The Role And Mechanism Of ATF2 In Sorafenib-induced Ferroptosis Of Gastric Cancer Cells

Background:Gastric cancer(GC)is one of the most common malignant tumors of digestive system in China,and its incidence rate and mortality are both high.The GC patients in China account for about half of all cases of the world.However,the prognosis of GC patients is still not optimistic due to individual differences among them and the high heterogeneity of GC itself.Ferroptosis was only officially named in 2012 and is distinguished from the traditional form of programmed cell death by apoptosis,pyroptosis,necrosis,and autophagy.The essence of ferroptosis is an intracellular redox state imbalance,which occurs with iron overload,accumulation of lipid reactive oxygen species(lipid ROS),rupture of mitochondrial shrinkage,and ultimately cell death.Inducing tumor cells to undergo ferroptosis has proven to be of great potential in cancer therapy.Sorafenib is a tyrosine kinase inhibitor that is currently mainly used for clinical targeted therapy of advanced liver cancer.Meanwhile,its role as a ferroptosis inducer is also reported by many studies to play a tumor suppressor role in a variety of tumors,including GC.However,the status of sorafenib as an inducer of ferroptosis has recently been questioned.Currently,there are few studies involving ferroptosis in GC with sorafenib,and it remains to be clarified whether sorafenib can induce ferroptosis in GC cells.Multiple members of the activating transcription factor family were confirmed to play important roles in the regulation of ferroptosis.However,there is only one study on activating transcription factor 2(ATF2),and the specific role and related mechanism of ATF2 in sorafenib induced-ferroptosis has not been reported.Therefore,this study aimed to explore the role of ATF2 in sorafenib induced-ferroptosis in GC cells and the downstream molecular mechanisms,thereby providing a new strategy for the clinical treatment of GC.Methods:In the first part,the protein expression level of ATF2 in GC cells and tissues was detected by immunohistochemistry and Western blot.We further analyzed the relationship between the expression level of ATF2 and the clinicopathologic parameters and prognosis of GC patients.Then,the expression of ATF2 was overexpressed or knocked down by lentivirus transfection.The CCK-8 assay,Transwell migration and invasion assays were used to evaluate the effect of ATF2 expression on the proliferation,migration and invasion of GC cells.In the second part,we divided the cells into three groups: DMSO,sorafenib and sorafenib + ferrostatin-1.AGS and MGC803 cells were treated for 24 hours respectively,and then the morphological changes of cells were observed under the microscope.The ferroptosis level of GC cells was evaluated by detecting the contents of total ROS,lipid ROS,MDA and GSH.The changes of mitochondrial microstructure of GC cells treated with sorafenib were observed by transmission electron microscopy.Next,the expression of ATF2 in GC cells treated with sorafenib was detected by Western blot and immunofluorescence staining.Finally,after regulating the expression level of ATF2,the related indicators of ferroptosis in GC cells treated with sorafenib were detected.In the third part,the whole genome DNA binding sites and downstream potential transcription targets of ATF2 were identified by RNA-seq and Ch IP-seq,and further verified by Ch IP-q PCR.Then,the co-immunoprecipitation(Co-IP)experiment was used to detect the interaction between the downstream target gene of ATF2 and SLC7A11(a key regulatory molecule of ferroptosis).Use si RNA to knockdown the expression of the target gene,and then detect the protein stability of SLC7A11 and the changes of ferroptosis related indicators in GC cells.In the fourth part,the nude mice were randomly divided into four groups as follows:sh-Ctrl,sh-ATF2,sh-Ctrl?+?sorafenib and sh-ATF2?+?sorafenib.The effects of ATF2 knockdown alone and combined with sorafenib treatment on the growth of GC were evaluated at the animal level by establishing the tumor xenograft model.Results:We found that ATF2 was abnormally high expression in both GC cells and tissues,and was significantly correlated with lymph node metastasis(P = 0.003)and TNM stage(P = 0.034).The prognosis of patients with high expression of ATF2 is significantly worse than that of patients with low(Logrank P = 0.011).The expression level of ATF2 is an independent prognostic factor for patients with GC.ATF2 knockdown can significantly inhibit the proliferation,migration and invasion of GC cells.Sorafenib can induce ferroptosis in GC cells,which is accompanied by the activation of ATF2.ATF2 knockdown can promote sorafenib-induced ferroptosis in GC cells,while overexpression of ATF2 shows the opposite result.Mechanistically,using Ch IP-Seq and RNA-Seq,we identified HSPH1 as a target of ATF2 and further validated it by Ch IP-q PCR analysis.HSPH1 can interact with SLC7A11 and increase its expression through,at least partially,increasing SLC7A11 protein stability.In addition,HSPH1 knockdown partially reversed the effect of ATF2 overexpression on sorafenib-induced ferroptosis in GC cells.Finally,the results from the tumor xenograft model showed that ATF2 knockdown in combination with sorafenib treatment conferred the strongest suppression of tumor growth,and ATF2 knockdown increases the sensitivity of GC to sorafenib in vivo.Conclusion:ATF2 knockdown can significantly promote sorafenib-induced ferroptosis in GC cells,and enhance the anti-tumor effect of sorafenib on GC in vitro and in vivo.