| BackgroundGastric cancer,as the fifth most common malignant tumor and the third leading cause of cancer death worldwide,poses a serious threat to human life and health.Although a variety of medical treatments have been widely used,the mortality of patients with gastric cancer is still as high as 50%.Therefore,understanding the underlying mechanism of gastric cancer occurrence and treatment tolerance is particularly important for effective treatment of gastric cancer.Activating transcription factor 2(ATF2)is a member of the basic Leucine zipper(bZIP)family of DNA-binding proteins,which plays an important role in cancer radiotherapy and chemotherapy.It has been reported that ATF2 has dual functions(oncogenic activity or tumor suppression function)in tumors with different genetic contexts,while the role of ATF2 in gastric cancer is yet to be elucidated.ObjectiveIn this study,we aim to dissect the role of ATF2 in gastric cancer and its effect on the chemosensitivity of gastric cancer cells.To clarify the relationship between the function of ATF2 in gastric cancer and cellular p53 genetic status.Finally,to further understand the molecular mechanism of ATF2 in the tumor,and finally provide a potentially theoretical basis for the treatment of gastric cancer.MethodsATF2 expression in gastric cancer(GC)was detected by immunohistochemistry and its cellular localization was performed by immunofluorescence assay.The genetic status of ATF2 and p53 were analyzed by cBioportal database and potential proteins interaction between ATF2 and p53 were predicted by STRING database.GST-pull down and immunoprecipitation assay were used to clarify the interaction between ATF2 and p53.Western blot assay was performed to detect the protein expression levels of ATF2 and p53 in different gastric cancer cell lines with or without cisplatin treatment.The effects of ATF2 on the proliferation and colony formation ability of gastric cancer cells with different p53 genetic states were detected by cell proliferation assay and cell colony formation assay.The effect of over-expressing ATF2 on the growth of tumor xenografts was investigated by in-vivo experiments.Cell necrosis in tumor tissue was detected by HE staining.Statistical analysis of all data was performed by SPSS 21.0 statistical software measurement.All data were presented as mean ± SEM.The Kaplan-Meier method and Log-rank test were used to analyze the overall survival of patients with gastric cancer.Differences between the control group and the experimental group were compared by paired t test.p value<0.05 is regarded as statistically significant(α=0.05).Results(1)The expression of ATF2 in GC tissues is related to the overall survival time of patients,especially those who received chemotherapy;Patients with high ATF2 expression have a poorer prognosis.IHC results showed that ATF2 was decreased in about 43%(22/51)of human GC tissues.Results of Kaplan-Meier and Log-rank survival analysis showed that the overall survival prognosis of GC patients with high ATF2 expression was poor(p<0.01)and patients those who received chemotherapy,with high ATF2-expressing,had a worse overall survival(p=0.044).However,ATF2 expressed high or low had no obvious effect on the survival time of patients who undergone surgically only(p=0.19).(2)The proliferation and cell colony formation abilities of ATF2 ATF2-overexpressing MGC-803,HGC-27 and AGS GC cells were different after cisplatin treatment,and the expression of p-ATF2(phosphorylated ATF2)was inconsistent in these three cell lines.Western blot analysis showed that cisplatin significantly induced the expression of p-ATF2 in MGC-803 and HGC-27 cells,while had little change in AGS cells.Cell proliferation and colony formation experiments showed that cisplatin could obviously inhibit cell proliferation(p<0.05)and cell colony formation abilities of ATF2 over-expressing AGS and MGC-803 cells,but had no significant difference in HGC-27 cells.(3)ATF2 could bind with p53 potentially,and p53 status effects the proliferation ability and the expression of p-ATF2 in ATF2-overexpressing HCT-116 cells after cisplatin treated.MGC-803 and HGC-27 are p53 mutant cells(wherein p53 in HGC-27 cells is dysfuctional),while AGS is p53 wild type cells.The results of STRING analysis and Venny software showed that both ATF2 and p53 could bind with a variety of molecules,and among the 202 proteins which had published to interact with ATF2,there were 53 proteins could interact with p53.Western blot results showed that cisplatin significantly induced the expression of p-ATF2 in HCT-116-p53-/--ATF2-WT cells(p<0.01),but has no obvious change in HCT-116-p53+/+-ATF2-WT cells.In addition,results of cell proliferation experiment showed that cisplatin could markedly inhibit the proliferation capacity of HCT-116-p53+/+cells with over-expressing ATF2-WT(p<0.05),and ATF2-T71E(p<0.01)compared with the vector group.However,in HCT-116-p53-/-cells,the resistance of over-expressing ATF2-WT(p<0.01)and ATF2-T71E(p<0.05)subtypes were enhanced after cisplatin treatment,and the proliferation capacity of cells is obviously increased.(4)ATF2 could interact with p53 in-vivo and in-vitro,and ATF2 bound with p53 transactivation domain(TAD).Immunofluorescence assays showed that ATF2 and p53 were co-localized in the nucleus.Discovery Studio molecular docking software analysis revealed that there was a potential binding region between the crystal structure of ATF2 and p53.GST-pull down assay demonstrated that Flag-p53 protein could interact with ATF2-GST protein in vitro.And Co-IP experiment further confirmed that ATF2 could bind with p53 TAD domain,but this interaction will disappear when the TAD domain was deleted(dTAD).(5)p53 has high rate of mutation and splicing in human GC.The cBioportal database analysis showed that p53 had the highest mutation rate(up to 50%)in GC tissue(n=395).And p53 mutation in GC tissues were mainly missense mutations,among which R175,R248,R273 are hotspot sites at its DNA binding domain.Analysis of p53 transcripts in GC tissues showed that the alternative splicing rate of p53 with N-terminal TAD domain deletions in tumor tissues was significantly increased(p<0.01)compared with the normal para-cancer tissues.(6)Higher ATF2 promotes the growth of GC cells xenograft tumor with p53 dysfunction.The results of animal experiments showed that,the tumor sizes of HGC-27 ATF2 over-expressing group were larger than that of HGC-27 vector control group after cisplatin treatment.And HE staining showed that,compared with the ATF2 over-expressing group,cisplatin caused obviously necrosis in tumor tissues of the vector control group.ConclusionOur research shows that the higher ATF2 expression in GC tissuses was involved in poor 5-year overall survival in patients with GC undergoing chemotherapy.The function of ATF2 in GC cells was associated with the p53 genetic status;Higher ATF2 could enhance the sensitivity of p53 wild-type GC cells to cisplatin,but also promote the cisplatin resistance in p53 dysfunctional GC cells and finally leading to the growth of GC xenograft tumor cells.In conclusion,this study has initially unveiled the molecular function of ATF2 in gastric cancer after cisplatin treatment and provide a new rational theoretical basis for clinical chemotherapy of gastric cancer. |
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