Snail/FOXK1/Cyr61 Signaling Axis Regulates EMT And Metastasis Of Colorectal Cancer

Background and Objection:Accumulating evidence reveals that epithelial to mesenchymal transition(EMT),a well-recognized process by which cells from a differentiated epithelial state could convert into a dedifferentiated migratory mesenchymal phenotype,which plays an essential role in the regulation of embryogenesis,organ fibrosis,wound healing and cancer metastasis.The crucial events of EMT include decreasing the cell-cell adhesion molecule E-cadherin;increasing more plastic mesenchymal proteins such as vimentin,N-cadherin,and deregulating the canonical WNT/?-catenin signaling pathway.Emerging evidence indicates that EMT is of vital importance in obtaining invasive and migratory ability in colorectal cancer(CRC).A number of transcription factors(TFs),such as Twist,Snail,Slug,and zinc finger E-box binding homeobox 1(ZEB1)/2,are considered as the major inducers of EMT that repress E-cadherin directly or indirectly in CRC cells.Thus,CRC patients may benefit from targeted therapies that inhibit EMT.Forkhead box k1(FOXK1)consists of the winged-helix DNA-binding domain and the N-terminal and C-terminal transcriptional domains.FOXK1 as one of the transcription factors are rising as a novel and promising of proteins in EMT.Previous evidence had indicated that FOXK1 acts as a tumor oncogene in colorectal cancer.We recently found that FOXK1 induced tumor cell EMT,maintained the invasive potential of gastrointestinal cancer.Snail also is a zinc-finger transcription factor that triggers EMT by directly repressing E-cadherin expression.Snail overexpression in different epithelial cells leads to dramatic conversion towards a fibroblastic phenotype at the same time that E-cadherin expression is lost,and invasive and migratory properties are acquired.Snail binds to the E-box sequence CANNTG in the promoter regions of gene.However,the effects on the EMT procedure between FOXK1 and Snail for CRC have not been investigated.Cysteine-rich 61(Cyr61)is a member of the CCN(Cyr61/CTGF/Nov)protein family,which consists of Cyr61(CCN1);connective tissue growth factor(CTGF/CCN2),nephroblastoma-overexpressed(Nov/CCN3).Cyr61was found to be up-regulated in many cancers,such as breast,esophagus and colorectal cancers.Several studies reported Cyr61 promotes EMT and tumor metastasis.Using DNA microarray analysis in the present study,we found that FOXK1 up-regulated Cyr61 expression in CRC cells.However,the molecular mechanism by which FOXK1 regulates Cyr61 expression remains unknown.In this study,we present evidence that FOXK1,which is a direct transcriptional target of Snail,promotes CRC invasion and metastasis by transactivating Cyr61 expression.In addition,Snail/FOXK1/Cyr61 signaling axis is associated with CRC metastasis and indicates poor prognosis.Methods:We evaluated that messenger RNA(mRNA)and protein expression levels by quantitative RT-PCR,western blot,immunofluorescence,tissue microarrays(TMA)and immunohistochemistry(IHC)assays.The migration and invasive abilities of colorectal cancer(CRC)cells was assessed in vitro.Tumour metastasis was conducted in nude mice in vivo.Results:A positive correlation was observed between the expression patterns of FOXK1 and snail in CRC.Luciferase promoter reporter and chromatin immunoprecipitation assays demonstrated that Snail directly binds to and activates the human FOXK1 gene promoter.Moreover,Snail-FOXK1 axis promotes epithelial-mesenchymal transition(EMT)-mediated colorectal cancer invasion and metastasis.FOXK1 and Snail expression were correlated with tumor progression and represented significant predictors of overall survival in CRC patients.Furthermore,overexpression of FOXK1 induced EMT by transactivating Cyr61 expression.Promoter assays showed that Cyr61 was a direct transcriptional target of FOXK1.Down-regulation of Cyr61 decreased FOXK1-enhanced HCC migration,invasion,and metastasis.Additionally,FOXK1 expression was positively correlated with Cyr61 expression and was associated with poorer prognosis.Conclusions:Snail/FOXK1/Cyr61 signaling axis regulates EMT and metastasis of CRC...