A Novel Tumor Suppressor ZBTB1 Regulates Tamoxifen Resistance Through HER2 Transcriptional Repression In Breast Cancer Cells

Objective: The tumor suppressor ZBTB1 is a necessary regulatory factor for DNA damage repair and determines the development of lymphoid tissues.However,it is still unclear the role of ZBTB1 in the development of cancer.Here,we will study the effect of ZBTB1 on the proliferation of breast cancer cells through in vivo and in vitro experiments,and decipher the drug resistance reversal effect and specific mechanism of ZBTB1 on MCF-7 / TamR cells.Methods: 1.The effect of ZBTB1 on the proliferation of breast cancer cells(1)Observe the TCGA database,breast cancer tissue immunochemical staining(IHC)experiment and 90 patients with high-level ZBTB1 and low-expression ZBTB1 breast cancer patients follow-up survey to observe the expression of ZBTB1 in breast tissue and the impact on patient survival.(2)Construct breast cancer cell lines that over-express and under-express ZBTB1,and use Western blot to verify the protein expression of ZBTB1 in the constructed cell line;through MTT experiment,soft agar clone formation experiment and to detect the over-construction Effects of ZBTB1 cell lines expressing and underexpressing on cell proliferation ability and sphere formation ability.(3)Overexpression of ZBTB1 cells and control cells were injected into the second pair of fat pads in the abdomen of nude mice to establish a model of subcutaneous xenograft tumor in nude mice to study the effect of ZBTB1 on subcutaneous xenografts in mice.2.Reversal of drug resistance of ZBTB1 on MCF-7/TamR cells(1)Western blot experiments were used to detect and compare the expression of ZBTB1 in MCF-7 cells and MCF-7/TamR cells.Transfect the cells with ZBTB1 overexpression plasmid to construct MCF-7/TamR cell line overexpressing ZBTB1,and use MTT method and dual luciferase reporter gene experiment to detect MCF-7/TamR cells over tamoxifen after overexpression of ZBTB1 Sensitivity and the effect of ZBTB1 on ERα transcription activity.(2)MCF-7/TamR cells overexpressing ZBTB1 and control cells were injected into the second pair of fat pads in the abdomen of nude mice to establish a model of subcutaneously transplanted tumors in nude mice,and mice were treated with tamoxifen for 2 weeks while measuring tumor volume To study the effect of ZBTB1 on tamoxifen resistance in breast cancer.3.ZBTB1 reverses the specific mechanism of breast cancer cell tamoxifen resistance(1)Western blot experiments were used to detect and compare the expression of HER2 protein in MCF-7 cells and MCF-7 / TamR cells.Up-regulate the expression of ZBTB1 in MCF-7 / TamR cells,and observe the effect of ZBTB1 on the expression of HER2 gene at the protein level and RNA expression level.(2)ChIP chromatin immunoprecipitation(ChIP)experiment and co-immunoprecipitation experiment were used to detect the effect of ZBTB1 on the transcriptional regulation of HER2 gene and the interaction between ZBTB1 and ERα protein.Results: 1.TCGA data and IHC test show that ZBTB1 is lowly expressed in breast cancer tissues.The overall survival of patients with high expression of ZBTB1 is higher than that of the low expression group,suggesting that low expression of ZBTB1 is a sign of poor treatment of breast cancer patients.2.ZBTB1 overexpression plasmid transfected MCF-7 and T47 D cells,which increased the protein expression of ZBTB1 in two groups of cells,significantly inhibited the cell proliferation of two groups of cells,reduced the ability of two groups of cells to form balls,and also significantly inhibited the subcutaneous The growth of tumors and the tumorigenic ability of MCF-7 cells.Silencing the expression of ZBTB1 relieves the cell proliferation inhibitory effect of ZBTB1 on MCF-7 and T47 D cells.3.Western blot and MTT experiments showed that ZBTB1 was significantly under-expressed in MCF-7/TamR cells,up-regulated ZBTB1 expression,and restored the sensitivity of MCF-7/TamR cells to tamoxifen.Nude mouse subcutaneous transplantation tumor model test showed that overexpression of ZBTB1 significantly reduced the tumor volume and weight of mice.In addition,the dual luciferase reporter gene experiment found that overexpression of ZBTB1 reduced ERα transcription in MCF-7/TamR cells.4.Compared with MCF-7 cells,HER2 is highly expressed in MCF-7/TamR cells,up-regulating the expression of ZBTB1 in MCF-7/TamR cells,significantly inhibiting the protein and RNA expression of HER2 gene in MCF-7/TamR cells.ChIP experiment found that under the action of TAM drugs,the re-expression of ZBTB1 significantly inhibited the recruitment of AIB1 and MED1 in the promoter region of HER2 gene in MCF-7/TamR cells,and increased the recruitment of co-suppressors N-CoR and HDAC1.Moreover,ZBTB1 binds to ERα protein and occurs at the ERα binding site in the promoter region of HER2 gene.Conclusion: ZBTB1 is lowly expressed in breast cancer tissues,has the ability to inhibit the proliferation of breast cancer cells,and is a new inhibitory factor that mediates the development of breast cancer.In MCF-7/TamR cells,ZBTB1 can regulate the transcription of HER2 gene to regulate the sensitivity of MCF-7/TamR cells to tamoxifen.