The Roles Of Abl-Abi-1-WAVE2 Signaling Pathway In Oncogenesis And Tumor Development

Objective to prove the existence of Abl-Abi-1-WAVE2 signaling pathway and studyits roles in oncogenesis and tumor development.Methods To prove the existence of Abl-Abi-1-WAVE2 signaling pathway and studyits role in cancer cell polarized motility, the Abi-1/WAVE2 complex was checked inthe model of melanoma B16 cell motility stimulated by Fibronectin (FN). The directbinding domains of Abi-1 and WAVE2 were elucidated by site mutation. In the modelof melanoma B16 cell motility stimulated by Fibronectin (FN), the location ofABl/Abi-1/WAVE2 and actin polymerization were observed by immunofluoresentmicroscopy, and their functions in this model were studied by site mutation. Furthermore, the cultured B16 cells were tmnsfected with Abi-1-siRNA construct and thechanges of actin polymerization, lamellipodium formation and cell spreading wereobserved after Abi-1 depletion.To study the role of Abl-Abi-1-WAVE2 signaling pathway in oncogenesis, thelocation of ABl/Abi-1/WAVE2 during cytokinesis were observed by confocalimmunofluoresent microscopy. Then, Abi-1 deficiency in NIH3T3 cells and MCF-7cells were induced by Abi-1 SiRNA. The cell proliferation and the expression of P53,CDC25, CDK1 and cyclin B1 were checked in MCF-7 cells after Abi-1 depletion, andclone formation assay in soft agar and neoplasia formation test in athymic mice wereinvestigated.Results In proving the existence of Abl-Abi-1-WAVE2 signaling pathway andstudying its role in cancer cell polarized motility, (1)The association of Abi-1 andWAVE2 was found in model of FN stimulated B16 cells. Abi-1 binds to WHD domainof WAVE2 with its Coiled-coiled domain. (2) In the model of melanoma B16 cellmotility stimulated by Fibronectin (FN), Abi-1 directs WAVE2 translocation to the leading edge of membrane after cell stimulation and induces actin polymerization toform membrane ruffles and lamellipodia. While deleted the WHD domain of WAVE2to destroy the binding of Abi-1 and WAVE2, WAVE2 recolcated to nucleus, and cellpolarization and lamellipodia can't be induced in these cells. (3)Abi-1 recruits Ablinto WAVE2 complex. Abl/Abi-1/WAVE2, Rac1 and polymerized actin colocalize atthe leading edge of membrane after cell stimulation. Abi-1 promotes Abl-mediatedWAVE2 Y150 tyrosine phosphorylation and the capacity of Abl to induce WAVE2tyrosine phosphorylation may promote assembly of the signaling complexes withinwhich WAVE2 is linked to activated Rac1. (4)Abi-1 deficiency by SiRNA impairsWAVE2 membrane translocation and cell spreading and migration. The morphologyof membrane changed to filopodia.In studying the role of Abl-Abi-1-WAVE2 signaling pathway in oncogenesis, (1)Abi-1, WAVE2, Abl, CDK1-15p and polymerized actin are co-localized in contractilering during cytokinesis. (2) G1 phase arrest in Abi-1 SiRNA transient transfected cellsand no obvious induced cell cycle specific apoptosis was checked. (3)When Abi-1stable deficiency in NIH3T3 cells and MCF-7 cells was induced by Abi-1 SiRNA,multiploid cells were observed in cells with Abi-1 depletion. (4) The expression ofP53 increased in cells with Abi-1 depletion, while CDK1 15-phosphorylation andCDC25 decreased. Cyclin B1 translocated to cytoplasm in multiploid cells and Ki-67in these cells keeps high expression. (5)Soft agar colony assay showed that thenumber of colonies had no obvious difference between Abi-1 depletion cells andcontrol cells, but multinucleated cells could be seen in 95% of the colonies from Abi-1depletion cells, while multinucleated cells were less than 1% in the colonies fromcontrol cells. Tumor formation assay in athymic mice showed that the size of thetumor formed from cells with Abi-1 depletion was larger than the control, and theweight of the tumor formed from cells with Abi-1 depletion was heavier than thecontrol. Conclusion Abi-1 promotes WAVE2 membrane translocation and Abl-mediatedtyrosine phosphorylation required for WAVE2 activation. Abl-Abi-1-WAVE2signaling pathway not only functions in cell polarized motility but also in contractilering formation during cytokinesis. Abi-1 depletion results in a significant induction ofmultiploid cells.