CDK4/6 Inhibitor Palbociclib Enhances The Sensitivity Of RB-Proficient Triple-Negative Breast Cancer To Cisplatin

Background: Triple-negative breast cancer(TNBC)is a highly aggressive and heterogeneous tumor,and improper intervention in terms of time or method may lead to poor prognosis.Cell cycle is a complex sequence event essential for cell replication and function,and its aberration has been considered as a hallmark of carcinogenesis.Palbociclib(PD),as a selective CDK4/6 inhibitor,combined with endocrine therapy has been proven to be an effective treatment for women with hormone receptor(HR)-positive/human epidermal growth factor receptor 2(HER2)-negative advanced or metastatic breast cancer.However,there are few studies on its combination with chemotherapy drugs in TNBC.At present,the killing effect of chemotherapy drugs is mainly enhanced by increasing the amount.The pharmacological interactions of different drugs and the efficacy of their different medication sequences are not clear.This study aims to explore whether PD can enhance the anti-tumor effect of cisplatin(CDDP)on TNBC,and the optimal drug combination scheme and corresponding molecular mechanism.Part one: The alterations of key genes in the CDK4/6 signaling pathway in TNBCObjective: To analyze alterations of key genes in CDK4/6-cyclin D1-RB-E2 F pathway in TNBC patients.Methods: The DNA copy number data of patients diagnosed with TNBC were screened from the TCGA database,and then alterations of key genes in the CDK4/6-cyclin D1-RBE2 F pathway were visualized through the c Bio Portal online tool.Results: DNA copy number data of 171 patients diagnosed with TNBC were screened from the TCGA database.The amplification or deletion of cyclin dependent kinase inhibitor2A(CDKN2A)was observed in 15% of TNBC patients using the visualization tool,and deletion was more common,which was accounting for 72% of these mutations.CCND1,CDK4,CDK6,and E2F1 genes,which are associated with cell cycle,showed 1%?5%alterations in amplification.23% of TNBC showed amplification,deletion or other mutations of retinoblastoma(RB),of which amplification accounted for 10.8%.Conclusion: The alteration of some genes in CDK4/6-cyclin D1-RB-E2 F pathway is very common in TNBC,and alterations of the genes above lead to disorder of CDK4/6pathway and uncontrolled cell cycle.CDK4/6 inhibitors may be effective on some patients with TNBC.Part two: Three drug regimens of PD and CDDP were initially establishedObjective: To investigate the anti-tumor effects of three common drug regimens of PD and CDDP.Methods: 1.The m RNA expressions of RB gene in all breast cancer cell lines in our laboratory were detected through q RT-PCR.2.Flow cytometry was used to detect the effect of PD alone on cell cycle and apoptosis.3.Flow cytometry was used to detect the effect of three common drug regimens of PD and CDDP on cell apoptosis.Results: 1.The expression level of RB in the HR positive breast cancer cell lines was higher.Among the TNBC cell lines,the expression level of RB in the MDA-MB-231 cell line was higher,and the expression level of RB in the MDA-MB-468 cell line was lower.2.PD alone for 24 h could significantly block the cell cycle in G1 phase and had no significant effect on cell apoptosis in MDA-MB-231 cells,while in the MDA-MB-468 cells,PD had no significant effect on cell cycle or apoptosis.3.Since PD alone has no significant anti-tumor effect,it is necessary to explore whether PD combined with CDDP has significant effects on TNBC cells.Three common drug regimens were established: PD and CDDP(synchronous treatment with PD and CDDP for24h),PD to CDDP(PD for 24 h followed by CDDP for 24h),and CDDP to PD(CDDP for24 h followed by PD for 24h).However,there was no significant effect on apoptosis of cells by any of the above methods.Conclusion: PD has cycle arrest effect on MDA-MB-231 cells,but not on MDA-MB-468 cells.There is no significant difference between the anti-tumor effects of CDDP alone and three common drug combinations of PD and CDDP on MDA-MB-231 cells.Part three: Establishment of three novel drug regimens according to the effect of PD on the cell cycleObjective: To set three novel drug regimens of PD and CDDP.Methods: By changing the duration of PD on MDA-MB-231 cells,the fine regulation of PD on cell cycle was detected by flow cytometry,and three new drug regimens were established according to the results.Results: With prolonged PD treatment,its effect in blocking the cell cycle in MDAMB-231 cells was gradually strengthened.When PD treatment continued for 48 h,the proportion of cells at G1 phase peaked,then withdrawn for 48 h,the ratio of cells in G1 phase began to decrease.According to the above data,we set three novel drug regimens:PD+CDDP,CDDP-PD and PD-CDDP.PD+CDDP treatment was performed by treating cells with PD for 48 h with concomitant CDDP treatment for the first 24 h.CDDP-PD treatment was performed by treating cells with CDDP for 24 h and then withdrawing CDDP for 48 h before PD exposure for 48 h.PD-CDDP treatment was performed by treating cells with PD for 48 h and then withdrawing PD for 48 h before CDDP exposure for 24 h.Conclusion: According to the regulation effect of PD on cell cycle,three novel drug regimens were established: PD+CDDP,PD-CDDP and CDDP-PD.Part four: The effects of PD-CDDP on cell proliferation,DNA damage and CDDP sensitivity of TNBC cellsObjective: To investigate the effects of novel drug regimens on TNBC cells and confirm the most effective one.Methods: 1.Flow cytometry was used to detect the effect of three novel drug regimens on cell apoptosis.2.Immunofluorescence technique was used to detect the damage of three novel drug regimens to cell DNA.3.CCK-8 assay and clone formation assay were used to detect cell proliferation under novel drug regimens.4.The 50% inhibitory concentration(IC50)and combination index(CI)of CDDP were calculated by Graph Pad Prism software and Chou-Talalay method.5.Nude mouse models were established.The tumor volumes and weights were measured regularly.The expression of tumor Ki-67 was detected by immunohistochemical technique.Results: 1.Compared with other drug regimens,PD-CDDP regimen increased the apoptosis of MDA-MB-231 cells more significantly compared with CDDP alone.2.PD-CDDP regimen could increase the DNA damage in MDA-MB-231 cells more significantly than CDDP alone,which was manifested as the increase of ?H2AX positive rate.3.PD-CDDP regimen could inhibit the proliferation ability of MDA-MB-231 cells more significantly than CDDP alone.4.The IC50 of CDDP in the PD-CDDP regimen was 35.33?mol/L in MDA-MB-231 cells,which was significantly lower than that in control group(57.09?mol/L).The difference was statistically significant.PD in the other regimens could not significantly enhance the sensitivity of cells to CDDP.5.The CI value of different drug regimens in MDA-MB-231 cells was calculated,and it was found that the CI value of PD-CDDP regimen was <1,indicating a synergistic effect.The CI value >1 indicated an antagonistic effect and CI value =1 indicated an additive effect.6.Nude mouse models were established with MDA-MB-231 cells.The tumor volumes and weights were significantly decreased through PD-CDDP regimen compared with the control group,and the expression of Ki-67 was also significantly reduced,further proving that PD-CDDP regimen had more significant anti-tumor effect than CDDP alone in vivo.Conclusion: Among the three novel drug regimens,PD-CDDP regimen shows more significant anti-tumor effects than CDDP alone in vitro and in vivo in MDA-MB-231 cells.Part five: the correlation between CDK4/6 pathway and the efficacy of PD-CDDP regimenObjective: To explore the mechanism of PD-CDDP regimen.Methods: 1.Lentivirus transfection technology was used to transfer sh RB and its control plasmid into MDA-MB-231 cells,and RB-overexpressed plasmid and its control plasmid into MDA-MB-468 cells.Stable transfected cell lines were constructed,and the expression of RB m RNA and protein were detected by q RT-PCR and Western blot(WB)to determine the transfection effect.2.The cell experiments of Part four were repeated with sh-MDA-MB-231 cells treated with PD-CDDP regimen.3.The cell experiments of Part four were repeated with Vector/OE-MDA-MB-468 cells treated with PD-CDDP regimen.4.WB was used to detect the changes of key proteins in CDK4/6-cyclin D1-RB-E2 F pathway in NC-MDA-MB-231 and sh-MDA-MB-231 cells treated with PD-CDDP regimen,and the apoptosis and DNA damage of cells were verified again at the protein level.Results: 1.Sh-MDA-MB-231 cells treated with PD-CDDP regimen showed no significant changes in cell cycle,cell apoptosis,DNA damage and cell proliferation compared with CDDP alone.2.Compared with CDDP alone,OE-MDA-MB-468 cells treated with PD-CDDP regimen were significantly arrested in G1 phase.Their cell apoptosis and DNA damage were significantly increased,and cell proliferation ability was significantly reduced.3.In NC-MDA-MB-231 cells,the expression level of p-RB protein and downstream E2F1 transcription factor in PD-CDDP group was significantly lower than that in CDDP alone group.In sh-MDA-MB-231 cells,the above changes were not observed.4.The expression of cleaved PARP and ?H2AX were also increased in the PD-CDDP group more significantly than CDDP alone in NC-MDA-MB-231 cells,while there were no such effects in sh-MDA-MB-231 cells.Conclusion: PD increases the sensitivity of TNBC cells to CDDP in an RB-dependent manner.PD-CDDP only has more anti-tumor effects than CDDP alone in TNBC cells with high RB expression through the CDK4/6-cyclin D1-RB-E2 F pathway.