The E3 Ubiquitin Ligase NEDD4-1 Is Involved In The Development And Chemoresistance By Regulating Expression Of PTEN In Lung Adenocarcinoma

Background Lung cancer is the leading cause of malignancy mortality all over the world,and its incidence has been increasing rapidly in rescent years.Non-small cell lung cancer(NSCLC)accounts for 80%of all malignancy tumors of lung,and adenocarcinoma accounts for 60%of NSCLC,which has become the most common hisitological type of pulmonary tumors.The main treatment of lung cancer is surgery and chemotherapy,in recent ten years,molecular targeted therapy has improved the prognosis of patients with lung adenocarcinoma.However,the prognosis of lung adenocarcinoma is still poor,even in patients with stage I tumor whose 5 year survival rate was also less than 30%after operation.Because most of the patients are diagnosised in the advanced stage,the prognosis are even more poor.Nowadays,although TKI targeted therapy can improve the survival,resistance to chemotherapy or TKI is still an important cause of metastasis,relapse and poor prognosis.Finding novel targets for molecular therapy is a useful approach to improve the prognosis of lung adenocarcinoma.The mechanism of tumor oncogenesis and drug resistance of lung adenocarcinoma is complex,including disorders of a variety of tumor suppressor genes and oncogenes.At present,the relationship between cell signaling pathway and tumor development or drug resistance has become a new focus of lung cancer research.The PTEN-PI3K/Akt cell signaling pathway is one of the most important intracellular signaling pathways,which can be activated by various growth factors.PI3Kactivation induces Akt phosphorylation which can activates many downstream target genes,such as BAD,mTOR,FKHR,GSK3 beta and MDM2,so as to promote cell survival,growth,proliferation and malignant transformation.PTEN(phosphate and tension homology deleted on Chromsome ten,PTEN),inhibitory molecules of this pathway,can antagonising PI3K/Akt cell signal pathwayactivitiy through dephosphorylateing the second messenger PIP3 to PIP2.In lung adenocarcinoma,the loss expression rate of PTEN is about 30～50%,which is the most common early molecular events of early stage of lung adenocarcinoma.Despite of gene mutation and deletion,the post-translational modification is antother important inactivation mechanism of PTEN.Ubiquitin modification process is the regulation way of PTEN degradation in cytoplasm proteasome system and protein stability of PTEN after translation.It has attracted much attention to PTEN-PI3K/Akt pathway because of the central role in many signal transduction pathways.At present,loss of PTEN protein and activation of PI3K/Akt signaling pathway are important mechanisms for promoting tumor cell malignant transformation and tolerance to chemotherapy in lung adenocarcinoma.NEDD4-1,an E3 ubiquitin ligase,is a member of the NEDD4 family.NEDD4-1 induces PTEN ubiquitination with the cooperative protein Ndfipl.Polyubiquitin of PTEN promote its cytoplasmic degradation and monoubiquitin can induce its import into the nucleus.Therefore,it regulates the stability and subcellular localization of PTEN,which may be the main the regulatory mechanism of PTEN post-translational modification.A number of studies have demonstrated that high expression of NEDD4-1 and loss of expression of PTEN exist in most human solid tumor tissues including lung adenocarcinoma,and are closely related to tumor progression and chemosensitivity.Recent studies have found that NEDD4-1 is an independent prognostic factor for certain malignancies,such as prostate cancer and breast cancer.However,the expression and prognostic significance of NEDD4-1 in lung adenocarcinoma is unknown.Studies on the mechanisms involved in regulating PTEN expression and the relationship between NEDD4-1 and chemoresistance are rarely reported.Aim Investigate the expression of E3 ubiquitin ligase NEDD4-1 in lung adenocarcinoma tissues,and analyse its association with multiple clinical pathological factors,and to investigate the patients prognosis.Study the effects of NEDD4-1 on cells growth,apoptosis,migration,invasion ability,phenotype of EMT and chemotherapy sensitivity by transfecting siRNA to silence NEDD4-1 in lung adenocarcinoma A549 cells and A549/DDP cells;preliminary analyze the regulation mechanism of NEDD4-1 to PTEN-PI3K/Akt signaling pathway;and to investigate the drug resistance related protein such as P-glycoprotein,LRP and MRP;research the effect on the expression of NF-kappa B and AP-1 transcription factors.This work will provide theoretical basis for lung cancer targeting gene therapy and reversal of drug resistance.Methods We retrospectively investigated the expression and significance of NEDD4-1,PTEN and p-Akt proteins in 135 paired ADC and adjacent non-cancerous tissue specimens using immunohistochemistry,at the same time,the expression of NEDD4-1 in 20 cases of fresh lung adenocarcinoma was studied by Western Blot and RT-PCR.We evaluated the relationship between NEDD4-1 expression and clinicopathologic characteristics and prognosis.The effects of NEDD4-1 on proliferation,migration,and invasion were examined in A549 cells and in A549/DDP in vitro by transfection of siRNA to NEDD4-1.The chemosensitivity to drugs of lung ADC cells was assayed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-suifophenyl)-2H-tetrazolium)(MTS).The migration and invasion changes of lung adenocarcinoma cells transfected with siNEDD4-1 were detected by transwell cell tests and apoptosis was detected by flow cytometry.Reverse-transcription quantitative polymerase chain reaction(RT-PC)and western blotting analyses were used to determine PI3K/Akt activity.Detect the expression of apoptosis protein BAD,Bcl-2,Caspase and MDM2 as well as the expression of EMT phenotype protein E-cadherin and Vimentin with Western Blotting;Study on the distribution of PTEN changes in the cell with laser scanning confocal microscopy;Detect effects on PTEN ubiquitination state by immunoprecipitation.The expressions of multidrug resistance proteins such as P-gp,LRP,and MRP were also detected.The occurrence and development of NEDD4-1 and lung adenocarcinoma,the relationship with PI3K/Akt cell signaling pathway and drug resistance were analyzed.Results 1.NEDD4-1 was significantly overexpressed in lung ADC tissues,whereas its staining in normal lung epithelial cells was weak or negative(P<0.05).NEDD4-1 overexpression was associated with lymph node metastasis,advanced tumor-node-metastasis stage,chemotherapy resistance,and prognosis(P = 0.020,0.001,and 0.001,respectively),but not with other clinicopathologic characteristics.The loss rate of PTEN in lung adenocarcinoma was significantly higher than that of the non-neoplastic lung tissues(P<0.01).Its expressin was closely related to lymph node metastasis,TNM staging,histological type,EGFR status and chemotherapy response(P= 0.030,0.010,0.019 and 0.001,respectively).The expression of P-Akt in lung adenocarcinoma was significantly higher than that in peripheral lung tissue,and correlated with the clinical stage and chemotherapy tolerance(P<0.05).The expression of NEDD4-1 was negatively correlated to PTEN expression(r =-0.632,P=0.01),and positive correlation between NEDD4-1 and p-Akt(r =0.679;P = 0.05)Multivariate analysis showed that NEDD4-1 and PTEN were independent prognostic factors in patients with lung adenocarcinoma.2.NEDD4-1 knockdown in vivo decreased proliferation,migration,and invasion and improved apoptosis and chemosensitivity in A549 cells.The growth inhibition rate of A549 cells induced by SiNEDD4-lwas 42.97±3.04%,Transwell assay showed that cell invasion and migration ability were significantly decreased and agarose colony formation ability also decreased.Western blot assays showed that NEDD4-1 silencing increased the expression level and reduced the ubiquitination level of PTEN,while luorescence signal in cytoplasm and nucleus showed different degree enhancement by confocal.The activation of p-Akt decreased after NEDD4-1 silencing in A549 cells,suggesting the blocking of PI3K/Akt pathway,which regulates expression of various downstream target proteins,such as increasing expression of apoptosis protein BAD and mTOR,decreasing expression of transfectors of NF-kBp65.3.NEDD4-1 knockdown also significantly increased sensitivity to chemotherapeutic drugs such as cisplatin and paclitaxel in A549/DDP cells by MTS and the IC50 value decreased.Flow cytometry results showed that was apoptosis of cells increased after treatment by chemotherapy drugs with NEDD4-1 knockdown.Rhl23 experiments showed that the Rh123 effluent was lower in experimental group than that of the control.Western blot showed that enhanced PTEN expression and inhibited PI3K/Akt activity.Moreover,the expression of P-gp,LRP and MRP protein was decreased significantly and we also found the down-regulation of nuclear transcription facors NF-kb p65 and AP-1(all P<0.05).Conclusion 1.The results confirmed that the expression of NEDD4-1 and p-Akt in lung adenocarcinoma was significantly up-regulated than that in adjacent lung tissues,but PTEN expression was inactivation.The expression NEDD4-1 was negatively correlated with PTEN and positively correlated with p-Akt.All expression of them were associated with the adverse prognosis of lung adenocarcinoma and chemoresistance.It was suggested that NEDD4-1 may be closely related to the oncogenisis and development of lung adenocarcinoma,and that its high expression is closely related with the resistance phenotype of these patients.NEDD4-1 may be one of the predicting factors of the prognosis and potential targe of patients with lung adenocarcinoma.2.NEDD4-1 silencing by siRNA can effectively reduce the expression of NEDD4-1 and increased the expression of PTEN in the cell cytoplasm.SiNEDD4-1 inhibited cell growth,cell invasion and migration ability of A549,suggesting that high NEDD4-1 expression can promote lung cancer cell growth,survival,invasion and metastasis,which means NEDD4-1 is related to tumor recurrence,metastasis and prognosis in lung adenocarcinoma.In addition,the expression of BAD increased and the expression of mTOR and NF-kBp65 decreased,suggesting that high expression of NEDD4-1 might promote the invasion and metastasis of lung cancer cells and maybe play a important role in lung adenocarcinoma as a proto-oncogene.3.SiNEDD4-1 transfection silenced NEDD4-1,and decreased ubiquitination level of PTEN and increased PTEN protein expression in the cytoplasm and nucleus.SiNEDD4-1 transfection decreased the phosphorylation level of p-Akt and its downstream transcription factor NF-KbP65 and mTOR.It was suggested that NEDD4-1 showed oncogenetic effects through ubiquitination of PTEN and activation of PI3K/Akt pathway to induce cell growth,survival and the chemotherapy tolerance in lung adenocarcinoma.4.In lung adenocarcinoma cells,the sensitivity of A549/DDP cells to cisplatin and paclitaxel were increased by NEDD4-1 silencing,and the expression of multidrug resistance protein decreased,such as P-gp,LRP and MRP.It suggestes that NEDD4-1 knockdown can improve the sensitivity of the lung adenocarcinoma cells to chemotherapeutic drugs through regulating the apoptosis proteins and EMT related proteins or reducing the expressions of drug resistance related proteins.SiNEDD4-1 can partially reversed the chemoresistance of A549/DDP cells,which suggests that NEDD4-1 may become a molecular target protein to reverse drug resistance of lung cancer cells.The study of small molecule inhibitors of NEDD4-1 will provides new ideas and methods for the treatment of lung adenocarcinoma.In conclusion,the down-regulation of the expression of NEDD4-1 in lung adenocarcinoma,thereby inhibited the activity of PTEN and activation of PI3K/Akt cell signaling pathway,which promotes the growth of lung cancer cell invasion and migration,but also increase the lung cancer cell sensitivity to chemotherapy drugs,and part of the increased expression of lung resistance protein,then the occurrence and development of lung adenocarcinoma and its high expression,so it is related with poor prognosis.Therefore,we believe that NEDD4-1 may be a novel molecular target for the treatment of lung adenocarcinoma and provide a new approach for clinical treatment.