| Objective: Breast cancer is the most common malignant tumors in women,and its incidence is increasing year by year.Combined therapy of CDK4/6 inhibitors and endocrine therapy is the first choice for the treatment of advanced breast cancer patients with HR positive and HER2 negative.However,the resistance in the course of treatment seriously affects the survival benefit of patients.Therefore,in-depth study of the mechanism of CDK4/6 inhibitor resistance in breast cancer is an urgent scientific problem to be solved.Cancer / testicular associated antigen MAGEC2(Melanoma-associated antigen C2)is a protein expressed in testis and cancer tissues,and is highly expressed in many cancers,including breast cancer,and is related to its poor prognosis.However,it has not been reported that MAGEC2 regulates the resistance of CDK4/6 inhibitors in breast cancer.The results of this study will reveal the new mechanism of CDK4/6 inhibitor resistance of breast cancer cells from the new perspective of MAGEC2,and further elucidate the molecular mechanism that MAGEC2 activates PI3K/AKT/mTOR signal pathway to mediate CDK4/6 inhibitor resistance in breast cancer.The results of this study will provide new ideas for elucidating the molecular mechanism of CDK4/6inhibitors resistance in breast cancer,and provide theoretical and experimental basis for exploring new targets that can effectively reverse the CDK4/6 inhibitor resistance of breast cancer.Methods:1.Identification of resistance of breast cancer cells resistant to CDK4/6 inhibitors:The sensitivity of resistant cells and parent cells to CDK4/6 inhibitors was detected by CCK-8,cloney formation and flow cytometry,and the differential expression of cell cycle-related genes between resistant cells and parent cells was detected by Western blot and q RT-PCR.2.Verification of the differential expression of MAGEC2 and its bioinformatics analysis: Using Western blot and q RT-PCR to verify the high expression of MAGEC2 in CDK4/6 inhibitor resistant cells compared with parent cells in RNA-Seq results,TCGA database and Kaplan-Meier plotter database were used to analyze the differential expression of MAGEC2 in cancers and paracancerous tissues,breast cancer subtypes,pathological stages of breast cancer,and the effect of MAGEC2 on distant metastasis survival of breast cancer patients.3.Analysis of MAGEC2 expression and its clinical significance in breast cancer tissue samples: The clinical samples of 124 patients with advanced HR+/HER2-breast cancer treated with CDK4/6 inhibitors from 4 centers were collected,and the relationship between MAGEC2 expression detected by immunohistochemical staining and clinical benefits of these patients was comprehensively statistically analyzed.4.Cytology experiment: The model of overexpression and knockdown of MAGEC2 in breast cancer cells was constructed by lentivirus,and the relationship between the differential expression of MAGEC2 and resistance of CDK4/6 inhibitors was analyzed by CCK-8,cloney formation and flow cytometry,and the related pathway of MAGEC2 mediated CDK4/6 inhibitor resistance in breast cancer was predicted by enrichment analysis,which was further verified by Western blot.Cloney formation assay was used to verify whether pathway inhibitors could restore the sensitivity of breast cancer cells to CDK4/6 inhibitors.5.In vivo experiment: Tumor-bearing mice were used to verify the role of MAGEC2 in resistance of CDK4/6 inhibitors in vivo.Results:1.Identification of resistance of breast cancer cells resistant to CDK4/6 inhibitors:The sensitivity of CDK4/6 inhibitor resistant cells to CDK4/6 inhibitors was significantly lower than that of their parent cells,and the expression of resistance markers of CDK4/6 inhibitors,such as RB1,CDK6 and Cyclin E,were significantly different between the two kinds of cells.2.Verification of MAGEC2 differential expression: Compared with parent cells,MAGEC2 in CDK4/6 inhibitor resistant cells increased significantly at m RNA and protein levels.MAGEC2 was highly expressed in cancer tissues compared with adjacent tissues in many cancers,and higher in Luminal B type breast cancer than in Luminal A type breast cancer,and higher in M1 stage breast cancer than in M0 stage breast cancer.The distant metastasis-free survival time of patients with high expression of MAGEC2 was shorter than that of patients with low expression.3.Expression of MAGEC2 in breast cancer tissue samples and its clinical significance analysis: MAGEC2 was positive in 21.77% of advanced breast cancer tissues,and the progression-free survival of positive patients treated with CDK4/6inhibitors was shorter than that of patients with negative expression of MAGEC2,and the proportion of the best evaluation of Progressive disease was higher,and the positive expression of MAGEC2 in tissue samples corresponds to higher expression of Ki-67.MAGEC2 is an independent predictor of the risk of progression after CDK4/6 inhibitor treatment in patients with advanced HR+/HER2-breast cancer.4.Cytology experiment: After overexpression of MAGEC2 in MCF-7 and T-47 D cells,the IC50 of overexpression group was significantly higher than that of control cells.Compared with PBS treatment,the cloney formation ability and G1-S phase transition ability of Palbociclib treatment group were not significantly changed.At the same time,the PI3K/AKT/mTOR signal pathway of MAGEC2 overexpression group was significantly activated.After knocking down MAGEC2 in T-47 DR cells,the IC50 of knock-down group decreased significantly,and the cloney formation ability and G1-S phase transition ability of Palbociclib-treated cells were significantly lower than those of PBS treatment.At the same time,after MAGEC2 knockdown,PI3K/AKT/mTOR signal pathway was significantly inhibited,while T-47 DR compared with parent cells,PI3K/AKT/mTOR signal pathway was also significantly activated.In addition,the application of AKT inhibitor MK2206 could partially restore the sensitivity of MAGEC2 high expression cells to Palbociclib.5.in vivo experiment: At the in vivo level,CDK4/6 inhibitor-resistant cells could be significantly inhibited by CDK4/6 inhibitor after knocking down MAGEC2.Conclusion:1.MAGEC2 is highly expressed in breast cancer cells resistant to CDK4/6inhibitors and can predict the clinical benefits of CDK4/6 inhibitors in clinical specimens.2.MAGEC2 has the biological function of promoting the resistance of breast cancer to CDK4/6 inhibitors.3.MAGEC2 can activate PI3K/AKT/mTOR signaling pathway and promote the resistance of CDK4/6 inhibitors in breast cancer. |
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