Knockdown Of FOXO3a Induces Epithelial-Mesenchymal Transition And Promotes Metastasis Of Pancreatic Ductal Adenocarcinoma By Activation Of The β-catenin/TCF4 Pathway Through SPRY2

Background:Early invasion and metastasis are responsible for the dismal prognosis of pancreatic ductal adenocarcinoma(PDAC),and epithelial-to-mesenchymal transition(EMT)is recognized as a crucial biological progress in driving tumor invasion and metastasis,which plays an important role in it.However,the molecular mechanism by which EMT induces the metastasis of PDAC has not been fully elucidated.The transcription factor FOXO3 a is inactivated in various types of solid cancers and the loss of FOXO3 a is associated with EMT and tumor metastasis.This suggests that FOXO3 a can act as a potential biomarker for the prediction and therapy of tumor metastasis.As a regulator of receptor tyrosine kinase(RTK)signaling,Sprouty2(SPRY2)was recently proposed to be a tumor suppressor in a multitude of cancers since it exerts a crucial role in tumor cell proliferation,apoptosis,migration,and invasion.Interestingly,a study on mouse endothelium validates SPRY2 as a direct FOXO target gene that mediates endothelial cell morphogenesis and vascular homeostasis.Therefore,it is reasonable to hypothesize that SPRY2 might be modulated by FOXO3 a and is involved in FOXO3a-mediated EMT and metastasis.Objective:In this study,we sought to explore whether SPRY2 is involved in FOXO3a-mediated EMT and metastasis in PDAC.Methods:1.Immunohistochemistry was performed in 130 paired PDAC tissues and paracarcinomatous pancreatic tissues.2.Cell proliferation and apoptosis were assessed by cell counting kit and flow cytometry.3.Cell migration and invasion were evaluated with wound healing and transwell assays.4.The changes in m RNA and protein levels were estimated by q RT-PCR and western blot.5.BALB/c nude mice xenograft model was established to evaluate tumorigenesis and metastasis in vivo.Results:1.FOXO3 a expression was remarkably reduced in PDAC tissues,and correlated with metastasis-associated clinicopathologic characteristics and poor prognosis in patients with PDAC.Decreased FOXO3 a expression correlated with poor prognosis in PDAC cases.Clinicopathological analyses demonstrated that decreased FOXO3 a expression prominently correlated with depth of invasion,TNM stage,differentiated degree,lymph node metastasis,and distant metastasis in patients with PDAC.2.knockdown of FOXO3 a or SPRY2 promoted the proliferation and suppressed the apoptosis of PDAC cells.3.knockdown of FOXO3 a or SPRY2 induced EMT and promoted the migration and invasion of PDAC cells via activation of the β-catenin/TCF4 pathway.Moreover,silencing of SPRY2 reversed the suppressor effects induced by FOXO3 a overexpression on EMT-associated migration and invasion of PDAC cells,while blockade of β-catenin reversed the effects of FOXO3 a or SPRY2 loss.4.FOXO3 a knockdown decreased SPRY2 protein stability,whereas SPRY2 knockdown enhanced β-catenin protein stability.5.In vivo,FOXO3 a knockdown promoted the tumorigenic ability and metastasis of PDAC cells.Conclusions:Our study suggests that knockdown of FOXO3 a induces EMT and promotes metastasis of PDAC by activation of the β-catenin/TCF4 pathway through SPRY2.Thus,FOXO3 a may represent a candidate therapeutic target in PDAC.