Research On The Mechanism Of CHK1 In Targeted Therapy For Breast Cancer

BackgroundIn recent years,with the gradual increase in the incidence of breast cancer in China,it has leapt to the top of common malignant tumors in women,seriously affecting women's lives and health.Chemotherapy,one of the main treatments for breast cancer,accompanies the drug side effects and resistance inevitably.The molecular targeted intervention has become a breakthrough for optimizing or even replacing the traditional therapies.In previous studies,we confirmed that checkpoint kinase 1(CHK1)was associated with acquired resistance to neoadjuvant chemotherapy for breast cancer.CHK1 has been reported to be a promising target for therapeutic intervention in cancer.However,the research results of CHK1 targeted therapy in various laboratories are not consistent,and the regulatory mechanism of CHK1 in breast cancer is still unclear.Oestrogen-receptor(ER),progesterone-receptor(PR)and human epidermal growth factor receptor-2(HER2)are important molecular markers for evaluating the prognosis of breast cancer.At present,the relationship between the molecular heterogeneity of ER,PR and HER2 and the role of CHK1 targeted intervention has not previously been described in breast cancer.Therefore,it is necessary to conduct an in-depth discussion on the regulatory mechanism of CHK1 in the targeted therapy,and to clarify the significance of CHK1 in the treatment of breast cancer with various status of ER,PR and HER2.Starting with the relationship between CHK1 and ER,PR,HER2,this study revealed the application and mechanism of CHK1 targeted intervention in combination with adriamycin(ADR)chemotherapy and its single-agent antitumor effect to provide the medicine basis.MethodsWe analyzed CHK1 expression in breast cancer with different ER,PR and HER2 statuses and their co-expressed genes using The Cancer Genome Atlas(TCGA)database.We also compared the survival analysis results of CHK1 in breast cancer with different ER,PR and HER2 statuses using the Kaplan Meier Plotter.An ADR-resistant strain of MDA-MB-231,named MDA-MB-231/ADR,was constructed by in vitro drug-inducing assay.ER+/PR+/HER2-(MCF-7 and T47D)and ER-/PR-/HER2-(MDA-MB-231 and MDA-MB-468)cells were transfected with the pEnter-CHK1 plasmid,the mutant of CHK1 containing alanine in place of serines 317 and 345 or siRNA targeting CHK1 to perform the experiments in vitro.The knockdown and overexpression efficiency of CHK1 were detected by real-time quantitative PCR(RT-qPCR)and Western-Blot.To reveal the role of CHK1 inhibition in combination with chemotherapy,we performed the drug sensitivity assay and cell apoptosis analysis.Next,we measured the changes resulting from ADR treatment in levels of mRNA,protein and chemical modifications using RT-qPCR and Western-Blot.To find the mechanism by which CHK1 regulates ADR chemosensitivity,we pioneered a conjoint transcriptome analysis.Based on that,the potential targets were verified by western blot.In the study for the regulation of CHK1 by ADR,according to the microarray analysis,we performed screening process for histone methylation and deacetylation and siRNA assay.Moreover,we measured the single-agent antitumor activity of CHK1 inhibition in ER+/PR+/HER2-cells,including cell proliferation assay,cell cycle and apoptosis analysis.And according to the analysis of microarray data,the downstream of CHK1 was detected by western blot.Result1.Gene expression pattern and survival analysis of CHK1:Published data from TCGA showed that CHK1 was highly expressed in breast cancer and was correlated with the co-expression networks of ER and PR.Moreover,survival analysis as calculated using the Kaplan-Meier Plotter showed that low levels of CHK1 predicted better overall survival and recurrence-free survival in breast cancer.2.CHK1 inhibition enhanced chemosensitivity to ADR in ER-/PR-/HER2-breast cancer:The CCK-8 assay and apoptosis analysis using flow cytometry showed CHK1 knockdown enhances chemosensitivity to ADR in ER-/PR-/HER2-cells instead of ER+/PR+/HER2-ones.3.Activation of CHK1 by ADR depends on ER/PR status:RT-qPCR and Western blot showed that ADR promotes CHK1 expression and protein phosphorylation(Chk1-Ser-317 and Chkl-Ser-345)in ER-/PR-/HER2-cells;this was the opposite trend from ER+/PR+/HER2-.In addition,we measured the effects of over-expressed CHK1 and its kinase dead mutant(CHK1-S317A/S345A)on ADR sensitivity and confirmed that ADR toxicity was attributed to the kinase activity of CHK1.4.The mechanism by which CHK1 regulates ADR chemosensitivity in ER-/PR-/HER2-cells:Cell cycle analysis showed ADR induced G2/M arrest in ER-/PR-/HER2-cells.Based on the conjoint transcriptome atlas analyses and Western-Blot,we found that CHK1 inhibition caused cell cycle disorder by up-regulating UBE2S and down-regulating BubR1 and cyclin B1,and promoted apoptosis mediated MSX2 and BIM in ER-/PR-/HER2-cells with ADR treatment.5.CENPF-mediated transcriptional regulation of CHK1 by ADR:By microarray analysis,screening for gene expression regulation,siRNA assay,RT-qPCR and Western-Blot,we found that CENPF-CHK1 transcriptional regulation existed in breast cancer,and interestingly,ADR suppressed it in ER+/PR+/HER2-cells and enhanced it in ER-/PR-/HER2-cells which leading to the differences in the mRNA level of CHK1 for these two types breast cancer cells.6.CHK1 inhibition caused a direct anticancer effect in ER+/PR+/HER2-cells:The CCK-8 assay,EdU assay,colony formation assay and apoptosis analysis showed that CHK1 inhibition dramatically weakened proliferation and promoted the apoptosis rate in ER+/PR+/HER2-cancer cells but not in ER-/PR-/HER2-ones.The clinical relevance of CHK1 to recurrence-free survival in breast cancer with heterogeneous ER/PR status analyzed by the Kaplan Meier Plotter also supported our results.7.Fas,p21 and Eg5 mediated the regulation of ER+/PR+/HER2-cells' survival by CHK1:Based on microarray analysis,cell cycle distribution detection and Western-Blot,we found that,with CHK1 suppressed,Fas and p21 were upregulated while Eg5 was downregulated in ER+/PR+/HER2-cells,which triggered the occurrence of the cell cycle arrest and apoptosis.ConclusionIn breast cancer,CHK1 acts two distinct roles;one is characterized by conditional induction,and the other is essential for cell survival.And the conversion of CHK1'role mainly depends on ER/PR status which determines that CHK1 inhibition is a sensitizer for ADR toxicity in ER-/PR-/HER2-breast cancer and an independent damage factor in ER+/PR+/HER2-breast cancer.In ER-/PR-/HER2-breast cancer,CHK1 can be induced by ADR,which in turn affects chemosensitivity by regulating cell cycle arrest mediated by the mitotic checkpoint complex(MCC)-anaphase-promoting complex/cyclosome(APC/C)-cyclin B1 axis and apoptosis induced by MSX2 and Bim.However,in ER+/PR+/HER2-breast cancer,due to significant suppression for CENPF-mediated transcriptional activation of CHK1 induced by ADR itself,CHK1 inhibition fails to sensitize ADR toxicity.Interestingly,the role of CHK1 is reversed to be that essential to survival in ER+/PR+/HER2-breast cancer,mainly manifesting as direct antitumor activity of CHK1 inhibition mediated by p21,Eg5 and Fas.Therefore,we suggested that accurately positioning the role of CHK1 in vious ER/PR status was the key to the reasonable targeted medication.