The Role Of Osimertinib Combined With PD0332991 In EGFR-TKIs Resistant Lung Cancer Cells

Background and purpose:lung cancer is one of the most common malignant tumors.Targeted therapy has become the most important treatment method for advanced non-small cell lung cancer patients,with strong specificity and mild adverse reactions,which greatly improves the prognosis of non-small cell lung cancer patients.However,most non-small cell lung cancer patients inevitably develop drug resistance after targeted therapy,leading to disease progression.Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)is a major challenge in the targeted therapy of non-small cell lung cancer.CDK 4/6 inhibitor PD0332991 can specifically inhibit the activity of CDK 4/6 and the progress of cell cycle.It has a significant effect in various malignant tumors such as breast cancer and multiple myeloma.Combined with anti-tumor drugs,it has a synergistic effect,which can improve the sensitivity of tumor cells to a variety of anti-tumor drugs,increase the therapeutic effect and delay the occurrence of drug resistance.The purpose of this study:1.To explore the relationship between the development of non-small cell lung cancer acquired resistance to EGFR-TKIs and abnormal cell cycle regulation;2.To explore the effect of CDK4/6 inhibitor PD0332991 combined with Osimertinib on acquired drug-resistant lung cancer cell line H1975AR;3.To explore the differential gene expression associated with acquired resistance to the third-generation EGFR-TKIs Osimertinib.Methods:Western blot was used to observe the expressions of CDK4,CDK6,CyclinD,Rb,P-Rb(S780),P-Rb(S795)and E2F1 proteins in normal bronchial epithelial cell lines,non-small cell lung cancer cell lines,EGFR-TKIs sensitive lung cancer cell lines and EGFR-TKIs acquired drug-resistant lung cancer cell lines.CCK8 assay,FACS,Transwell assay,and EdU cell proliferation assay were used to explore the effects of combined drug on the cell viability,cell cycle,cell apoptosis,cell migration,and cell proliferation of Osimertinib acquired drug-resistant lung cancer cell line H1975AR.Osimertinib acquired drug-resistant lung cancer cell line H1975AR was constructed as an animal experimental model to observe the effect of combined drug administration on tumor growth.Immunohistochemical staining Ki-67 was used to detect the proliferation of tumor cells.High-throughput sequencing explored the differential genes associated with the mechanism of drug resistance.Results:in this study,different expression of CDK4/6-CyclinD-Rb/E2F signaling pathway was found in EGFR-TKIs sensitive lung cancer cell lines and EGFR-TKIs acquired drug-resistant lung cancer cell lines,which confirmed that the occurrence and development of non-small cell lung cancer acquired resistance to EGFR-TKIs were related to abnormal cell cycle regulation.PD0332991 combined with Osimertinib could increase its inhibition on the cell viability of Osimertinib acquired drug-resistant lung cancer cell line H1975AR,induce cell apoptosis,lead to cell cycle S phase arrest,inhibit cell migration and proliferation,and inhibit tumor growth and tumor cell proliferation.RNA was extracted from Osimertinib,PD0332991 and combined treatment of H1975AR for RNA-seq.The sequencing results showed that the differentially expressed genes were mainly enriched in the cell adhesion pathway after combined treatment,and the Versican(VCAN)showed the most significant difference.Using siRNA interference technology,CCK8 experiments,Transwell experiment,EdU proliferation experiment,it was found that after knocking down VCAN,the sensitivity of Osimertinib acquired drug-resistant lung cancer cell line H1975AR to Osimertinib increased,the cell migration ability decreased,and the cell proliferation ability was significantly inhibited,suggesting that the acquired resistance of third-generation EGFR-TKIs osimertinib may be related to abnormal expression of VCAN gene.