The Mechanism Of Drug Resistance Of CDK4/6 Inhibitors In The Treatment Of Breast Cancer

As a high-risk malignant tumor,breast cancer is one of the important diseases that cause women’s death.The incidence and mortality of breast cancer are increasing year by year in the world,which has aroused widespread concern of mankind.Although the most effective treatment for breast cancer is surgical treatment,the number of patients with advanced breast cancer or recurrence after surgery remains high,and more effective treatments for these patients is urgently needed.With the increasing in-depth study of breast cancer,scientists have made a series of important research results,some of which have been applied to clinical treatments and achieved certain therapeutic effects.In recent years,especially after the diagnosis and treatment of diseases entered the molecular era,doctors formulated different treatment schemes according to more detailed molecular types of breast cancer,and achieved better therapeutic effects.At the same time,the development of new drugs to treat breast cancer has also made great progress.Among these new drugs,there are some star drugs,such as: trastuzumab,palbociclib,olaparib,and the like.Cyclin-dependent protein kinase 4/6(CDK4/6)inhibitors are one of the new drugs used in the clinical treatment of breast cancer in recent years.It has achieved good therapeutic effect at the initial stage of its use.However,with long-term and extensive use,it is inevitable that many patients with congenital or acquired drug resistance appear,which greatly limits the application of this drug and causes heavy health and economic burden.Therefore,it is of great significance to study the drug resistance of CDK4/6 inhibitors in the treatment of breast cancer,and it is also one of the important directions of breast cancer research.In this study,multiple sets of transcriptome data of breast cancer cells resistant and sensitive to CDK4/6 inhibitors were used for differential analysis.The differential genes of multiple data sets were crossed and overlapped,and the target genes were preliminarily screened.MCF7 and ZR75-1 cell lines resistant to CDK4/6 inhibitor were employed.The expression levels of candidate genes were verified by real-time quantitative PCR in sensitive and resistant cells.It was preliminarily determined that the Carboxypeptidase vitellogenic like(CPVL)may be a key factor regulating the resistance of breast cancer cells to CDK4/6 inhibitors.We verified that CPVL overexpression could promote the survival and clonogenic ability of breast cancer drug-resistant cells in vitro.Since CDK4/6 inhibitors inhibit tumor cell proliferation by blocking cell cycle progression,we next performed cell cycle experiments.We verified that overexpression or knockdown of CPVL could promote or inhibit the progression of drug-resistant cells from G 1 to S phase.On this basis,we also prove that overexpression or knockdown of CPVL can inhibit or promote apoptosis of drug-resistant cells.These results suggest that CPVL may be involved in the regulation of the resistance of breast cancer cells to CDK4/6 inhibitors.To further determine the molecular mechanism of CPVL regulating breast cancer cell resistance to CDK4/6 inhibitors,we downloaded the transcriptome dataset of breast cancer patients from The cancer genome atlas(TCGA)database,and found that the expression level of CPVL in breast cancer tissues may be related to the phosphatase and tensin homolog(PTEN)signaling pathway using gene set enrichment analysis(GSEA).Western blot analysis showed that CPVL could negatively regulate the expression of PTEN protein and its downstream cell cycle related proteins.CCK-8 assay and plate colony formation assay confirmed that CPVL promoted the resistance of breast cancer cells to CDK4/6 inhibitors by regulating PTEN protein.Further,CPVL affected cell cycle progression and apoptosis by regulating PTEN protein.In conclusion,we found that CPVL promotes breast cancer cell resistance to CDK4/6 inhibitors by regulating PTEN protein.Next,we investigated the molecular mechanisms by which CPVL is regulated in breast cancer drug-resistant cells.First,we predicted the transcription factors that may bind to the promoter region of CPVL via the bioinformatics web site.The transcription factor CCAAT enhancer binding protein beta(CEBPB)may activate the transcription of CPVL by RT-PCR and Western blot.The binding site of CEBPB to the promoter of CPVL was also identified.Since Deoxyribonucleic acid(DNA)methylation is one of the important ways to regulate gene expression,we focused on whether CPVL promoter region is methylated.We predicted the existence of methylation island in the promoter region by analyzing the sequence characteristics.Interestingly,the region of CEBPB binding to CPVL promoter was located in the methylation island.Further studies demonstrated that Thymine DNA glycosylase(TDG)negatively regulates the methylation level of methylation islands in the promoter region of CPVL by Co-immunoprecipitation of chromatin and methylation-specific PCR.TDG could also promote the transcription of CPVL under CEBPB-dependent conditions,upregulating the resistance of breast cancer drug-resistant cells to CDK4/6 inhibitors.We demonstrated that the CPVL/PTEN axis regulates breast cancer resistance to CDK4/6 inhibitors in vivo.The results showed that knockdown CPVL could inhibit the growth of subcutaneous tumor in mice,and the addition of PTEN inhibitor counteracted the antitumor effect of knockdown CPVL,which proved that CPVL could also promote tumor proliferation by inhibiting PTEN in mice.In conclusion,we found the molecular mechanism of CPVL/PTEN axis regulating the resistance of breast cancer cells to CDK4/6 inhibitors.These results provide a new strategy for exploring and solving the drug resistance problem in the process of CDK4/6inhibitor treatment of breast cancer.