CBLB Inhibits Dynamin-2-mediated Cell Pseudopodia Formation And Invasive Metastasis Of Breast Cancer By Stabilizing SNX18 Through Ubiquitination

Background and objectiveThe E3 ubiquitin ligase CBLB plays an important role in the pathogenesis and progression of a variety of malignancies,but its role in breast cancer remains unclear.Previous work in the study group has demonstrated that CBLB is poorly expressed in breast cancer tissues and cells,and that low levels of CBLB are associated with a worse prognosis in breast cancer patients;overexpression of CBLB inhibits proliferation and metastasis of breast cancer cells,and CBLB interacts with the autophagy-associated protein SNX18 and promotes its ubiquitination modification[1-2],but whether CBLB affects proliferation,migration,and invasion of breast cancer cells through SNX18 remains to be investigated.Hence,this research aims to further define the role of CBLB in proliferation,migration,and invasion of breast cancer cells,and to elucidate the molecular mechanism by which CBLB stabilizes SNX18 via ubiquitination and affects autophagy and pseudophagy formation.MethodThe effects of knockdown of CBLB on migration and invasiveness of MCF7 breast cancer cells were examined by Transwell assays in established MCF7 breast cancer cell lines stably knocked down CBLB.Identification of CBLB and SNX18 endogenous interactions using endogenous immunoprecipitation and immunofluorescence colocalization.Regulation of SNX18 mRNA and protein expression by CBLB using RT-qPCR and Western blot analysis.Effect of CBLB on SNX18 protein stability was verified by cycloheximide tracing.To investigate the effects of CBLB on SNX18 ubiquitination through ubiquitination experiments and to identify key sites where CBLB-mediated ubiquitination and SNX18 accept ubiquitination.Identification of SNX18 and its cell-mediated autophagy pathway in breast cancer metastasis by Transwell assay.Effect of ubiquitin-modified upregulation of SNX18 on autophagy of breast cancer cells in CBLB using immunofluorescence co-localization,Western blot,and cytoskeletal staining.Validation of CBLB effects on SNX18 recruitment to Dynamin-2 and Dynamin-2-mediated pseudopodia formation using immunoprecipitation and immunofluorescence colocalization assays.ResultThere is a significant increase for the capability of CBLB in knock-down against migration and invasion of breast cancer cells.CBLB TKB domain interacts with SNX18 LC fragment,CBLB and SNX18 colocalized in the cytoplasm.CBLB up-regulated SNX18 ubiquitylation and protein expression through the K63 ubiquitin chain,and C373 was a critical site for CBLB-mediated SNX18 ubiquitination.K391 is a key site for SNX 18 acceptance of ubiquitination modifications.CBLB promotes perinuclear recruitment of SNX18 to autophagy-associated proteins LC3,Atg9A,and Atg16L1,and enhances cell-wide autophagy levels by stabilizing SNX18 through ubiquitination.Overexpression of SNX18 reversed the decrease in autophagy and promoted migration induced by knockdown of CBLB in breast cancer cells.CBLB inhibits Dynamin-2-mediated pseudopodia formation and migration invasion of breast cancer cells by promoting SNX18 recruitment of Dynamin-2.ConclusionCBLB binds to the LC segment of SNX18 via its TKB domain and upregulates SNX18 expression levels via C373 and K63-dependent ubiquitylation modifications,promoting the recruitment of SNX18 to the perinuclear domain of Dynamin-2 and autophagy-associated proteins LC3,Atg9A,and Atg16L1,thereby promoting autophagy,and changes in the localization of Dynamin-2 significantly reduce the pseudopodia formation of breast cancer cells,thereby inhibiting migration and invasion of breast cancer.This study further refines the molecular mechanisms of the CBLB-SNX18-Dynamin-2 regulatory axis and provides an initial insight into the role of autophagy in breast cancer metastasis,providing new insights for targeted therapy of breast cancer.