Slit2-Robo1 Signaling Induces E-cadherin Phosphorylation And Epithelial-mesenchymal Transition Through Activated Src

Colorectal carcinoma(CRC)is one of the most common types of cancer worldwide.The death rate of CRC without metastasis has been falling in recent years.However,the therapeutic effects for distant metastatic patients with CRC are limited.The molecular mechanism underlying tumor metastasis of CRC is still unclear.Studies show that,EMT is an early event in the process of cancer metastasis,and the loss of E-cadherin leading to cell-cell adhesion disintergrates plays a critical role in EMT.E-cadherin is regulated by various factors,Slit2 binding to Roundabout 1(Robol)have been shown to induce EMT through Hakai-mediated E-cadherin ubiquitination and degradation.However,how Slit2-Robol signaling are mechanistically linked to E-cadherin phosphorylation,which results in its ubiquitination by Hakai,is still unclear.In this study,we investigate the mechanism of E-cadherin phosphorylation by Slit2-Robol.Methods1.We generated the human colorectal epithelial carcinoma cell lines of HCT116 cells stably transfected with the plain vector(HCT116/V),the plasmid of Robol(HCT116/Robol)or Slit2 plus Robol(HCT116/Robo1/Slit2).Then we observed the activity of Src and phosphorylation of E-cadherin induced by Slit2-Robol signaling.Using short hairpin RNA(shRNA),we next tested whether knockdown of c-Src could reverse EMT by abrogating E-cadherin phosphorylation,ubiquitination and degradation elicited by Slit-Robo signaling.2.To understand how Slit2 binding to Robol induces Src-mediated tyrosine phosphorylation of E-cadherin,we tested whether Slit2 could facilitate association of Robol with Src and E-cadherin in colorectal epithelial cells by immunoprecipitation.To test whether Robol could bind to E-cadherin,we co-transfected human embryonic kidney 293(HEK293)cells with the plasmids of full-length Robol and full-length and deletion mutants of E-cadherin fused with the myc tag.We then verified E-cadherin binding to Robo1 by co-transfecting HEK293 cells with the plasmids of full-length E-cadherin and full-length Robo1 or its deletion mutants.We next examined the interactions among Robo1,E-cadherin and c-Src using purified recombinant proteins with a His tag fused to human Robo1 cytoplasmic motifs(His-CC1 to 3)and glutathione S-transferase(GST)fused to c-Src(GST-Src)or the cytoplasmic domains of N-cadherin or E-cadherin(GST-N-cad or GST-E-cad).3.To test the biological significance of our newly identified trimeric complex of Src-Robol-E-cadherin,we constructed plasmids of plain vector(V)or the cDNA sequence of the intracellular CC3 motif of human Robol(CC3)fused with a FLAG tag.Following transient transfection,we detected the association of Robo1 with Src and E-cadherin,as well as Src activity and E-cadherin phosphorylation,ubiquitination and degradation.Furthermore,we tested the migration of these cells and observed their shape by immunofluorescent staining.Results1.Pretreatment of HCT116/Robol cells with hSlit2 caused tyrosine phosphorylation of immunoprecipitated E-cadherin and activated Src.c-Src knockdown drastically reduced E-cadherin phosphorylation and degradation.2.Only in HCT116/Robo1/Slit2 cells,Robol was bound to endogenous E-cadherin and Src,E-cadherin was also bound to Src.But in HCT116/V cells,E-cadherin was not bound to Src.In transfected HEK293 cells,Robo1 was co-immunoprecipitated with full-length E-cadherin and mutant E-cadherin lacking ectodomains,but not mutant E-cadherin lacking cytoplasmic domains;E-cadherin co-immunoprecipitated full-length Robol and mutant Robol lacking intracellular CC0,CC1 and CC2 motif,but not mutant Robo1 lacking intracellular CC3 motif.Using recombinant proteins,we found that GST-E-cad,but not GST-N-cad,bound directly to His-CC3;GST-Src-SH3/SH2 bound avidly to His-CC3.3.CC3 overexpression inhibited immunoprecipitated Robol from binding to E-cadherin and Src,inactivated Src,increased E-cadherin expression,mitigated E-cadherin phosphorylation,ubiquitination and degradation,altered cell morphology from the mesenchymal-like shape into the epithelial-like shape,reduced the migration of HCT116/Robol/Slit2 cells.Conclusion1.Slit2-Robol signaling induces Src activation,which mediates E-cadherin phosphorylation,in human colorectal epithelial cells.2.In the trimeric complex of Src-Robol-E-cadherin,the cytoplasmic CC3 motif of Robol acting as a adaptor binds simultaneously to the cytoplasmic SH3 and SH2 domains of c-Src and the cytoplasmic domains of E-cadherin.3.CC3 overexpression inhibited Robo1 from binding to E-cadherin and Src,inactivated Src,increased E-cadherin expression,mitigated E-cadherin phosphorylation,reverse EMT.