The Effects And Mechanisms Of NDRG1 On The "Stemness" Of Colorectal Cancer

Objectives N-myc downstream-regulated gene 1(NDRG1),has been identified as an important metastasis suppressor for different tumor types,i.e.colorectal cancer(CRC),prostate cancer and pancreatic cancer.In our formal studies,downregulation of NDRG1 increase the ability of CRC cells to migrate and invade through enhanced Epithelial-Mesenchymal Transition(EMT).Increase evidence indicates that tumor cells may display "cancer stem cell-like"(CSC)properties(or stemness)during the process of EMT.However,there is no evidence whether NDRG1 could influence the"stem cell-like" properties of CRC cells.Thus,this current study aims at investigating the effects and probable mechanisms of NDRG1 on the "stemness" of colorectal cancer,which is based on this unsolved scientific question.Methods 1.The CRC cells,namely the HT29 and HCT116 cell lines,which were stably transfected to either over-express NDRG1(labeled "NDRG1")or silence NDRG1(labeled as "sh NDRG1"),as previously used in our studies.To examine the relationship between NDRG1 and these CSC-related properties,we performed a number of assays to assess sphere formation,metastasis,soft-agar colony formation,chemoresistance in vitro.Also,we examine the effect of NDRG1 on tumorigenesis in vivo,using a series of diluted HT29 cells.2.Detect the effect of NDRG1 on the expression of colorectal-CSC surface markers(i.e.CD44 and CD133)using flow cytometry(FCM),as well as the transcription of other intracellular stem-cell markers(i.e.OCT4,NANOG,SOX2,ALDH1),using RT-PCR.3.Observe the effect of NDRG1 on the subcellular localization of ?-catenin using immunofluorescence.Detect the expression of ?-catenin in different cell components(i.e.whole cell lysates,membrane components and nuclear components)by Western Blot,as well as the expression of ?-catenin-downstream proteins(i.e.c-myc and cyclin D1).To illustrate the function of NDRG1 in WNT signaling,TOP/FOP Flash assay was adopted to detect the transcriptional activation of nuclear ?-catenin.4.In order to verify whether NDRG1 inhibits CSC-related properties of CRC cells via regulating ?-catenin,we downregulated ?-catenin expression in sh NDRG1 and sh Con cells(including HCT116 and HT29)with sh RNA and determined whether these properties can be reversed cause by NDRG1 low-expression.5.Detect the expression of NDRG1 in clinical CRC tissues and corresponding normal mucosas,and analyze the relationship between NDRG1 expression and clinicopathological data.Investigate the co-localization and correlation among NDRG1,nuclear ?-catenin and membrane CD44 using the continuous CRC tissue slides.Results 1.NDRG1 over-expression(i.e.HCT116 and HT29 cells)inhibits CSC traits(i.e.sphere formation and self-renewal ability;invasive ability;colony formation and chemoresistence)in vitro,while down-regulation of NDRG1 markedly enhances these properties.Also,NDRG1 over-expression markedly reduces tumorigenesis in vivo,relative to the control group.2.NDRG1 overexpression remarkablely reduces the expression of CSC marker,CD44,on the cell membrane,while NDRG1 knockdown enhances its expression.However,changes in NDRG1 expression show no influence on CD133 expression.In addition,NDRG1 overexpression significantly inhibits the transcription of OCT4 and NANOG,while low-expression of NDRG1 markedly increases the transcription of SOX2.However,NDRG1 expression does not significantly affect ALDH1 transcription.3.NDRG1 over-expression promotes the location of ?-catenin on the cell surface and inhibits its translocation to the nucleus,which leads to a reduced transcriptional activity and decreased expression of Wnt-downstream proteins(i.e.c-myc and cyclin D1).While down-regulation of NDRG1 displays the opposite effect.4.Down-regulation of ?-catenin(sh ?-catenin)can decrease nuclear ?-catenin transcriptional activity,and also reverse these stemness properties induced by NDRG1 low-expression(sh NDRG1).However,cells(i.e.sh NDRG1 and sh Con HCT116 and HT29)transfected with control plasmid(sh ?-catenin Con)display no remarkable changes in these CSC-related phenotypes.5.NDRG1 expression in normal colorectal tissues is significantly higher than that in CRC tissues.Also,compared with NDRG1 negative CRC cases,NDRG1 positive CRC cases indicate a better prognosis.CRC samples with low NDRG1 expression always accompany with high nuclear ?-catenin and membrane CD44 expression,while those samples with low ?-catenin and CD44 expression correlate with high NDRG1 expression.Conclusions This study indicates that NDRG1 attenuates CSC characteristics and tumorigenesis of CRC in vitro and in vivo.Moreover,NDRG1 could function as a metastasis suppressor for CRC through inactivation of ?-catenin signaling and down-regulation of CD44.Identification of the role of NDRG1 as a metastasis suppressor provides a novel diagnostic biomarker and a therapeutic target for the treatment of CRC.