YAP1 Promotes Erlotinib-resistance In Lung Adenocarcinoma Cells And The Underlying Mechanism

Background and Objective:Nowadays,lung cancer has been the leading cause of cancer-related death worldwide.In China,the morbidity and mortality of lung cancer remain high,and rank the first in the male,only secondary to breast cancer in the female.Representing approximately 85% of lung cancer cases,non-small cell lung cancer(NSCLC)is the most common subtype.As the research at molucular and genetic levels is progressing,the molecular target therapy aiming at driver genes of lung cancer has made enormous progress.The epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)represented by erlotinib and gefitinib have been the standard first-line treatment in advanced NSCLC patients harboring EGFR active mutation.And the progress-free survival is much better than that obtained by traditional therapies such as chemoradiotherapy.Unfortunately,drug resistance in patients inevitably occurs in 6 to 12 months after treatment.So the mechanisms of NSCLC EGFR-TKIs resistance have become a difficulty in lung cancer treatment at present.Yes-associated protein(YAP),located on human chromosome 11q22,is the major transcriptional co-activator in Hippo signaling pathway,which contains two molecular subtypes: YAP1 and YAP2.They regulate the expression of downstream gene by combining transcription factors.As research continues,the YAP1 gene in drug resistance has gradually become a hot area of research.Some researches have shown that YAP1 is highly expressed in NSCLC especially in lung adenocarcinoma.Meanwhile,YAP1 is up-regulated in NSCLC patients with cetuximab resistance.The AXL gene is an oncogene first isolated from leukemia cells.The protein encoded by this gene is a member of the receptor tyrosine kinase(RTK)family.It can bind to the protein growth arrest-specific 6(GAS6)to activate the downstream signaling pathway.Studies have revealed that the overexpression of AXL is related to the EGFR-TKIs resistance,and YAP1 that regulates AXL expression is also involved in the formation and development of malignant tumors such as hepatocellular carcinoma.However,it is not clear whether YAP1 regulates acquired erlotinib resistance in NSCLC EGFR-TKIs via AXL signaling pathway.Based on these facts,we plan to explore the effect of YAP1 and the potential mechanism underlying erlotinib resistance in lung adenocarcinoma using PC-9 cells and acquired erlotinib resistant PC-9(PC-9/ER)cells.This research uncovers a new effective target for cancer therapy and it provides a theoretical foundation accelerating the anti-cancer drugs development.Materials and Methods:1.Our team have established PC-9/ER cells.Based on them and PC-9 cells,we observed the morphology changes and used CCK-8 assay to detect PC-9/ER drug resistance.2.In PC-9 and PC-9/ER cells,qRT-PCR and Western blot were used to measure the expression changes of YAP1,AXL and p-AKT.And the location of YAP1 and AXL were measured by the methods of inderect immunofluorescence.3.Lentivirus carring YAP1-RNAi and negative control vector was packaged to infect PC-9/ER cells.The expression changes of YAP1,AXL and p-AKT were analyzed by qRT-PCR and Western blot before and after infection.CCK-8 assay was used to detect the changes in drug resistance.4.In PC-9 and PC-9/ER cells,we constructed lentivirus vector overexpressing AXL stably to rescue the AXL which is the target gene of YAP1.The expression changes of YAP1,AXL and p-AKT were analyzed by qRT-PCR and Western blot before and after infection.The changes in drug resistance were analyzed by means of CCK-8 assay.Results:1.PC-9/ER was developed by using high-dose(1-5?M)pulses of erlotinib and continued by means of low-dose(0.1?M)erlotinib.CCK-8 assay was used to measure the half maximal inhibitory concentration(IC50)and drug resistance index.The IC50 of PC-9 and PC-9/ER cell were(0.10±0.02)?M and(8.68±1.17)?M(P<0.05).The drug resistance index was 90.92±14.06.Compared with the PC-9 cells,the mRNA and protein of YAP1,AXL and p-AKT had higher expressions in PC-9/ER cells.2.We constructed a lentivirus vector harboring RNAi sequence targeting the YAP1 gene and infected the PC-9/ER cells.The inhibitory efficiency of YAP-RNAi was about 52% and the protein levels of YAP1,AXL and p-AKT were down-regulated.The IC50 of PC-9/ER decreased from(8.57±0.65)to(1.28±0.16)?M(P<0.05).Resistance index reduced to one seventh of the original.3.We constructed a lentivirus vector to overexpress AXL gene in PC-9 cells.The mRNA of AXL increased 34 folds and the protein levels of AXL and p-AKT were up-regulated accordingly.The IC50 of PC-9 cells increased from(0.11±0.02)to(1.41±0.36)?M(P<0.05).4.We constructed a lentivirus vector to interfere with YAP1 and overexpress AXL simultaneously in PC-9/ER cells.Compared with the PC-9/ER cells only infected with lentivirus vector harboring RNAi sequence targeting the YAP1 gene,the mRNA of AXL increased 2.6 folds and the protein levels of AXL and p-AKT were up-regulated accordingly.The IC50 of PC-9/ER cells increased from(1.23±0.15)to(2.90±0.27)?M(P<0.05).Conclusions:1.YAP1 gene is overexpressed in PC-9/ER cells compared with PC-9 cells.Overexpressing YAP1 contributes to erlotinib resistance in lung adenocarcinoma by activating AXL tyrosine kinase and p-AKT.2.Knockdown of YAP1 can partially restore the sensitivity to erlotinib in PC-9/ER cells and down-regulate the expressions of AXL and p-AKT simultaneously.3.Overexprssion of the AXL gene can enhance the resistance of PC-9 cells to erlotinib and upregulate the expression of downstream proteins AXL and p-AKT.4.Rescuing AXL tyrosine kinase further demonstrates that YAP1 regulates the role of target gene AXL in erlotinib resistance in lung adenocarcinoma.