The Mechanism Study Of Acquired Resistance Of EGFR-TKIs And Methods To Reverse It In NSCLC

Lung cancer is the first mortality desease in the world.Traditional treatments include surgery,radiotherapy and chemotherapy,but theses three treatments lack specificity.The emergence of targeted therapy has significantly improves the survival time and quality of life.But EGFR-TKIs acquired resistance has been a huge problem in the application.So know exactly the mechanism of acquired resistance of EGFR-TKIs,find ways to reverse EGFR-TKIs acquired resistance and potently research the cell cycle associated with acquired resistance are important.First,our previous results of gene analysis of three gefitinib-resistanct cells(PC-9/AB2,PC-9/AB11,PC-9/BB4)and gefitinib-sensitive cell(PC-9)showed that the cell cycle-related pathway Rb-E2 Fs signal pathway had the most sinigicantly changes.We used immunohistochemical staining(IHC)to demonstrate that lung adenocarcinoma is associated with Rb-E2 Fs signaling pathway in clinical specimens.Furthermore,western blot showed that PC-9,PC-9 / AB2 and HCC-827 cell cycle-related proteins showed abnormal expression of different types and levels compared with normal bronchial epithelial cells.So the occurrence of lung cancer and gefitinib acquired resistance are associated with dysregulation of cell cycle.Secondly,we used MTT assay,EdU assay,cell cycle and apoptosis to research the combination effect with cell cycle inhibitor,PD 0332991,and gefitinib in PC-9 and PC-9/AB2 cells.MTT assay: the survival reate in combination group is obviously low than other groups.EdU assay: the proliferation rate is 3.7%(PC-9)and 2.1%(PC-9/AB2)respectively and clearly lower than other single drug group.Apoptosis: the combination groups showed obvious apoptosis(23.66% in PC-9/AB2)than gefitinib groups(13.2% in PC-9/AB2).Cell cycle: The combination groups showed 30.6%(PC-9)and 23.75%(PC-9/AB2)cell in G1 phase compared with single drug group.These data had identified PD 0332991 could reverse the acquired resistance of gefitinib in vitro.Next we used gene chip analysis,real-time PCR and western-blot to approve the effect is work through the cell cycle regulation with Rb-E2 Fs passway.Subcutaneous tumor model in nude mice was used further verify the combination effect of the combination therapy with PD 0332991 and gefitinib.The IHC results showed Ki-67 positive rate is 3%,TUNNEL positive rate is 67% and CD34 positive rate is 4%,they all had significance.So PD 0332991 could potent the effect of geitinib and revers acquired resistance of gefitinib.Three lung cancer patients with gefitnib acquired resistance and metastasis in bone or brain after oral administration of gefitinib.We give these three patients both PD 0332991 and gefitinib orally.After three weeks,two of them had been in well control stage,one of them had well response,the metastasis in brain had disappeared and the pain in bone had controlled.The last,for the research of cell cycle regulation and lung cancer acquired resisitanc,we had also found the long non-coding RNA HOTAIR could regulate the cell cycle progression and lung cancer cell proliferation,invasion and migration.In the 67 pairs of lung cancer tissues,100% of tumor tissues had HOTAIR expression and only 42.65% corresponding normal lung tissue expressed HOTAIR.It’s indicating that HOTAIR is associated with lung cancer.The further data in NSCLC cell had shown that the percentage of cells in G1 phase had decreased and in S phase had increased in the NSCLC cell line transfected with HOTAIR.Also in the same cells,the proliferation increased about 20%,invasion increased about 30%,migration increased about 50%.When knockdown the expression of HOTAIR in the NSCLC cell,the results also have significant.Then western-blot and TOP/FOP flash reporter had used to know Rb-E2 Fs pathway,EMT and β-catenin pathway involved the function of HOTAIR in NSCLC.Last in mouse model verified the results in vitro.In conclusion,we had identified EGFR-TKIs acquired resistance is associated with dysregulation of cell cycle by Rb-E2 Fs pathway.Cell cycle inhibitor PD 0332991 could use to potent the effect of gefitinib and reverse the acquired resistance of gefitinib.In the further research of cell cycle in lung cancer,we had found lnc RNA HOTAIR could regulate the cell cycle,proliferation,invasion,migration,and this regulation in NASCL,the regulation was also associated with Rb-E2 Fs,EMT and β-catenin pathway.We thought it could be a new cell cycle inhibitor targeted site.