The Notch-1/AP-1/miR-451 Signaling Axis Plays A Role In The Mechanisms Of Chemoresistant Phenotype In Human Lung Adenocarcinoma Cells

Background and ObjectiveWith the development and application of three generations chemotherapetic drugs including pacilataxel,docetaxel,gemcitabine and vinorelbine,the clinical response in non-small cell lung cancer(NSCLC)treatment was improvement.Among that,the objective response rate(OR)of docetaxel combined with platinum for advanced NSCLC was increased to nearly 40%,disease progression-free survival(PFS)reached nearly 4 months.However,there are still more than half of patients unable to benefit from chemotherapy as above,resulting in tumor progression.Chemoresistance is the most significant obstacle of the clinical chemotherapeutic efficacy in advanced lung adenocarcinoma(LAD).The mechanism involved in genetic change with multiple tumor drug resistance related proteins,and it can regulate various physiological and pathological processes by complicated signaling axis network.In recent years,studies were focused on molecular markers which could guide clinical individualized targeted therapy.Unfortunately,ET study published in 2014 ESMO conference and other two previous studies showed in 2013 ASCO annual meeting of the United States were all indicated the excision repair cross-complementing 1(ERCC1)protein could not predict the platinum-based chemotherapy efficacy and prognosis in NSCLC.The negative results of clinical studies undoubtedly alerted us that chemotherapy guided by molecular markers were still needed further detected.Therefore,research on clarify the mechanism of chemotherapy resistance and discover the important molecular targets still faces enormous challenges.Notch signaling pathway especially Notch-1 plays a remarkable role in cell fate determination and cell signal communication in organisma multicellularis,including stem cell maintainance,tumor differentiation,proliferation,apoptosis and chemoresistance.The Crosstalk was existed between Notch signaling pathway and miRNAs.This study aimed to explore the molecular targets which participated in human LAD chemoresistant phenotype formation,gene expression was artificial regulated by singliang pathway specific inhibitor or gene transfection,the effect of Notch receptor Notch-1 on chemoresistance in lung adenocarcinoma was validated in vitro and in vivo.On the basis of our previous studies on miRNAs in lung adenocarcinoma,crosstalk between Notch pathway and miRNAs was deeply investigated.Then,the function of miR-451 in chemoresistant lung adenocarcinoma was explored.After explicited activator protein 1(AP-1)as an key point with the help of bioinformatics analysis and biological methods application,tried to elucidate the underlying molecular mechanism of Notch-1/AP-1/miR-451/MDR-1 signaling axis involved in regulating chemoresistant phenotype formation in lung adenocarcinoma.Materials and Methods1.A number of 101 patients diagnosed with LAD were collected to detected Notch-1 expression.Among that,only 39 patients were enrolled because of adjuvant chemotherapy based on taxanes was received.Notch-1 expression was detected by immunohistochemistry staining.2.All cases were closely followed up and the overall survival time was identified from surgery date to the latest follow-up or death.The correlation between Notch-1 and clinicopathological factors or prognosis was assessed by statistical analysis.3.The human chemoresistant LAD cells SPC-A1/DTX and H1299/DTX were established previously.Combined with cDNA array results,real-time PCR and Western Blot were performed to detect Notch-1 expression.The time-dependent induction assay was designed to treat parental cells with docetaxel in different concentration(0,3,5?g/L)and concentration-dependent induction assays was observed to treat parental cells 5?g/L for different time(0,3,5,7days).Real-time PCR and Western Blot were employed to exam Notch-1 expression in various conditions.4.The expression of each receptor or ligand in Notch signaling pathway was identified with real-time PCR in order to test the efficiency of special Notch signaling pathway inhibitor DAPT.Chosen the most appropriate concentration for further study by increasing dose of DAPT treated screening experiment.The effect of DAPT in chemoresistant LAD cells in vitro was measured by CCK-8,colony formation and flow cytometric analysis in order to reflect chemosensitivity,proliferation ability,apoptosis process and cell cycle distribution.5.The xenograft tumor model subcutaneously injected by SPC-A1/DTX was employed to demonstrate the relationship between Notch signaling activity and chemoresistance in vivo.Four groups were randomly divided when the average tumor volume reached 100mm3,1mg/kg docetaxel and 100mg/kg DAPT were used respectively or collectively via intraperitoneal injection,normal saline(NS)was normalized as control.Hematoxylin and eosin(H&E)staining and immunohistochemistry staining(IHC)were performed to verify the tumor proliferation ability.6.Since Notch-1 was high expressed in SPC-Al/DTX and H1299/DTX in comparison with parental SPC-A1 and H1299.Notch-1 short hair RNA plasmid(pGPU6/sh-Notch-1)and Notch-1 overexpression plasmid(pcDNA3/Notch-1)were introduced and transfected into LAD cells.Stable transfected cells were selected by G418.The function of Notch-1 in chemoresistant LAD cells in vitro was measured by CCK-8,colony formation and flow cytometric analysis in order to reflect chemosensitivity,proliferation ability,apoptosis process and cell cycle distribution.7.Subcutaneous xenograft tumor model were established by chemoresistant LAD cells stably transfected with shRNA-Notch-1.Docetaxel was treated via intraperitoneal injection twice a day for 2 weeks.Tumors were finally observed under microscope with H&E staining and IHC analysis.8.Crosstalk was existed between miRNAs and Notch signaling pathway,in particular Notch-1.Combined with results of miRNA array and our previous research on five miRNAs(miR-200b,miR-100,miR-451,Let-7c and miR-650),q-PCR was performed to detected expression of each miRNA after Notch-1 inhibition.MiR-451 was selected for further study.The gain of function assays of miR-451 was investigated by CCK-8,colony formation and flow cytometric analysis.Rescue experiment was exhibited by western blot after co-transfected with shRNA-Notch-1 and miR-451 inhibitor.9.Three open acess online miRNA databases(Pictar,TargetScan,and MirBase)were applied to predict targets of miR-451.MDR-1 was identified as a preferred downstream target of miR-451 by Luciferase activity assay,MDR-1 expression were detected by Western blot after regulated by Notch-1 or miR-451 in LAD cells10.About?3kb in miR-451 upstream was analyzed as promoter by TFSEARCH(ver.1.3),and was further confirmed by Chromatin Immunoprecipitation(ChIP)and Luciferase activity assay.Rescue experiment was exhibited by western blot after co-transfected with GV144/c-Jun and miR-451 inhibitorResults1.Notch-1 could be used as a biomarker to detect in different pathological and histological subtypes in LAD for diagnosis and prognosis.According to the response to chemotherapy,complete response,partial response or stable disease were defined as higher sensitivity.Progressive disease was defined as lower sensitivity.Notch-1 was upregulated in lower sensitivity group2.In patients diagnosed with advanced LAD and treated with taxanes chemotherapy,positive Notch-1 expression was found to be close related to lymph node metastasis(p=0.023),recurrence(p=0.014)and poor prognosis(p=0.026).A significantly association was revealed among higher Notch-1 expression,advanced TNM stage and poor survival time,including disease-free survival(DFS)and overall survival(OS).Notch-1 was also observed to act as an independent poor prognostic factor in LAD.3.The cDNA gene arrays result was previously told that Notch-1 expression was 34.5 fold higher in SPC-Al/DTX than in parental SPC-A1.Notch-1 was remarkable increased in docetaxel-resistant cells when compared with parental ones.And Notch-1 was gradually elevated along with the concentration increased or time extension.4.DAPT effectively suppressed most of Notch receptors or ligands,including Notch-1.10?M DAPT was chosen as the most suitable concentration.DAPT showed synergistic effect with docetaxel,inducing an obviously reduce in IC50 value,inhibiting proliferation,increasing apoptosis and mediating G2/M phase arrest in chemoresistant LAD cells(p<0.05)while G1 and S phase was decreased simultaneously.5.The tumor volume curve and final tumor weight of chemotherapeutic and inhibition combination group were significantly smaller and lighter than every other groups.Downregulated Notch-1 was discovered after DAPT treated.Decreased positive rates of proliferation markers ki-67 or proliferating cell nuclear antigen(PCNA)and predominant nuclear fragmentation was observed in combination treated tissues.6.Notch-1 expression was sharply decreased in SPC-Al/DTX and H1299/DTX transfected "with sh-Notch-1,while increased in SPC-Al and H1299 transfected with pcDNA3/Notch-1.The IC50 of docetaxel in sh-Notch-1-transfected chemoresistant LAD cells were completely reduced when compared with control.Conversely,elevated IC50 value was induced by overexpressed Notch-1 in parental SPC-A1 or H1299.Colony formation assays indicated Notch-1 could promote proliferation ability and chemoresistant phenotype.In addition,Notch-1 mediated an increased in G1 phase and G2/M phase of cell cycle and a dramatic reduced in S phase(p<0.05).Inhibition of Notch-1 resulted in a dramatic increase of apoptosis in docetaxel-resistant LAD cells.7.Final xenograft tumor weight formatted by SPC-A1/DTX and H1299/DTX transfected with shRNA-Notch-1 was surprisingly smaller in compared with SPC-A1/DTX and H1299/DTX transfected with control vector.Tumor growth curve derived from shRNA-Notch-1 transfected chemoresistant LAD cells were shown gently growth tendency when compared with control group.Decreased positive rates of proliferation markers(ki-67 and PCNA)and predominant nuclear fragmentation were observed in shRNA-Notch-1 transfected chemoresistant LAD cells.8.MiR-451 was sharply increased in SPC-Al/DTX transfected with sh-Notch-1 and it was downregulated in docetaxel resistant LAD cells compared with parental cells.MiR-451 could lead the IC50 value decreased,proliferation capacity reduced,apoptosis rate increased and predominat G2/M phase arrested in SPC-A1/DTX and H1299/DTX.Moreover,miR-451 was inversely interacted with Notch-1.Inhibition of miR-451 could partly reverse the effect mediated by Notch-1 suppression9.MDR-1 was a direct downstream target of miR-451,the expression was higher in SPC-A1/DTX than that in SPC-Al.Meanwhile,MDR-1 expression was heightened in parental SPC-A1 with miR-451 inhibited or Notch-1 overexpressed,and was reduced in SPC-A1/DTX transfected with pcDNA3/miR-451 or sh-Notch-1.The DAPT execution easily caused MDR-1 decreased,and MDR-1 expression could be partly restored in miR-451 inhibited simultaneously.10.Five complementary binding sites of Activator proteinl(AP-1)were analyzed in miR-451 promoter.AP-1 has already been demonstrated as a functional target of Notch-1,and its activation was associated with the phosphorylated level of c-Jun C-Jun phosphorylation was enhanced in SPC-Al/DTX treated with DAPT or sh-Notch-1 plasmid,it was also evaluated by luciferase assays after transfected with pGL3-miR-451 promoter.The probably corresponding region that c-Jun combined with miR-451 promoter is located in 705-957bp,986-1252bp,2402-2697bp and 2706-3005bp.MDR-1 could be restored by miR-451 inhibitor when c-Jun was upregulated in advanced.Conclusions1.Notch-1 could function as a potential biomarker and play a vital role in chemoresistant phenotype in human lung adenocarcinoma both in vitro and in vivo.Inhibition of Notch-1 induced an obviously IC50 reduction,proliferation suppression,apoptosis enhancement and mediated G2/M phase arrest in chemoresistant LAD cells.2.MiR-451 was inversely regulated by Notch-1,and it acted as a tumor suppressor and associated with chemosensitization,proliferation,apoptosis and cell cycle redistribution.3.MDR-1 was a direct target of miR-451,and AP-1 could probably bind in miR-451 promoter.Notch-1/AP-1/miR-451 axis dysregulation contributed to chemoresistant phenotype in lung adenocarcinoma cells.4.To our knowledge,this study is the first to reveal the critical role of Notch-1/AP-1/miR-451 axis in human lung adenocarcinoma and it will provide a novel strategy for reversing chemoresistance and clinical individualized treatment of lung adenocarcinoma.