotherapy and surgery are key components of the NSCLC treatment regimen.Although various treatment methods are used clinically,the overall survival rate of lung cancer is still not ideal.Recent breakthroughs in targeted therapy and immunotherapy have revolutionized the treatment of lung cancer.However,with the prolongation of treatment time,targeted drug resistance has become another worldwide problem.Therefore,it is imperative to explore new targeted molecules.Glutathione peroxidase(glutathione peroxidases,GPXs)is a kind of peroxidase widespread found in animals,its main function is to reduce excess reactive oxygen species(ROS)into non-toxic compounds and catalyze the decomposition of H2O2 into water,thereby reducing various peroxide oxidation damage to cells.GPX4 is one of the most important members of the enzyme family and has been found to be the only antioxidant enzyme responsible for the elimination of lipid hydrogen peroxide in cells.In addition,inhibition of GPX4 leads to ROS accumulation and lipid peroxide production,which ultimately leads to cell death.GPX4 is distributed on various membrane structures in cells and is responsible for removing various lipid peroxides.Recent studies have found that down-regulation of GPX4 expression could increase the sensitivity of tumor cells to chemotherapy drugs by inducing ferroptosis in tumor cells.Studies have shown that intracellular antioxidant enzymes are associated with treatment outcomes,particularly drug resistance,and are overexpressed in many cancer types.Glutathione peroxidase(GPXs)is a member of the antioxidant enzyme family.GPX4 has been reported to be overexpressed in a variety of tumor types and associated with prognosis,such as breast cancer and liver cancer.GPX4 has also been reported to be associated with tumor metastasis and invasion,such as glioma.However,the expression and role of GPX4 in lung cancer are still not clear,and whether GPX4 is related to metastasis,invasion and prognosis of lung cancer is still not elucidated.This aim of our study is to explore the expression of GPX4 in lung cancer and its role in tumor metastasis,invasion and prognosis.Methods1.We retrospectively analyzed the clinicopathological data and corresponding pathological specimens including survival and death status of 432 cases of lung cancer surgery patients from 2009 to 2014 in Tianjin Cancer Hospital.To detect the expression of GPX4 in lung cancer tissues,immunohistochemical staining was adopted then.We retrospectively analyzed the relationship between GPX4 and clinical parameters.Cox proportional hazards analyses were used to evaluate the clinical value of GPX4.2.We detected the expression of GPX4 in multiple lung cancer cell lines.To explore the role of GPX4 and its downstream,lentiviral expression vector was used to knock down the expression of GPX4,Furthermore,GPX4 was next overexpressed with GPX4 plasmid on the basis of the original GPX4 knockdown cells,and changes in GPX4 and its downstream pathway were detected by western blot.3.To clarify the effect of GPX4 in cell biological function,we used lentiviral expression vector to generate GPX4 knockdown HCC827 ShRNA cells and PC9 ShRNA cells and restoring GPX4 expression with a mutated GPX4 plasmid.MTT experiment were used to detect cell proliferation ability,we used cell cloning experiment to detect cell viability,wound healing assay and transwell experiment were performed to test the change of cell migration and invasion ability.Results1.GPX4 overexpression accounted for 41.9% in tumor tissue samples of 432 patients with lung cancer after surgery.In addition,our study analyzed the relationship between the GPX4 expression and the clinicopathological parameters of 432 patients,and our results showed that the GPX4 expression was related to smoking index(P = 0.012).Univariate survival analysis showed that high GPX4 expression was closely correlated with disease prognosis(P = 0.014,HR 1.483,95% CI 1.082-2.032).Multivariate survival analysis showed that high GPX4 expression(P = 0.014,HR 1.487,95%CI 1.085-2.038)and clinical stage(P = 0.001,HR 1.518,95%CI 1.297-1.777)were independent predictors of poor prognosis in patients with lung cancer.2.GPX4 overexpression was only found in lung cancer cell lines with EGFR mutation.Western blot results showed that the expression level of EGFR was down-regulated in lung adenocarcinoma cell lines with relatively high GPX4 expression(HCC827 and PC9)treated with GPX4 lentiviral vector.What’s more,when GPX4 overexpressed plasmids were transferred into cells,the expression level of EGFR was significantly up-regulated samely.In addition,the EGFR-related downstream signaling moleculers such as ERK,AKT pathway as well as STAT3 all changed correspondingly.3.MTT results showed that,compared with the control group,the expression level of GPX4 protein in HCC827 and PC9 cell lines infected with lentivirus targeting GPX4 gene was down-regulated,and the cell proliferation ability was significantly reduced,while the cell proliferation ability of the cells restored the expression of GPX4 protein was partially restored.The results of plate clonal formation experiment showed that the cell clonal formation ability of GPX4 was significantly decreased after the down-regulation of GPX4 expression,while the cell survival ability was significantly increased after the recovery of GPX4 expression.The results of cell wound healing experiment,transwell migration and invasion experiment showed that the cell migration and invasion ability was significantly decreased after the down-regulation of GPX4 expression,while the cell migration and invasion ability increased significantly after the recovery of GPX4 expression.Conclusion1.The prognosis of patients with high GPX4 expression in lung cancer tissues is poor,suggesting that GPX4 can be used as a clinical indicator for the prognosis of lung cancer.2.In lung cancer cell lines with positive EGFR mutation,GPX4 can regulate the expression of EGFR and thereby regulate the expression of its downstream molecules by regulating the expression of EGFR.3.GPX4 can regulate the proliferation,survival,migration and invasion of lung adenocarcinoma cells.