Cytoplasmic P120ctn Promotes Tyrosine Kinase Inhibitor Resistance In Lung Cancer Cells By Activating PAKs And Mediated EGFR Downstream Activation

Objective: Lung cancer is one of the most common malignant tumors worldwide.Non-small cell lung cancer(NSCLC)comprises 85–90% of all lung cancer cases.Significant advances in the elucidation of cancer biology and its molecular mechanisms have supported the development of molecular-targeted therapeutic agents against NSCLC.Targeted therapy involving the epidermal growth factor receptor(EGFR)-specific tyrosine kinase inhibitors(TKIs)gefitinib/Iressa? and erlotinib/Tarceva? as first-line drugs is recommended for the treatment of lung cancers with somatic mutations in EGFR.These small molecule-targeted therapeutic agents substantially improve survival,but patients eventually acquire resistance to them,but the manifestation of drug resistance during treatment is a major hindrance to efficacious EGFR-TKIs administration against lung cancer.Although the methods to overcome the drug resistance is gradual development following the new-generation TKIs,but it is important to choose the real patients for the treatment of first-line drugs,because it is essential for the patients to choose the economical and suitable treatment for them.The p120-catenin(p120ctn)is a member of the armadillo gene family.It regulates E-cadherin/catenin complex stability and small GTPase(RhoA,Cdc42,and Rac1)activation in a cell-dependent manner.Thus,it influences various cancer processes such as cell adhesion,survival,invasion,and metastasis.Recent results show that P120 ctn also has an important function of regulating EGFR signaling pathway,which suggests that P120 ctn may be involved in the EGFR-TKI resistance mechanism of lung cancer cells,but there is no direct research in human solid tumors.Elucidation of the putative molecular roles of p120 ctn in drug resistance mediation may also clarify its functions in lung cancer and uncover new biomarkers for targeted molecular lung cancer therapy.Detailed investigation of the TKI resistance mechanism may lead to improvements in prognostic accuracy and treatment strategies for patients with lung cancer.Methods: In this study,the expression pattern of p120 ctn was examined in patients with the EGFR gene mutation in 170 lung adenocarcinoma(Exon 19 mutation with 81 cases,Exon 21 mutation with 89 cases),and p120 ctn was found to have different patterns of expression even in the same mutation type.The therapeutic effect of EGFR-TKIs was investigated in these patients,and patients with an abnormal expression of p120 ctn were found to be more likely to have drug resistance.A gefitinib resistant lung cancer cell line “Hcc827-GR” was established in a step-wise manner from 0.1 to 1 mM,and alterations in the p120 ctn expression pattern were also observed in vitro.We aimed to detect the correlation between the expression pattern of p120 ctn in solid tumors and the therapeutic effect of EGFR-TKIs.Secondly,we explored the expression pattern of phosphorylated PAKs in NSCLC and their potential value as drug targets for treating cancer.The expression and prognostic significance of phosphorylated group I PAKs were evaluated in 182 patients with NSCLC.Immunohistochemical analysis revealed low group I PAK expression in normal lung tissues and increased expressed in the cytoplasm,particularly in lung squamous cell carcinoma.Western blot was used to detect the expression of group I PAK immortalized human bronchial epithelial HBE,human lung adenocarcinoma A549(wild-type EGFR/mutant K-Ras),NCI-H1299(wildtype EGFR/mutant N-Ras),lung SCC LK-2(wild-type EGFR),and SK-MES-1(wild-type EGFR).We used group I PAK inhibitor(IPA3)to specifically decrease group I PAK activity in human lung cancer cell lines.By MTT and flow cytometry in lung cancer cell line,we found that decreased group I PAK activity inhibited cell proliferation and combined IPA3 and EGFR-TKI(gefitinib)treatment inhibited cell proliferation in an obvious manner.In the third,IHC detected p120 ctn Y335 phosphorylation in lung cancer specimens cytoplasmically expressing p120 ctn.We analyzed the relationship between cytoplasmic p120 ctn location and p120 ctn Y335 expression.Immunoprecipitation,immunoblotting,and RT-qPCR evaluated p120 ctn translocation,PTP-PEST,and p120 ctn Y335 in response to TKI treatment.Immunoblotting,protein activity analysis,and functional experiments(MTT?Transwell)examined the roles of cytoplasmic p120 ctn in regulating ERK and AKT activity and downregulating PAKs and Cdc42/Rac1 in HCC827 and H3255 cell lines.Results: 1.Patients with an abnormal expression of p120 ctn were observed to have a decreased curative effect of EGFR-TKIs,compared with patients with a normal expression of p120ctn(P<0.05).In the resistant cells Hcc827-GR,p120 ctn expression was upregulated in the cytoplasm compared with the parental Hcc827 cell.2.phosphorylated group I PAKs were undetectable(-)or weak(+)in normal bronchial epithelium.However,68.13 % of NSCLC samples had abnormal phosphorylated group I PAK expression,and expression was localized mainly in the cytoplasm in lung SCC samples(76.64%).Compared with the localization in lung SCC,56 % of lung adenocarcinoma samples showed moderate to strong group I PAK staining.Phosphorylated group I PAK expression correlates with,high TNM stage,of lung cancer abnormal group I PAK expression was associated with Histology(P= 0.003),lymph node metastases(P=0.024)and high tumor-node-metastases(TNM)stage(P= 0.015)in NSCLC patients and correlated with poor prognosis(P=0.017).The combination of group I PAK phosphorylation inhibitor(IPA3)and gefitinib strongly inhibited cell growth obviously than treatment with IPA3 or gefitinib alone.Combined IPA3 and gefitinib causes an increase in the proportion of G1-phase cells and a reduction in S-phase cells.3.Patients with NSCLC cytoplasmically expressing p120 ctn showed significantly increased p120 ctn Y335 phosphorylation.When first-generation TKI resistance was induced,p120 ctn translocated from the membrane to the cytoplasm.PTP-PEST was inhibited,PAKs were upregulated,and Cdc42/Rac1 was activated.Gefitinib treatment downregulated PTP-PEST protein in HCC827 and H3255 cells in a dose-dependent manner.PTP-PEST downregulation enhanced p120 ctn Y335 site phosphorylation and increased membrane-to-cytoplasm p120 ctn translocation.Gefitinib treatment induced cytoplasmic p120 ctn to activate ERK and AKT via different PAKs.PAK1/PAK4 downregulation attenuated ERK and AKT activation by cytoplasmically expressed p120 ctn.PAK2 downregulation did not weaken AKT activation but impeded ERK activation by cytoplasmically expressed p120 ctn.After Cdc42/Rac1 inhibition,cytoplasmic p120 ctn expression could not activate PAKs.Conclusions:1.the expression pattern of p120 ctn is associated with acquired resistance to EGFR-TKIs in lung cancer.2.The overexpression of Pak group I plays an important role in the development of lung cancer.In the lung cancer cells with low sensitivity to gefitinib,combination with IPA3 can significantly improve the sensitivity of lung cancer cell lines to gefitinib.3.Phosphorylation of P120 ctn y335 site may be one of the main reasons for cytoplasmic expression of P120 ctn in lung cancer.4.gefitinib inhibits ERK activity in the EGFR downstream signaling pathway,destabilizes the PTP-PEST structure,downregulates PTP-PEST protein,attenuates p120 ctn Y335 dephosphorylation,and promotes p120 ctn Y335 site phosphorylation.In this way,p120 ctn dissociates from the cell membrane and is shuttled to the cytoplasm in lung cancer cells subjected to TKI.5.Cytoplasmic p120 ctn activates various PAKs via Cdc42/Rac1 activation,continuously activates ERK/AKT in EGFR downstream,and promotes EGFR-TKI resistance in lung cancer cells.Results of the current study will help in clinically screening the lung cancer patient's subpopulation who would benefit from first-generation of EGFR-TKIs therapy.