Direct Regulation Of HOXD9 By Snail Promotes Metastasis In Colorectal Cancer

Background and Objection:HOX genes belong to the superfamily of homeobox genes that encode for transcription factors with important roles in development[1,2].HOX genes contain a conserved 183 bp sequence and encode nuclear proteins called homeoproteins.HOX proteins control diverse cellular processes by regulating the expression of many downstream target genes[3,4].Thus,it is typical for an individual homeoprotein to confer pleiotropic effects on cell behavior,including alterations in proliferation,survival,migration,and invasion.HOX genes are a family of 39 transcription factors that are divided into four clusters(HOXA,HOXB,HOXC,and HOXD)[5-8].These clusters are located on different chromosomes in tandem.HOXD9 is one of the HOXD genes located closest to the 3’ end of the chromosome,and it is involved in cancer development and progression[9,10].Studies have demonstrated that HOXD9 is overexpressed in most human esophageal squamous cell carcinomas and that it contributes to both cell proliferation and/or cell survival[9].Recently,it has been shown that HOXD9 promotes epithelial-mesenchymal transition(EMT)and cancermetastasis by ZEB1 regulation.In contrast,ZEB1 knockdown inhibits HOXD9-induced invasion and EMT in hepatocellular carcinoma cells.However,the role of HOXD9 genes in colorectal cancer(CRC)development and progression remains unknown.The Snail family of zinc-finger proteins comprises transcription repressors that are involved in the development of vertebrate and invertebrate embryos,as well as in tumor progression[11,12].This family leads to broad biological functions,such as cell differentiation,cell motility,cell cycle regulation and apoptosis[13,14].Moreover,Snail triggers EMT by reducing E-cadherin expression and increasing Vimentin expression and by binding to the E-box nucleotide sequence,CANNTG,in the promoter regions of target genes[15,16].Overexpression of Snail in epithelial cells can change epithelial cells from mesenchymal phenotype to fibroblast phenotype.At the same time,expression of E-cadherin decreases or even loses.Vimentin expression increases significantly,which makes cells more conducive to invasion and distant metastasis.We have previously reported that the FHL2 gene negatively regulates the transcription of E-cadherin through interaction with Snail[17].Studies have shown that there is a binding site of transcription factor Snail in the promoter region upstream of human FOXK1 gene,Snail can bind to it and activate downstream regulation of FOXK1.Furthermore,Snail can promote epithelial to mesenchymal transition(EMT)and increase the ability of invasion and metastasis of colorectal cancer cells together with FOXK1.A large number of clinical datas show that co-expression of FOXK1 and Snail significantly reduces prognostic survival time in patients with colorectal cancer[11].However,the effects of Snail-regulating target genes,including HOXD9,on the EMT process in CRC remain to be defined.The present study reported the first evidence that overexpression of HOXD9 is closely related to poor prognosis and overall survival in patients with colorectal cancer.Moreover,Snail regulates HOXD9 expression by transcriptional activation in human CRC cells,thereby promoting cell invasion and metastasis in vitro and in vivo.Methods:The protein expression levels of HOXD9 were measured by Western blot,immunofluorescence and immunohistochemistry(IHC)assays.Moreover,HOXD9 promoter activity was studied by luciferase and ChIP assays.Therefore,the EMT and the potential of proliferation,migration and invasion of colorectal cancer(CRC)cells were evaluated by many ways.Results:HOXD9 expression was upregulated in CRC compared to matched normal tissues.High HOXD9 expression was significantly associated with American Joint Committee on Cancer(AJCC)stage,tumor differentiation and metastasis,and it was predictive of poor survival.Functionally,HOXD9 promoted proliferation,migration and EMT processes in CRC cells.In addition,TGF-β1 induced HOXD9 expression in a dosedependent manner,and HOXD9 knockdown inhibited TGF-β1-induced EMT.Mechanically,Snail regulated the expression of HOXD9 by binding directly to the E-box response element and activating the HOXD9 proximal promoter.Inhibition of Snail attenuated the invasive mesenchymal phenotype and metastatic potential of HOXD9-overexpressing CRC cells,indicating that Snail is a major mediator of HOXD9-induced EMT and metastasis.In addition,HOXD9 and Snail were positively correlated at the expression level in CRC tissue samples.In vivo,Snail promoted HOXD9-mediated metastasis via orthotopic implantation.ConclusionsThe present study provided evidence of a key signaling pathway involving Snail and HOXD9 that promotes metastasis in CRC.