Mechanisms Of EGFR-TKIs Acquired Resistance Induced By NEDD4 In Non-small Cell Lung Cancer

Background and Objective:Lung cancer is the most common cause of cancer-related death worldwide.In China,its incidence and mortality is high,and has a tendency to further increase.Non-small cell lung cancer(NSCLC)is the most common pathology classification of lung cander,accounting for 80-85% of all the cases,with a 5-year survival rate of < 20%.Patients with activating mutation in epidermal growth factor receptor(EGFR)gene,exon 19 deletion or exon 21 L858 R mutation,occur in about 55% of all NSCLC cases in China,with a good reactivity to the treatment of EGFR-tyrosine kinase inhibitors(EGFR-TKIs).Thus,TKIs,such as erlotinib and gefitinib,have been widely used to treat these patients,often with excellent therapeutic results.Significantly prolong the progression-free survival(PFS).However,despite initial remission,recurrence within one or two years(median,12 months)with an inevitable acquired resistance to EGFR-TKIs.Therefore,explorations of the exact mechanisms and solutions to improve the sensitivity to TKIs are urgently required.A variety of reasons cause EGFR TKIs acquired resistance in NSCLC,the most common causes are EGFR T790 M mutation,MET gene amplification,HGF over-expression and PTEN decrease.in our previous study,decreased PTEN was found in HCC827/ER cells,NSCLC cells resistant to erlotinib.Gene of phosphate and recogniton does on chromsome ten(PTEN)is the secondary tumor suppressor gene,located in the chromosome 10q23,transcript an mRNA with 515 kb.PTEN protein is a central negative regulator of the PI3K/AKT signaling cascade and influences multiple cellular functions.It is reported that loss of PTEN is associated with TKI resistance and downregulation of PTEN was also identified in clinical specimens obtained from patients who acquired resistance to EGFR-TKIs.Evidence shows that NEDD4 is a Proto-Oncogenic Ubiquitin Ligase for PTEN,negatively regulate PTEN abundance via catalyzing poly-ubiquitination of PTEN and leading to proteolysis of PTEN protein in cells.It also has been reported that negative regulation of PTEN by NEDD4-mediated polyubiquitination is involved in tumorigenicity.Inverse relationships between the expression of NEDD4 and PTEN also have been observed in human NSCLC.However,whether this process is related to NSCLC secondary EGFR TKIs resistant is rarely reported.In this study,HCC827 cells,harboring 19 exons deletion,and HCC827/ER cells,resistance to erlonitib which is induced by HCC827 cells,were taken for the research,to explore the underlying mechanisms of the acquired erlotinib resistance induced by NEDD4.This research will uncover a new effective target for overcome EGFR-TKIs acquired resistance in NSCLC.Materials and methods1.HCC827 cell line was purchased from ATCC(ATCC?CRL-2868?).HCC827/ER cells were developed by Dr.Jing Han using HCC827 cells.The sensitivity of two kinds cells was detect and compared Using Cell Counting Kit-8(CCK 8).2.The expression of NEDD4,PTNE,and pAkt in HCC827 and HCC827/ER cells was measured using qRT-PCR and Western blot.HCC827/ER cells were treated with MG132 for 12 h,the change of PTEN protein was measured.3.Knockdown NEDD4 expression in HCC827/ER cells,the changes of NEDD4,PTEN,p-Akt,and the sensitivity of the cells were measured using qRT-PCR,Western blot,and CCK 8.4.Nude mouse transplantation tumor model was established,to further explored the change of sensitivity to erlotinib in HCC827/ER cells with NEDD4 knockdown.Results:1.The CCK-8 assay revealed the sensitivity of the HCC827 cells and HCC827/ER cells to erlotinib.HCC827/ER was clearly more resistant to erlotinib than HCC827,The IC50 of HCC827 and HCC827/ER cell were(0.10±0.12)μM and(11.6±0.90)μM(P < 0.05).The drug resistance index was 118.23±23.77.2.Increased NEDD4 and decreased PTEN at both mRNA and protein levels was found in HCC827/ER,compared with the levels in HCC827 cells.Increased p-Akt protein level was found in HCC827/ER cells.MG132 partially restore the expression of PTEN protein in HCC827/ER cells.3.Knockdown NEDD4 expression in HCC827/ER cells,partially restore the sensitivity of HCC827/ER cells to erlotinib,and up-regulate PTEN expression at protein level but not mRNA level,and inhibit PI3K/AKT signaling path way.The IC50 of HCC827/ER(LV-siNEDD4)and HCC827/ER(LV-EV)cells were(0.72±0.12)μM μM and(13.88±1.96)μM(P < 0.05).4.Decreased expression of NEDD4 enhanced the sensitivity to erlotinib and increased the treatment benefit from erlortintb in nude mice.Conclusions:1.NEDD4 over-expression plays an important role in erlotinib acquired resistance in NSCLC.2.PTEN degradation induced by NEDD4 promotes erlotinib acquired resistance in NSCLC cells.3.NEDD4 decrease partially restore PTEN protein level and the sensitivity to erlotinib in NSCLC cells.4.In vivo,NEDD4 decrease enhance the sensitivity to erlortintb in nude mice burdened NSCLC cells,confirmed that NEDD4 promotes erlotinib acquired resistance in NSCLC.