Kaempferitrin Overcome Gefitinib Resistance In NSCLC By Targeting A Novel Marker Of AurkC

Background:Lung cancer has a tremendous impact on the global public health.Previous investigations have revealed that mutations of epidermal growth factor receptor(EGFR)occur in 55%of Asian lung adenocarcinoma,while 75%of these patients are tolerated to EGFR-tyrosine protein kinase inhibitors(EGFR-TKIs).Although EGFR-TKIs has achieved great success in improving the overall survival and progression-free survival of patients with advanced NSCLC,resistance to FGFR-TKIs has become a big concern in clinical trials.Therefore,discovering new therapeutic markers or mechanisms of drug resistance will facilitate understanding of NSCLC heterogeneity,which is beneficial for developing new treatments to s overcome drug resistance and mitigate the survival of NSCLC patients.Aneuploidy and chromosomal instability are prone to produce clones with tumor characteristics and aggravate the tumor heterogeneity,leading to drug resistance and recurrence in cancer.Aurora kinase family members play crucial roles in mitotic entry,spindle assembly and cytokinesis,which are closely associated with chromosome aneuploidy and genomic instability.High clinical failure rates of developing drugs targeting Aurk A and Aurk B have frustrated researchers over the past decades.However AurkC has only been found in testicular and tumor tissues without expressions in other tissues.Hence,AurkC is supposed to be a promising drug target for chemotherapeutics.Objective:1.To clarify whether AurkC is a novel diagnostic and therapeutic marker for EGFR-TKI resistance.2.To explore the expression regulation mechanism of AurkC and tumor resistance-specific signaling pathways in TKI resitant NSCLC.3.To determine kaempferitrin is a specific inhibitor of AurkC and its therapeutic effect to overcome TKI-resistant NSCLC.Methods:1.Clinical specimens of NSCLC patients with EGFR activation mutation(128 cases)and paracancerous(140 cases)were collected and prepared into tissue microarray(TMA).Immunohistochemical staining(IHC)was performed to detect the expression levels of Met and AurkC proteins.Combined with the immunohistochemical score and clinical data of the patients,Kaplan-Meier method was used to analyze whether there was a correlation between the overall survival of the patients and the expression level of AurkC protein,and Pearson's Correlation Coefficient was used to analyze whether the expression level of AurkC protein was correlated with the expression level of MET protein.2.Western Blot was used to detect the protein levels of Aurks and MET in gefitinib sensitive(HCC827)or resistant cell line(HCC827GR).3.The levels of AurkC and MET were reduced or overexpressed in HCC827GR cells and HCC827 cells by gene silencing or overexpression.Western Blot was used to detect the changes in apoptosis-related molecules(Cleaved-PARP),cell cycle-related molecules(Cyclin B1,P27),and key molecules in the known signaling pathways of AurkC and MET.Then,TMT labeling quantitative proteomics was used to identify unknown regulatory molecules of AurkC.MTT method and soft agar colony formation were used to detect the cell proliferation in anchor-independent growth ability.Flow cytometry was used to detect the number proportion of>4N cells,cell apoptosis and cell cycle in the AurkC knockout or overexpression cell lines.4.Docking model,Kinase profiler assay,in vitro kinase experiment and in vivo drug binding experiments were applied to determine wether kaempferitrin binds and inhibits the activity of AurkC.5.Gefitinib resistant cell lines were treated with kaempferitrin or gefitinib alone or in combination,respectively.MTT assay and softagar colony assay were used to detect cell survival rate and cell anchorage-independent growth.The levels of Cleaved PARP were detected by Western Blot.Cell apoptosis and cell cycle were tested by flow cytometry.6.The NOD-Prkdc^scid Il2rg^null(NPSG)mice were used to establish xenograft animal model,the subcutaneous xenograft tumors of gefitinib-resistant cells were treated with kaempferitrin or gefitinib,or in combination by caudal intravenous injection,respectively.The size of the tumors and body weight were observed.Results:1.AurkC was highly expressed in NSCLC patients with EGFR-activated mutations and HCC827GR cells with gefitinib resistance.The prognosis of patients with high AurkC expression level is poor.2.Pearson correlation analysis indicated that AurkC was positively correlated with MET expression.However,knocking out AurkC or MET would not affect eachother's protein expression.3.Knockout of AurkC or inhibition of its activity could inhibit the proliferation and anchorage-independent growth of HCC827GR cells,and promote G2 cell cycle arrest and apoptosis of NSCLC cells.Overexpression of AurkC could promote theproliferation and anchorage-independent growth of HCC827 cells,and increase the proportion of aneuploidy chromosomal cells.4.The phosphorylation of A variety of protein kinases,cycle-related proteins(CDK13(S413));MAPK signaling pathway related molecules(MAP2K4(S80),MAPK4K5(S336),ERK1/2(Y204/Y187);transcription factors(RREB-1(S1475/S42/S1653/S175/S1575)),HOXA3(S184))were decreased in AurkC knockout cells,and many new phosphorylation sites such as MET(S996/S1236/S1020),EGFR(S1104)were found.5.Kaempferitrin could inhibit the proliferation and anchorage-independent growth of HCC827GR cells,which highly expressed AurkC.6.Kaempferitrin could bind to AurkC and inhibit the activity of AurkC in vitro and ex vivo.7.The combined use of kaempferitrin and gefitinib inhibited the proliferation and anchorage-independent growth in HCC827GR cells.And the inhibition of the subcutaneous tumorigenic growth in HCC827GR cells was observed.Conclusion:1.AurkC is a potential novel marker of gefitinib-resistant in NSCLC.2.Drug resistance induced by AurkC may be associated with MAPK,cell cycle,cell acetylation related protein signaling pathway etc.3.Kaempferitrin is an inhibitor of AurkC and combination treatment with gefitinib can overcome the resistance of gefitinib.