The Investigation Of WTAP On Regulating Pancreatic Cancer Malignant Phenotype And Associated Molecular Mechanism

BackgroundPanceatic cancer(PC),charactorized by insidious onset and rapid progression,is the most lethal malignancy among digestive tract tumors.During the past decades,no substantial progress was achieved in the treatment of PC,making radical resection remain the only treatment for cure.However,due to its high invasiveness and resistance to chemotherapy,the overall 5-year survival of PC is only up to 9%.Although the oncogenic role and its underlying mechanism has been confirmed in several malignancies,the relationship between WT1 associated protein(WTAP)and the onset/progression of PC still remain unkown.Our preliminary results identified WTAP as a potential oncogene in PC.Therefore,detailed investigation of the relationship between WTAP and the carcinogenesis/progression of PC is necessary and may provide insight for new treatment targets and evidence to improve PC prognosis.ObjectiveThe aim of this study is to investigate the role of WTAP on the malignant phenotype and prognosis of PC,and to unveil the underlying mechanism.MethodsWTAP expression was preliminarily assessed in twenty PC tissues and the invasion/migration of MIA PaCa-2 cells was examined after transient WTAP knock-down.Mono-clonal stable transductions were established using lentivirus.Cell proliferation assay,cell cycle assay and orthotopic xenograft model were performed to assess PC cell proliferation in vitro and in vivo.Transwell assay together with orthotopic xenograft model and cytotoxicity assay together with cell apoptosis assay were adopted to assess PC cell mobility and chemoresistance to gemcitabine respectively.Quantitative real-time PCR(qRT-PCR)was conducted to screen the potential downstream target of WTAP which was further confirmed in PC tissues by using Gene Expression Proling Interactive Analysis(GEPIA).Western blot were used to detect the expression of WTAP,focal adhesion kinase(Fak)and important components in Fak signaling pathway.Fak mRNA stability assay and RNA immunoprecipitation(RIP)were conducted to identify the interaction between WTAP and Fak.GSK2256098 was employed in the Fak inhibition assay.The relationship between WTAP expression and clinicopathological factors as well as its prognostic value were evaluated by immunohistochemistry(IHC)staining.Statistical methods included the Cox proportional hazards regression analysis,Mann-Whitney U test,Kaplan-Meier survival analysis,Pearson chi-square test and Student's t test.A P value of<0.05 was considered as statistically significant.Results1.The regulation of WTAP on malignant phenotype of PCWTAP expression was higher in PC tissues when compared with non-tumor tissues(P<0.05).Transient WTAP knock-down could inhibit in vitro migration/invasion in MIA PaCA-2 cells(P<0.05).In WTAP bi-directionally regulated MIA PaCa-2 and BxPC-3 cells,WTAP overexpression could promote migration/invasion and chemoresistance to gemcitabine(P<0.05).Consistently,the half inhibitory concentration(IC50)of gemcitabine in WTAP overexpression group was significantly higher than that in the control group(P<0.05).In contrast,WTAP knock-down had an opposite effect(P<0.05).However,no difference was observed on cell proliferation(both in vitro and in vivo)and cell cycle when WTAP was up-regulated or down-regulated(P>0.05).2.The molecular mechanism of WTAP in regulating malignant phenotype of PCThe qRT-PCR screening result showed that Fak mRNA significantly decreased in MIA PaCa-2 cells after transient WTAP knockdown(P<0.05).In WTAP bi-directionally regulated MIA PaCa-2 and BxPC-3 cells,WTAP overexpression could increase Fak mRNA and Fak expression(P<0.05).In addition,the phosphorylation of Fak(Tyr397),Src(Tyr416),AKT(Ser473)and Erkl/2(Thr202/Tyr204)were significantly elevated,although the total Src,AKT and Erkl/2 remained unchanged.On the contrary,when the WTAP was knocked down,opposite results were observed.In order to confirm the important role Fak-PI3K-AKT and Fak-Src-GRB2-Erkl/2 signaling pathways played in WTAP-induced malignant phenotype of PC,Fak inhibition assay was carried out.We found that GSK2256098 could significantly decrease the phosphorylation of Fak(Tyr397),Src(Tyr416),AKT(Ser473)and Erk1/2(Thr202/Tyr204)while the total Src,AKT and Erk1/2 remained unchanged.Moreover,the metastasis and chemoresistance in WTAP overexpression group was effectively inhibited by GSK2256098(P<0.05).Further mRNA stability assay and RIP assay demonstrated that WTAP could bind to and stabilize Fak mRNA(P<0.05).3.The prognostic value of WTAP in PCWTAP expression in PC tissues was significantly higher than that in non-tumor tissues(P<0.05).WTAP expression was positively correlated with gender,T stage and TNM stage(P<0.05).Kaplan-Meier survival analysis indicated that the overall survival of patients with high WTAP expression was significantly shorter than that of patients with low WTAP expression(P<0.05).Multivariate analysis demonstrated that high WTAP expression was an independent indicator for poor prognosis of PC patients(P=0.039,HR=1.855).Conlusions1.WTAP could promote in vitro/vivo metastasis and increase chemoresistance to gemcitabine in PC.2.WTAP could bind to and stabilize Fak mRNA,which,as a result,activated Fak-PI3K-AKT and Fak-Src-GRB2-Erkl/2 signaling pathways.3.GSK2256098(Fak inhibitor)could reverse WTAP-induced cell migration/invasion and chemoresistance to gemcitabine in PC.4.The expression of WTAP in tumor tissues was significantly higher than that in non-tumor tissues.High WTAP expression was an independent indicator for poor prognosis in PC patients.