The Effect Of ATF2 On BET Inhibitor Resistance In Triple Breast Cancer And The Underlying Molecular Mechanism

Breast cancer originates from the glandular epithelium of the breast and is the most common malignant tumor in women.With the continuous change of risk factors such as delayed birth,reduced number of births,overweight and obesity,and lack of exercise,the incidence of breast cancer is increasing year by year.According to the latest global cancer burden data released by the International Agency for Cancer Research of the World Health Organization,it is estimated that the number of breast cancer in the world increased by 2.26 million in 2020,more than 2.2 million of lung cancer.Breast cancer replaced lung cancer and became the largest cancer in the world.Accounting for 24.5% of the total incidence of malignant tumors in women,680000 women died of breast cancer,accounting for 6.9% of all female malignant tumor mortality,ranking fifth in the number of cancer deaths in the world,resulting in a heavy social burden.Triple-negative breast cancer(TNBC)accounts for 12%～25% of all breast cancers.Due to the lack of effective targeted treatment,conventional chemotherapeutic drugs are still the first-line choice for advanced triple-negative breast cancer.Therefore,TNBC is still a disease with poor prognosis and full of challenges.Bromodomain and Extraterminal domain(BET)inhibitors can inhibit the binding of BRD4 protein to chromatin and block the transcription of oncogenes,which show strong anti-tumor activity in TNBC and are expected to become targeted drugs for TNBC.However,clinical studies have shown that the effect of single drug application of BET inhibitors is limited,and drug resistance is the main problem,and the specific mechanisms have not been completely clarified.Phosphorylated activating transcription factor-2(ATF2)can be expressed as a transcription factor and participate in tumorigenesis and drug resistance.Studies have shown that ATF2 participates in the drug resistance of cancer cells to chemotherapy through the JNK pathway,and is also related to the poor prognosis of breast cancer.However,it is not clear whether ATF2 plays a role in drug resistance of BET inhibitors.To explore whether ATF2 is involved in regulating the resistance of TNBC to BET protein inhibitors and its molecular mechanism.In this study,two different BET inhibitors were selected to act on TNBC cell lines to detect the expression of ATF2 and to determine whether BET inhibitors had an effect on the activation of ATF2 in TNBC cells.Then,two BET inhibitors were added to ATF2 overexpressed and silenced TNBC cell lines to detect cell viability,clone formation ability and cell death.The effects of tumor formation in vivo and sensitivity to BET inhibitors and other biological functions preliminarily explored the molecular mechanism of ATF2 regulating the resistance of BET inhibitors in TNBC cells.Part One:The Regulation of ATF2 in BET inhibitors-resistant Triple Negative Breast Cancer CellsMethods1.BET inhibitors JQ1 and I-BET-151 and JNK1/2 inhibitors SP600125 were selected to act alone or in combination on triple negative breast cancer MDA-MB-231 cell lines.The phosphorylation levels of JNK1/2 and ATF2 were detected to determine whether BET inhibitors had an effect on ATF2 activation in triple negative breast cancer cells.2.Increase the dose of BET inhibitors(JQ1 and I-BET-151),and CCK8 cell proliferation assay was used to detect the cell viability of triple negative breast cancer cell lines MDA-MB-231 and BT-549,which were overexpressed by ATF2.3.After treatment with BET inhibitor JQ1,clone formation ability of triple negative breast cancer MDA-MB-231 cell line with overexpression of ATF2 was detected by colony formation assay.4.Triple negative breast cancer cell lines MDA-MB-231 and BT-549 were treated with BET inhibitors(JQ1 and I-BET-151).The changes of cell death in triple negative breast cancer cell lines MDA-MB-231 and BT-549 which were overexpressed by ATF2 were analyzed by 7-AAD/Annexin-V assay.5.The recombinant lentivirus overexpressing ATF2 was prepared and packaged,and the breast cancer cell line stably overexpressing ATF2 and blank control was established by infecting the logarithmic triple negative breast cancer cell line MDA-MB-231.In vivo tumor formation test was used to detect the stable overexpression of ATF2 and the body weight of mice treated with BET inhibitor JQ1 and the blank control.Further immunohistochemistry was performed to detect the expression of phosphorylated ATF2.6.Si RNA was used to down-regulate the expression of ATF2,and the sensitivity of triple negative breast cancer to BET inhibitor was observed by cell viability test and cell death analysis.7.Statistical analysis: All experiments were repeated three times and expressed as mean ± SD.Student’s t test was used to compare the differences between the two groups.p<0.05 was regarded as statistically significant.Statistical analysis was analyzed using the Statistical Package for Social Sciences(SPSS)software(version20.0).Results1.The levels of phosphorylated ATF2 was significantly induced by BET inhibitors.In addition,BET inhibitors also increased the phosphorylated JNK1/2expression.JNK1/2 inhibitors could significantly inhibit the induction of ATF2 phosphorylation by BET inhibitors(p<0.05).2.qRT-PCR and Western Blotting results showed that overexpression of ATF2 vector in MDA-MB-231 and BT-549 cells could significantly up-regulate the m RNA and protein expression of ATF2.After 48 hours treatment with BET inhibitor in ATF2 overexpressed triple negative breast cancer cells,the cell viability was significantly decreased by BET inhibitor in dose-dependent manner than that of the control group(p<0.05).3.Colony formation assay showed that the clone number of MDA-MB-231 cells in the overexpression ATF2 group which treated with BET inhibitor was significantly higher than that in the control group in the triple negative breast cancer(p<0.05).4.Flow cytometry results showed that the cell death rate of MDA-MB-231 and BT-549 cells in ATF2 overexpression group was significantly lower than that of control group(p<0.05).5.The tumor formation test in mice showed that the tumor formation volume of MDA-MB-231 cells in overexpression ATF2 group(231/ATF2)was significantly larger than that in mice treated with BET inhibitor JQ1 compared with control vector group(231/Ctrl)(p<0.05).The sensitivity of overexpressing ATF2 group(231/ATF2+JQ1)to BET inhibitor was significantly lower than that of control group(231/Ctrl+JQ1)(p<0.05).There was no significant change in body weight in overexpressed ATF2 group(231/ATF2+JQ1).These results of immunohistochemistry showed that after treatment with JQ1,the expression of phosphorylated ATF2 was significantly increased.6.Western blots showed that small interference RNA could effectively reduce the expression of ATF2.After treatment with BET inhibitor,the cell viability of MDA-MB-231 cells silenced by ATF2(Si ATF2)was significantly lower than that of the control group(p<0.05),and the cell death rate was significantly higher than that of the control group(p<0.05).ConclusionBET inhibitors activate triple negative breast cancer cell line ATF2 through JNK1/2 pathway.Overexpression of ATF2 can significantly reduce the effect of BET inhibitors on the cell viability of triple negative breast cancer,enhance the clone formation ability of MDA-MB-231 cells,and inhibit cell death induced by BET inhibitors.ATF2 promotes the resistance of triple negative breast cancer to JQ1 in vivo,and after treatment with JQ1,the expression of phosphorylated ATF2 was significantly decreased.Silencing ATF2 can restore the sensitivity of MDA-MB-231 cells to BET inhibitors.Part Two:The Molecular Mechanism of ATF2 Regulating BET Resistance in Triple Negative Breast Cancer CellsMethods1.MDA-MB-231 cells with overexpressing or silencing ATF2 were treated with increasing dose of JQ1 and I-BET-151.And then we detected the changes of end products of lipid peroxidation during ferroptosis : the oxidized form glutathione(GSSG),lipid peroxide end product malondialdehyde(MDA),intracellular lipid reactive oxygen radical(ROS)level.2.MDA-MB-231 and BT-549 cells with stable ATF2 overexpression were used to detect the protein and m RNA levels of ATF2 and NRF2 by Western blot and RT-PCR respectively.The m RNA expression of ATF2 and NRF2 in breast cancer tissues were obtained from the TCGA database.The expression correlation of ATF2 and NRF2 were analyzed by Pearson analysis.Pearson correlation coefficients r and P values were indicated.3.qRT-PCR methods were used to detect the NRF2 and ATF2 m RNA levels and Western Blotting methods were used to detect the NRF2 and ATF2 protein expression of ATF2-depleted MDA-MB-231 cells treated with BET inhibitor JQ1.4.Western Blotting method was used to detect the expression of NRF2 protein in Si NRF2 of MDA-MB-231 cells,the changes of ferroptosis index and cell death were detected in overexpressing ATF2 and knockdown NRF2 of MDA-MB-231 cells which treated with BET inhibitor JQ1.5.Statistical analysis: All experiments were repeated three times and expressed as mean ± SD.Student’s t test was used to compare the differences between the two groups.p<0.05 was regarded as statistically significant.Statistical analysis was analyzed using the Statistical Package for Social Sciences(SPSS)software(version20.0).Results1.BET inhibitors significantly increased GSSG and MDA levels in a dose-dependent manner,whereas overexpression of ATF2 significantly prevented the increase of lipid peroxidation products induced by the two BET inhibitors.Moreover,we also showed ATF2 significantly inhibited lipid ROS production by BET inhibitors(p<0.001).Depletion of ATF2 remarkably increased the levels of GSSG,MDA and lipid ROS by BET inhibitors(p<0.001).2.Overexpressed ATF2 significantly increased NRF2 protein and m RNA expression.By contrast,knockdown of ATF2 obviously reduced NRF2 protein and m RNA expression.By analysis of TCGA database,we identified significantly positively relationship between NRF2 and ATF2 m RNA expression in 1104 breast cancer tissues.3.To verify that BET inhibitors JQ1 induced NRF2 expression was associated with ATF2,NRF2 was detected in ATF2-depleted MDA-MB-231 cells treated with JQ1.Results showed ATF2 siRNA significantly attenuated NRF2 expression induced by JQ1(p <0.05).Overexpressed ATF2 significantly increased NRF2 protein and m RNA expression.By contrast,knockdown of ATF2 obviously reduced NRF2 protein and m RNA expression.4.NRF2 siRNA significantly decreased NRF2 expression.The induction of GSSG,MDA and lipid ROS levels by JQ1 were significantly decreased by ATF2,whereas these ferroptosis markers were further raised by NRF2 siRNA.Notably,in NRF2-depleted MDA-MB-231 cells,overexpression of ATF2 failed to significantly attenuate JQ1-stimulated ferroptosis markers levels.Similarly,results from detection of cell death also showed that NRF2 siRNA blocked ATF2-inhibited ferroptosis(p<0.05).ConclusionOverexpression of ATF2 inhibited ferroptosis induced by BET inhibitors in triple negative breast cancer.ATF2 inhibited BET inhibitors-induced ferroptosis by increasing NRF2 expression.ATF2 suppressed ani-tumor effects of BET inhibitors in a negative feedback manner by attenuating ferroptosis.In conclusion,based on the fact that the application of BET inhibitors alone was limited in clinical studies,this study investigated the molecular mechanisms involved in the regulation of BET inhibitor resistance in triple-negative breast cancer by ATF2,and confirmed that the BET inhibitors JQ1 and I-BET 151(I-BET)activated the JNK/ATF2 pathway in MDA-MDA-MB-231(MDA-MB-231)in breast cancer cells,and that overexpression of ATF2 resisted the anticancer effects of BET inhibitors both in vitro and in vivo,i.e.ATF2 was involved in tumour drug resistance.Moreover,the correlation between ATF2 and NRF2 expression was found by searching and analyzing the TCGA database verified that ATF2 blocked BET inhibitor-induced iron death in triple-negative breast cancer cells through upregulation of NRF2 expression and inhibited the antitumor effects of BET inhibitors in a negative feedback manner,i.e.the JNK/ATF2/NRF2 functional axis mediated BET inhibitors through the mechanism of iron death inhibition in TNBC.These results are the first to elucidate the mechanism by which ATF2 inhibits the antitumor effect of BET inhibitors by inhibiting iron death in a negative feedback manner,and provide a theoretical basis for the treatment of BET inhibitor resistance in triple-negative breast cancer.