Suppressive Effect Of CDK7 Inhibitor THZ1 Combined With Nutlin-3 On Breast Cancer P53^wt Cells Survival And Its Mechanism

Background:According to statistics of 2020,female breast cancer has become one of the malignant tumors with the highest incidence rate around the world,overpassing lung cancer.In China,the number of newly added cases for female breast cancer increased to 420,000 in 2020,topping among the incidence of female malignant tumors.Now the treatment has converted from sore surgery excision to comprehensive treatment,i.e.radiotherapy,endocrine therapy,molecular targeted therapy,immunotherapy or a combination of all therapies.The treatment for breast cancer is oriented to be diverse and accurate and has made impressive progress now.However,some treatments of the cancer don’t turn out well as expected.Therefore,molecular typing and new therapeutic targets need to be further explored.During the last decade,great progress has been achieved in the discovery and development of cycle-dependent kinase（CDK）inhibitors.It is hard for the first-generation pan-CDK inhibitors to make the balance between effectiveness and safety due to its multi-target active feature which implies they could not inhibit one CDK particularly.Later,the dual selective CDK4/6 inhibitors manifest a sign of controllable toxicity in the clinic application,which contributes to promoting CDK research towards selective inhibition.Recently,CDK4/6 inhibitors have been authorized to treat breast cancer patients who are hormone receptor-positive metastatic in clinic.CDK7 inhibitors manifest inhibition on breast cancer in vivo and in vitro,but the research data are not sufficient and need further exploration.THZ1 is a covalent inhibitor of CDK7 with dual functions in the cell.It not only suppresses CDKs inhibitory to regulate cell cycle,but blocks the activation of RNA polymeraseⅡ（RNAPⅡ）by suppressing the interaction between transcription factor IIH（TFIIH）and CDK7 to control cell transcriptional process.THZ1 is a small molecular inhibitor with exceptional anti-cancer potential,which is highly valued by researchers due to its outstanding anti-tumor effects on triple negative breast cancer（TNBC）in vitro.In our previous study,we discovered that compared with breast cancer p53^wtcells,breast cancer p53 mutant-type(p53^mut)cells were more sensitive to THZ1,which shows the up-regulation of tumor-suppressive p53^wtprotein of the inhibiting CDK7 and the down-regulation of carcinogenic p53^mutprotein level.Although without further study in the mechanisms,these results present that whether intracellular p53 mutates or not might lead to different outcomes of CDK7 inhibition.According to data in 2012,about 30%breast cancer cells carried p53^mut,meaning there were a lot of breast cancer cells manifested as p53^wt.This study explored methods to improve the sensitivity of THZ1 in breast cancer p53^wtcells,so as to expand the application range of THZ1.Nutlin-3 is a small molecular MDM2 inhibitor.By preventing MDM2 and p53from combination,it decreases p53 ubiquitination degradation mediated by MDM2 to stabilize intracellular p53.It is reported that Nutlin-3 induces p53 accumulation in non-genotoxic pathway,thus activating cell cycle arrest and apoptosis,making it a potential alternative to chemotherapy.Similar with other agents utilized in cancer treatment,Nutlin-3 is unlikely to be applied as a monotherapy.Studies verify that Nutlin-3 synergized with THZ1 suppress colon cancer-derived HCT116 cells,which not only decreases dose of sole drugs but attenuates the negative impact of“off-target”effects.Thus,the combination of Nutlin-3 and THZ1 applied in breast cancer cells may achieve stronger cancer inhibition effect than sole drug treatment.Objective:To explore the suppression capability of THZ1 combined with Nutlin-3 on breast cancer p53^wtcells viability,colony formation ability and apoptosis-induced effect of THZ1 and Nutlin-3 in breast cancer p53^wtcells;and to investigate the mechanism of THZ1 and Nutlin-3 combination on breast cancer p53^wtcells.Methods:1.Cell culture:Non-TNBC p53^wtcell lines（MCF7,ZR-75-1）,non-TNBC p53^mutcell line（SK-BR-3）,TNBC p53^wtcell line（DU4475）,TNBC p53^mutcell lines（MDA-MB-231,HS578T,HCC1937）were cultured in vitro.2.Cell viability measurement:The cells viability in each group was detected by cell counting kit 8（CCK8）assay.3.Colony formation ability measurement:Plant cells in 6 well plates and treat them with THZ1.10 days after reagents addition,cells were fixed,stained with crystal violet and taken picture.The effects of single drug treatment or combination treatment on colony formation ability were evaluated by comparing the formation of colony spots in each well.4.Apoptosis measurement:treated cells were collected and detected by flow cytometry.The results were displayed in pictures.5.Detection of proteins expression in breast cancer cells:p53,PARP,Cleaved PARP,Tom20,Histone H3 andβ-actin protein expression was measured by Western Blot assay.6.Statistical methods:Graph Pad Prime 8 software was used for statistical analysis and graph generating.One-way ANOVA and Student’s t test were used for statistical analyses to compare the differences between groups.P<0.05 indicates significant statistical difference,P<0.01 indicates extremely significant statistical difference.Results:1.The suppressive effect of THZ1+Nutlin-3 in breast cancer p53^wtcells viability and colony formation ability was more significant than Nutlin-3 or THZ1 sole treatment（p<0.05）.2.The apoptosis-induced effect of THZ1+Nutlin-3 in breast cancer p53^wtcells was stronger than Nutlin-3 or THZ1 sole treatment.3.The p53 expression and cleavage of PARP was higher in breast cancer p53^wtcells under THZ1+Nutlin-3 treatment than those under Nutlin-3 or THZ1 sole treatment.4.In breast cancer p53^wtcell nucleus and mitochondria,p53 protein level was higher under THZ1 combined Nutlin-3 treatment than Nutlin-3 or THZ1 sole treatment.5.In breast cancer p53^wtcells,the anti-tumor effect of THZ1 and Nutlin-3combination was weaker in cells which p53 expression was pre-inhibited than which p53 expression was not inhibited（p<0.05）.6.In breast cancer p53^wtcells,the anti-tumor effect of THZ1 and Nutlin-3combination was down regulated in cells pre-treated with p53 inhibitor compared with cells that did not receive p53 inhibitor pretreatment（p<0.05）.Conclusion:1.In breast cancer p53^wtcells,THZ1 combined Nutlin-3 inhibited viability,colony formation ability and induced apoptosis more significantly than sole drug treatment.2.In breast cancer p53^wtcells,THZ1+Nutlin-3 induced intracellular p53accumulation in nucleus and mitochondria.3.In breast cancer p53^wtcells,the anti-tumor effect of THZ1 and Nutlin-3combination was p53-dependent.