Wee1 Inhibitor AZD1775 Inhibits The Growth And Metastasis Of Esophageal Squamous Cell Carcinoma And Its Molecular Mechanism

Background:Esophageal cancer(EC),a common malignant gastrointestinal tumor,that occuring in the esophageal epithelium.EC is the seventh most prevalent types of cancer and ranks as the sixth leading cause of cancer associated death worldwide.EC has two histological types: Esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinom(EAC).ESCC,as the most predominant histological form of the disease(>95%),especially in the China.Although advanced therapeutics in ESCC had been achieved recently,including chemotherapy and radiotherapy,have been used,5-year overall survival rate of ESCC patients is still lower than 20%,and the quality of life of ESCC patients has not been significantly improved,mainly due to late diagnosis,metastasis,and frequent tumor recurrence.Therefore,it is necessary to urgently find new treatment methods to improve the prognosis of ESCC patients.AZD1775 is an orally available,high specificity small-molecule inhibitor for Wee1 tyrosine kinase activity in clinical trials.Wee1 is a tyrosine kinase that plays a crucial role in halting the G2/M cell cycle checkpoint for DNA repair.AZD1775 inhibits tyrosine 15 phosphorylation of CDK1 by inhibiting Wee1 kinase activity,there by inhibiting the repair of damaged DNA in tumor cells.AZD1775 exerts antiproliferative effect in ovarian cancer,non-small cell lung cancer,acute lymphoblastic leukemia,ovarian cancer,colorectal cancer or lung cancer cells.Whether AZD1775 is active against ESCC has not been reported previously.Objective:This project takes ESCC cells as experimental research objects,and explores the effects of Wee1 selective inhibitor AZD1775 on ESCC cells in vitro and in vivo and its molecular mechanism.Methods:The expression of Wee1 in ESCC clinical tumor tissue was detected by immunohistochemistry,and western blotting and q RT-PCR were used to detect the expression of Wee1 in ESCC cells.Western blotting or immunofluorescence analysis was used to assay the effect of AZD1775 on the expression of p-CDK1Y15,p-HH3S10,γH2A.X.PI staining was used to evaluate the effect of AZD1775 on ESCC cells(EC109 and KYSE150)cycle distribution.The in vitro effects of AZD1775 on ESCC cells proliferation were determined in cells using the MTT assay.Cell viability and possible synergy between AZD1775 and 5-FU/CDDP was measured by MTT assay and Chou Talalay combined index method.The effect of AZD1775 on the formation of ESCC cells by plate clone and soft agar assay.Annexin V-FITC / PI double staining was used to detect the effect of AZD1775 on the apoptosis of ESCC cells.Western blotting analysis was used to detect the effect of azd1775 on the expression of Bcl-2,Bax,Bcl-XL,XIAP,survivin,PARP,Caspase-3,cyclochrome C and AIF in ESCC cells.The dual Rh123 and Hoechst33342 staining by flow cytometry was used to examine the mitochondrial transmembrane potential of ESCC cells.Wound healing assay was used to detect the effect of AZD1775 on ESCC cell migration.Transwell assay was utilized to evaluate the effect of AZD1775 on ESCC cell migration and invasion.Western blotting can detect the effect of AZD1775 on the MMP-2 and MMP-9.sh RNA was used to knock-down Wee1 in ESCC cells,and was used to determine the effects on the proliferation,clone formation,migration and invasion of ESCC cells.Furthermore,the effects of AZD1775 on ESCC cell growth were determined by subcutaneously injecting KYSE150 cells in the right flank of nude mice.Western blotting and immunohistochemistry were used to detect the related proteins in tumor tissue.H&E staining was used to detect morphological changes of ESCC cells in tumor tissues of nude mice.The effects of AZD1775 on metastasis of ESCC cells were determined by injecting KYSE150 cells through the lateral tail vein of nude mice.Results:We showed that Wee1 was highly expressed in ESCC cell lines and clinical samples.In addition,we demonstrated that AZD1775 significantly inhibited effect against Wee1 kinase in both tested ESCC cells at nanomolar concentrations.Moreover,AZD1775 effectively inhibited the proliferation of ESCC cells and induced apoptosis through mitochondrial dependent pathway.AZD1775 inhibited the migration and invasion of ESCC cells and reduced the expression of MMP-2 and MMP-9.In addition,knocking down Wee1 by sh RNA phenocopied the inhibitory effects of AZD1775 on ESCC cell proliferation,colony formation,migration,and invasion.However,combination treatment between AZD1775 and 5-FU or CDDP resulted in a significant increase in the dead cells in both KYSE150 and EC109 cells.More importantly,the in vivo experiments also demonstrated that AZD1775 potently inhibited ESCC cell growth and metastasis.Conclusions:In the present study,we showed that Wee1 was overexpressed in ESCC cells.AZD1775,a selective Wee1 inhibitor,that significantly suppressed the proliferation,migration,invasion of ESCC cells in vitro.AZD1775 could remarkably attenuate the growth and lung metastasis of ESCC cells in nude mice.In addition,AZD1775 had a synergistic inhibitory effect on cell growth when combined with 5-FU or CDDP.Our results suggest that AZD1775 might be a potential therapeutic drug for the treatment of ESCC,that warrants further clinical investigation.