The Role And Mechanism Of Phosphatase PHLPP2 In Regulating Ferroptosis In Non-small Cell Lung Cancer Cell

ObjectiveThe objective of this study is to investigate the expression level of the phosphatase PHLPP2 and its role and mechanism in relation to ferroptosis in non-small cell lung cancer(NSCLC).Methods1.The expression level of PHLPP2 in tumor tissue and corresponding normal tissue of NSCLC in public databases,as well as its correlation with the key ferroptosisrelated gene expressions of SLC7A11,GPX4,and ACSL4,were analyzed using the online platforms GEPIA2 and cBioPortal.2.The correlation between the transcriptional expression level of PHLPP2 and the AUC value of the ferroptosis inducers in NSCLC cell lines from CCLE was analyzed using the DepMap Portal.3.Human NSCLC cell lines were cultured in vitro,and cell viability was assessed after treatment with a ferroptosis inducer using the MTT assay.Real-time PCR was performed to detect mRNA expression levels,Western blot analysis was conducted to detect protein expression,and lipid ROS production was evaluated by flow cytometry using C11-BODIPY staining.4.Stable PHLPP2 knockdown or overexpression NSCLC cell lines were generated through retrovirus and lentivirus infections.5.The independent sample t-test was employed to compare between the two groups.Results1.The expression of PHLPP2 in tumor tissues of lung squamous cell carcinoma and lung adenocarcinoma was lower than that in corresponding normal tissues from TCGA.It was negatively correlated with transcriptional expression and the key ferroptosis-related gene GPX4,with Spearman’s correlation coefficients of-0.336 and-0.254(P<0.05),respectively.PHLPP2 showed no significant correlation with SCL7A11 and ASCL4(P>0.05).2.The expression level of PHLPP2 was negatively correlated with the AUC values of the ferroptosis inducers RSL3,ML210,and ML 162 in NSCLC cell lines from CCLE.The Pearson’s correlation coefficients were-0.117,-0.161,and-0.102,respectively.3.PHLPP2 expression in H1650,H1299,H1975,HCC827,and A549 cell lines decreased in the following order,as confirmed by western blot.The proliferation rates of the cells upon RSL3 10μM treatment were as follows:H1650(44.41%±1.61%),H1299(13.17%±2.54%),H1975(25.05%±5.92%),HCC827(15.22%±2.35%),and A549(41.48±10.01%).RSL3 treatment induced PHLPP2 protein expression.4.Stable knockdown of PHLPP2 in H1650 cells significantly increased the semiinhibitory concentration(IC50)of RSL3.The IC50 values in the experimental group(knockout expression group)and control group were 0.52μM and 2.59μM,respectively.The level of lipid ROS production induced by RSL3 was significantly lower in the experimental group(14.76%±1.22%)compared to the control group(4.89%±1.81%)(P=0.005).The in hibitory effect of RSL3 on GPX4 was significantly weakened.5.Overexpression of PHLPP2 in A549 cell lines significantly decreased the IC50 value of RSL3 compared to the control group,which were 1.7μM and 0.23μM,respectively.The level of lipid ROS generation induced by RSL3 was significantly lower in the overexpression group(4.34%±0.39%)compared to the control group(15.36%±0.80%)(P<0.001).The inhibitory effect of RSL3 on GPX4 was enhanced.ConclusionOur study investigated the role of phosphatase PHLPP2 in ferroptosis sensitivity and its implications as a positive regulator in non-small cell lung cancer(NSCLC).The findings demonstrate that knockdown of PHLPP2 expression significantly inhibits cell proliferation induced by ferroptosis inducers and decreases lipid ROS production.Conversely,overexpression of PHLPP2 enhances cell ferroptosis and lipid ROS production in NSCLC cell lines.Mechanistically,the ferroptosis inducer upregulates PHLPP2 protein expression,and PHLPP2,in turn,promotes ferroptosis sensitivity by inhibiting the expression of GPX4.These results suggest that PHLPP2 can serve as a potential biomarker for ferroptosis sensitivity,and further investigations are warranted to explore targeting the ferroptosis pathway as a potential treatment strategy in NSCLC.