Molecular Mechanisms Of Tyrosine Kinase Inhibitor Resistance Induced By Cytoplasmic/Nuclear Translocation Of Epidermal Growth Factor Receptor

Objective: Of all the malignant tumors,lung cancer currently has the highest incidence and mortality,with 85–90% of these cancers being non-small cell lung cancers(NSCLCs)(1-3).About one-third or more of the patients with NSCLC showed epidermal growth factor receptor(EGFR)19 del(54%)or 21 L858R(43%)(4).Therefore,targeted therapy involving the use of EGFR-specific TKIs gefitinib/IRESSA and erlotinib/Tarceva as the first-line drugs is generally recommended for the treatment of lung cancers with somatic mutations in EGFR.These targeted drugs have better objective response rate(ORR)and progression-free survival(PFS)than chemotherapy(5,6).However,more than 50% patients are predicted to develop TKI resistance after the administration of these two drugs.Although studies have shown that more than half of the cases of TKI resistance are due to the EGFR mutation T790 M,the mechanism of TKI resistance in the remaining 30% cases remains unclear(7).The EGFR family plays an important role in tumor formation and development.High EGFR expression can promote the occurrence and development of cancer and result in poor prognosis(8).In the past 10 years,several articles have reported that EGF can stimulate the translocation of membranous EGFR(mEGFR)into the cytoplasm,followed by its entry into the nucleus by binding to importin-? through its nuclear localization sequence(NLS)(9,10).This suggests that EGFR localization change between the cell membrane,cytoplasm,and nucleus.It has also been suggested that EGFR acts as a transcription factor in plasma/nucleus shuttle and plays an important role in tumor progression(11).Over the past 10 years,several studies have confirmed the important mechanism of nuclear EGFR(nEGFR)function.nEGFR promotes the activation of seven oncogenes(CCND1,iNOS,B-Myb,Aura Kinase A,COX-2,C-Myc,and BCRP)and promotes the development of tumors and the emergence of TKI resistance(12,13).Moreover,nEGFR can regulate RNA stability,promote protein translation,and ultimately induce resistance to radiotherapy(14-16).However,it is not clear whether mutant EGF can enter the nucleus and its mechanism of nuclear translocation is the same as that of the wild type.Its role after nuclear translocation also remains unclear.Recently,several studies have reported TKI resistance caused by inhibition of the Hippo pathway(17-20).The Hippo pathway was first discovered and studied in Drosophila melanogaster(21).Its main components include the upstream molecules(Fat,NF2,WWC1,AMOT,etc.),central kinase complex(MST1/2-SAV1-LATS1/2-MOB),and downstream effector molecules(YAP,etc),and target genes of the Hippo pathway in the nucleus(CTGF,CCNE,DIAP,etc.)(22).When the Hippo pathway is activated,MST 1/2 phosphorylates SAV1,SAV1 functions as a partner of MST 1/2 in promoting LATS1 phosphorylation,ultimately resulting in YAP phosphorylation,which binds to the 14-3-3 protein and is eventually degraded(23);When the Hippo pathway is inhibited,YAP is not phosphorylated by the central kinase complex and it translocates into the nucleus,activates the target genes(CTGF,CCNE,etc.)with its co-activator(TEAD),and ultimately promotes the proliferation,invasion,and drug resistance of cancer cells(24,25).Although TKI resistance is known to be related to the inhibition of the Hippo pathway,the specific mechanism underlying Hippo pathway inhibition is not fully understood.In this study,we investigated(i)whether cEGFR inhibits the Hippo pathway via some mechanism and ultimately leads to the nuclear translocation of YAP;and(ii)whether the nuclear translocation of EGFR is associated with the nuclear translocation of YAP.Such a detailed investigation into TKI resistance mechanism is essential to improve the prognosis evaluation and treatment strategy formulation for patients with lung cancer.Furthermore,it will also provide an experimental basis for preventing TKI resistance and developing new effective targeted drugs.Methods: Twenty-one puncture specimens from patients with advanced lung adenocarcinoma complicated with TIKs somatic cell mutation without TKIs treatment were collected from Liaoning Cancer Hospital.Firstly,immunohistochemical staining was used to detect the localization and expression of EGFR in puncture specimens.The relationship between the different localization of EGFR in lung adenocarcinoma cells and progression-free survival(PFS)was analyzed by statistical analysis(Chi-square test).After that,we used Gefitinib and Erlotinib to induce drug resistance in HCC827 cell line.During the process of inducing drug resistance,we monitored the changes of EGFR localization in cells by confocal laser scanning.Two drug-resistant cell lines named HCC827/GR and HCC827/ER were successfully induced.MTT,colony formation,Transwell and cell apoptosis were used to detect the proliferation,invasion,anti-apoptotic ability and IC50 changes of drug-resistant cells.Next,we compared the changes of the main proteins of Hippo pathway and the expression of p-EGFR before drug resistance by Western Blot.The changes of YAP nucleation and Hippo pathway activity were monitored by immunolaser confocal and luciferase reporting fund.The SPSS22.0 statistical analysis software was used to analyze the experimental data by t-test,and P<0.05 showed that the results were statistically significant.Results: We found that in 21 patients with advanced lung adenocarcinoma who were not treated with TKIs,there were two different cellular and cytoplasmic localizations of mutant EGFR,and the different localizations could affect the progression-free survival(PFS)of patients.Chi-square test confirmed that the PFS of patients with EGFR localization in cell membrane was significantly longer than that of patients with EGFR localization only in cytoplasm(411 + 107.6 days vs 202.5 + 96 days).In vitro experiments,we first chose the same EGFR 19 del mutation,but the IC50 of PC-9 cell lines with different EGFR localization(EGFR localization in cytoplasm)and HCC827 cell lines(EGFR localization in cell membrane).MTT assay showed that the IC50 of PC-9 cell lines was significantly higher than that of Erlotinib cell lines(P < 0.001).We selected HCC827 cell lines to induce drug resistance of Gefitinib and Erlotinib.It was found that EGFR gradually transferred from cell membrane to cytoplasm and finally to nucleus during the induction of drug resistance.2.By comparing the changes of the main proteins of Hippo pathway,p-EGFR and its downstream proteins before and after drug resistance,we found that the expression levels of p-MST1,p-LATS1,p-YAP in Hippo pathway were down-regulated,while the expression levels of YAP and its oncogenes CTGF and CCNE were up-regulated.At the same time,the expression of p-EGFR and its downstream major proteins were still in a low level.3.In the process of inducing drug resistance,we found that EGFR first translocated from cell membrane into cytoplasm,and then inhibited the activity of Hippo pathway.Mass spectrometry analysis and immunoprecipitation showed that EGFR in cytoplasm could bind to SIK2.Immunocoprecipitation(quantitative)confirmed that the binding of SIK2 to SAV1 increased after drug resistance,while the binding of LATS1 to MST1 decreased.After interfering with EGFR in drug-resistant cells,the binding of SIK2 to SAV1 decreased,and the binding of LATS1 to MST1 increased.4.In the process of inducing drug resistance,we found that EGFR and YAP entered the nucleus at the same time.It was confirmed by immunofluorescence and immunoprecipitation that EGFR and YAP co-localized in the nucleus and plasma of drug-resistant cells,and they could combine with each other.After interfering with YAP,the number of EGFR entering nucleus decreased,but after transfecting YAP,the number of EGFR entering nucleus increased.5.In vivo experiments,we demonstrated that the sensitivity of drug-resistant cells to TKI would be restored to a certain extent after YAP or SIK2 were stably knocked out.Conclusion: 1.PFS of patients with positive EGFR membrane was significantly longer than that of those with cytoplasmic expression in gefitinib treatment;2.TKIs induced drug resistance of lung cancer cells with membrane/plasma translocation of EGFR protein;3.EGFR translocated into plasma inhibited the activity of Hippo pathway and induced TKIs resistance.4.EGFR in cytoplasm inhibits Hippo pathway activity through SIK2.5.EGFR was transported into the nucleus after binding with YAP;6.Erlotinib sensitivity was partly restored after stably interfering with SIK2 or YAP in HCC827/ER cells.These results provide experimental and theoretical basis for the sensitivity of non-small lung cancer patients to TKIs and the development of new targeted drugs.