Spred2 Regulates Epithelial Mesenchymal Transition In Colon Cancer Cells And Its Mechanism

Colon cancer is the common malignant tumors of digestive system.Colon cancer is the third common diagnosed malignant tumors among men,and second common diagnosed malignant tumors among women.Recently,with the progress and development of diagnosis and therapeutic technologies,colon cancer at early stage always could be cured or delayed,which significantly improved the life quality of colon cancer patients.However,efficient strategies for advanced and metastatic colon cancers are still lack.Distal metastasis is the major blockade for effective treatment of malignant tumors.It has been reported that distal metastasis could be detected in>90%cancer caused death cases.The development of molecular biology,genetics,genomics and proteomics promoted the progression of gene therapy,which might produce breakthroughs in treatment of tumor metastasis.To clarify the mechanisms of tumor distal metastasis and to screen potential targets are the basis and key for tumor treatment.Sprouty related with EVH1 domain（Spreds）is a member of sprouty related membrane proteins,which could negatively regulate Ras-Raf-ERK signaling pathway.In mammalian,the Spreds proteins contain three members,such as Spred1,Spred2 and Spred3.Our previous work has reported that Spred2 could regulate the proliferation and apoptosis in choronic myelocytic leukemic cells and hepatocytes,as well as inhibit the growth of tumors.However,the roles and mechanisms of Spred2 in initiation,development and metastasis of colon cancer are still largely unexplored.Transforming growth factor-β（TGFβ）is a multiple cytokine,which plays dual roles in the development and progression of tumors:at the early stage of the tumor,TGFβcould promote the apoptosis and inhibit the proliferation of tumor cells via TGFβ/SMADs signaling pathway;while atadvanced stage of tumor,aberrant activated TGFβsignaling could cross-talk with various pathways,such as vascular endothelial growth factor A（VEGFA）signaling pathway and hypoxia inducible factor（HIF-1α）signaling pathway.Importantly,TGF-βhas been known to be an effective inducer of epithelial mesenchymal transition（EMT）,which could promote the invasion and metastasis of tumor cells.It has been reported that receptor tyrosine kinase（RTK）antagonist could negatively regulated TGF-βsignaling,and Spred2 is a natural antagonist of RTK.It is interesting to investigate if Spred2 could regulated EMT of tumor cells and tumor metastasis via modulating TGF-βsignaling.In this study,we aimed to clarify the effects of Spred2 on biological characteristics of colon cancer cells,and to explore itspotential mechanisms.Meanwhile,we also try to investigate the roles of Spred2 and TGF-βsignaling in the regulating EMTof colon cancer cells,as well as to clarify the possible mechanism of Spred2 in regulating EMT.Hopefully,this study could provide novel and effective therapeutic targets and strategies for treating colon cancer.Firstly,We collected cancerous samples and corresponding para-cancerous samples from Beijing Friendship Hospital,a teaching affiliation of Capital Medical University,between 2009 and 2012.Thirty-one paired samples were processed for detecting mRNA expression of Spred1 and Spred2 by real-time reverse transcript polymerase chain reaction（RT-PCR）.Our results showed that Spred2 expression,but not Spred1 was down-regulated in tumor tissues,suggesting that Spred2 might play important roles in regulating development and progression of colon cancer.Secondly,to study the effects of Spred2 on EMT and biological characteristics of colon cancer cells,we grew recombinant adenoviruses,Ad5/F11p.Spred2 and Ad5/F11p.Null,The adenoviruses were purified by CsCl density gradient centrifugation.The viral particles and infective titers were analyzed by ultraviolet spectrophotometry and tissue culture infective dose（TCID50）.The purity of Ad5/f11p.Spred2and Ad5/f11p.Null were 1.3521 and 1.4025（OD260nm/OD280nm）;the viral particles were3.36×10¹11 VPs/mL and 4.95×10¹11 VPs/mL;the infective titers were 1.28×10¹⁰IU/mL and 2.5×10¹¹IU/m L,respectively.Meanwhile,results of real-time PCR and western-blotting also showed that Ad5/F11p.Spred2 could express Spred2 in SW480cells effectively.Our results suggested that our adenoviruses are high-quality,and they could be used for further studies.Thirdly,SW480 cells were infected with Ad5/f11p.Spred2 or Ad5/f11p.Null at various multiplicity of infection（MOI）.At 24h and 48h post-infection,cells were collected,and the apoptosis was analyzed by flow cytometry.And,Dye670 treated SW480 cells were infected with 50MOI Ad5/f11p.Spred2 or Ad5/f11p.Null,and the fluorescence intensity of Dye670 was detected by flow cytometry at 0h,24h,48h,72h and 96hafter infection.Moreover,after 50MOI adenovirus infection,the migration of SW480 cells were analyzed by wound healing and transwell assay.Our results suggested that Ad5/F11p.Spred2 could inhibit proliferation and migration of colon cancer cells,as well as dose-dependent and time-dependent induced apoptosis.Therefore,we concluded that Spred2 over-expression could inhibit the proliferation and migration,while promote the apoptosis of colon cancer cells.Fourthly,we analyzed the effects of TGF-βon the EMT of colon cancer cells.According to previous reports and our studies,we stimulated SW480 cells with10ng/ml recombinant human TGF-β,and then the effects of TGF-βon EMT was detected by morphological observation,western-blotting,immunofluorescence and laser confocal.We found that TGF-βsignificantly promote the migration of SW480,and also induced morphological changes:from epithelial phenotype to mesenchymal phenotype.And,as expected,TGF-βincreased the expression of downstream molecules,such as Smad2/3 and Smad4.Further analysis showed that TGF-βcould obviously reduced E-cadherin expression,while increasedN-cadherin and Vimentinexpression.Our results suggested that TGF-βis the pivotal molecule in EMT of colon cancer cells,which might be a potential mechanism of advanced tumor metastasis.Finally,we detected the effects of Spred2 on the TGF-beta/SMADs signaling pathway and EMT of colon cancer cells with different genetic background.SW480cells.and HCT116 cells were infected with 50MOI and 100MOI adenoviruses,respectively.At 24h and 48h after infection,proteins were extracted from corresponding cells.The EMT markers,TGF-β/SMADs signaling related molecules and MAPK/ERK signaling related proteins were detected by western-blotting or immunofluorescenceIn SW480 cells,compared with Ad5/F11p.Null,Ad5/f11p.Spred2 inhibited EMT related transcriptive factors,such as snail and slug.And,it also promoted E-cadherin expression and inhibited Vimentin expression,while it has no obvious influence on N-cadherin expression.Moreover,Ad5/f11p.Spred2 mediated Spred2 over-expression in SW480 cells could effectively inhibit phosphorylation of ERK,but had no significant effects on expression of Smad2/3 and Smad4.In HCT116 cells,Ad5/F11p.Spred2also could significantly inhibit EMT related transcriptive factors,such as snail and slug,as well as EMT markers,such as Vimentin and N-cadherin.However,compared to that in SW480 cells,Ad5/F11p.Spred2 not only inhibited phosphorylation of ERK,but also decreased expression of SMAD2/3 and SMAD4.The results described above suggested that Ad5/f11p.Spred2 could negatively regulated Ras-Raf-ERK signaling and TGF-β/SMADs signaling,which in turn inhibit the EMT and migration of colon cancer cells。In conclusion,Spred2 expression was significantly down-regulated in tumor tissues of colon cancer patients.And,Spred2 overexpression could inhibit proliferation and migration,and promote apoptosis of colon cancer cells.Further analysis suggested that Spred2 over-expresion negatively regulated Ras-Raf-EKR signaling pathway and TFG-βEMT signaling pathway,and inhibit the EMT of colon cancer cells.Therefore,Spred2 is a promising candidate target for treatment of colon cancer.