Mechanisms Of Ferroptosis In Drug Resistance Of Esophageal Squamous Cell Carcinoma And Effects Of DSG2 In Drug Resistance Of Lung Adenocarcinoma

In China,the morbidity and mortality of esophageal cancer ranks among the top.The main pathological type is esophageal squamous cell carcinoma(ESCC).For combination treatment with chemoradiotherapy or neoadjuvant chemotherapy,platinum regimens are still the cornerstone of ESCC treatment for now.However,the response rate of platinumbased therapy in the treatment of ESCC remains only 40-60%.Furthermore,it is common to develop drug resistance and tumor progress after a long period of treatment.Thus,it is very important to further study the resistance mechanism of ESCC to platinum drugs and to explore novel targets for reversing the resistance,which can improve the chemotherapy efficacy,survival,and prognosis of ESCC patients.As a novel programmed cell death,ferroptosis has been proved to play an important role in drug resistance of various cancers.The imbalance between oxidative damage and antioxidant defense,which results in the accumulation of lipid peroxidation,is fundamental to ferroptosis.Glutathione peroxidase 4(GPX4)is one of the most significant antioxidant proteins.It can catalyze the reduction of toxic phospholipid hydroperoxides into nontoxic phospholipid alcohols by using reduced glutathione(GSH)as a substrate,thus preventing the occurrence of ferroptosis.Several studies have shown that the survival of some drug-resistant cancer cells greatly depends on the high activity of GPX4.Moreover,the corresponding targeted drugs have the potential of becoming an important new strategy to overcome drug resistance.However,how ferroptosis pathway affects the sensitivity of ESCC to platinum drugs is barely investigated,and the underlying mechanisms remain unknown.Therefore,our current study is aimed to evaluate the role of the ferroptosis in ESCC resistance to cisplatin,and to explore its potential mechanisms.In this study,high-content screening was performed to evaluate the effects of ferroptosis-related genes on the sensitivity of ESCC cells to cisplatin,finding that the ferroptosis pathway plays an important role in ESCC resistance to cisplatin.In the screening,the downregulated heat shock protein family A member 5(HSPA5)was one of the genes most significantly enhancing the sensitivity to cisplatin.Then,we constructed stable HSPA5 cell lines by lentiviral infection,and conducted drug sensitivity experiments to verify that the HSPA5 knockdown enhanced the sensitivity to cisplatin,while its overexpression showed insignificant effects on the sensitivity.The increased cell death caused by HSPA5 knockdown after cisplatin treatment was not only partially reversed by the apoptosis inhibitor Z-VAD-FMK,but also partially alleviated by the ferroptosis inhibitor Ferrostatin-1.Furthermore,HSPA5 knockdown promoted Erastin-induced ferroptosis and increased the lipid peroxidation.Mechanistic studies found that silencing of HSPA5 resulted in a reducition in the protein expression and enzymatic activity of GPX4,and an increase in intracellular GSH content.Further experiments confirmed that knockdown of HSPA5 activated the PERK-eIF2α pathway and inhibited the translation of GPX4,thus downregulating its protein expression.In conclusion,this study revealed the important role of ferroptosis in the resistance of ESCC to cisplatin,and screening results demonstrated that HSPA5 might be a critical molecule affecting cisplatin sensitivity.Further mechanistic exploration showed that the knockdown of HSPA5 decreased the expression of GPX4 protein by activating the PERKeIF2α pathway,thereby increasing the vulnerability to ferroptosis and enhancing the sensitivity to cisplatin.This research enriched our understanding of the drug resistance mechanisms of chemotherapeutic regimens in treating ESCC,providing novel insights into overcoming drug resistance.Lung cancer is one of the most common malignancies in the world with a high rate of mortality.As the main subtype of lung cancer,lung adenocarcinoma(LUAD)incidence has increased significantly during the past 20 years,especially among females.The prognosis for late-stage patients is still very poor.Therefore,an in-depth investigation into pathogenesis,molecular genotyping,and new target discovery is of great importance for LUAD.Desmoglein-2(DSG2),a member of cadherin superfamily,has been known to mediate cell-cell adhesion and promote tumorigenesis of many malignant tumors.However,a complete understanding of its mechanism of action remains an important goal.In this study,we demonstrated that DSG2 was highly expressed in both LUAD cell lines and tissues compared to normal control,and its high expression was associated with poor prognosis in LUAD patients.To determine the functions of DSG2 in the development of LUAD,DSG2 stably over-expressed and silenced cell lines,including H538,H1975 and HCC827,were established by lentiviral transfection.Then,cell counting assays,transwell assays and IC50 detection were conducted in vitro.Notably,DSG2 over-expression promoted cell proliferation and migration and increased their resistance to EGFR tyrosine kinase inhibitor osimertinib,whereas DSG2 silencing could reverse the results.To further explore the underlying mechanisms of DSG2 promoting tumor malignancy,specific molecular assays were also conducted.Firstly,immunoprecipitation,immunofluorescence and cell fractionation results demonstrated that DSG2 stabilized EGFR in the cell membrane,while DSG2 knockdown significantly attenuated EGFR membrane localization.In all cell types,relative to control,DSG2 overexpression significantly increased EGFR Tyr845 phosphorylation but not Tyr 1068 phosphorylation.Moreover,DSG2 overexpression significantly increased Src activation through Tyr416 phosphorylation,and reduced inactivating Tyr527 phosphorylation.Then,Rac1 activation assays proved that Rac1 was activated by exogenous DSG2 overexpression.Finally,significantly increased activating PAK1 Ser423 phosphorylation was detected in DSG2 overexpressing cells.On the contrary,our immunoblot results demonstrated that silencing DSG2 inhibited the malignancy of tumor cells via suppressing the EGFR-Src-Racl-PAK1 signaling pathway.Notably,total expression of these proteins was not significantly altered by DSG2 overexpression or silencing relative to controls.Intriguingly,immunoprecipitation of EGFR confirmed binding between EGFR and Src,but binding dissociated after DSG2 silencing.Further siRNA silencing of EGFR and Src in DSG2overexpressing cells led to remarkable decrease in downstream signaling,which confirmed that DSG2 promoted PAK1 signaling by inducing EGFR and Src phosphorylation.In addition,EGF stimulation and osimertinib treatment further proved that DSG2 regulates the mentioned pathway in LUAD.After treatment with EGF for 24 h,DSG2overexpressing cells showed significant increases in EGFR Tyr845 and PAK1 Ser423 phosphorylation relative to untreated cells.Additionally,EGFR and PAK1 phosphorylation levels were much higher in DSG2-overexpressing cells relative to vector control cells for all cell types.As expected,24 h osimertinib treatment of vector control cells caused significant decreases in EGFR Tyr845 and PAK1 Ser423 phosphorylation relative to untreated cells.However,in DSG2-overexpressing cells,osimertinib effects were insignificant,causing either a slight decrease or no change to EGFR and PAK1 phosphorylation.Consistent with these findings,a nude mouse xenograft model using H1975 cells demonstrated that DSG2 promoted LUAD cell growth in vivo and increased osimertinib resistance.Of note,tumor burdens for all the mice were reduced by osimertinib treatment when compared to untreated mice.For mice injected with DSG2-silenced cells,osimertinib effects were more remarkable.Nevertheless,for DSG2 overexpression treated with osimertinib,their sizes were relatively larger than vector control.Also,IHC analysis demonstrated that phosphorylated EGFR Tyr845 and PAK1 Ser423 were induced in tumors by DSG2-overexpression,and suppressed in tumors by DSG2-silencing when compared with control groups.We demonstrate that DSG2 has a unique role in inducing Src activation by regulating the interaction between EGFR and Src for the first time.This effect facilitates downstream Src signaling,in particular by promoting the Racl-PAK1 pathway,causing LUAD cell proliferation,migration,and osimertinib resistance.