?-catenin SUMOylation Inhibits Breast Cancer Progression

As the focus protein in this paper,?-catenin,is one of the catenin protein family members.?-catenin is encoded by CTNNA1.?-catenin is down-regulated or lost in a wide range of tumor types,which associate with higher tumor malignancy.?-catenin is frequently down-regulated in human skin cance,and an ?-catenin-knockout skin squamous cell carcinoma mouse model showed up-regulation of many NF-?B downstream genes.In E-cadherin-negative basal-like breast cancer,down-regulating of ?-catenin lead to NF-?B hyperactivated,which promote progress of breast cancer.SUMOylation is a post-translational modification which often affects physical and chemical properties of target proteins,thus regulating substrate proteins related signaling.A computer analysis of proteins mass spectrometry and the ?-catenin protein sequence reveals two conserved SUMOylation sites.Therefore,we hypothesized that ?-catenin is a target of SUMO.The main works of the paper are as follows:1.We observed that ?-catenin is a target of SUMO using immunoprecipitation assay in HEK293 T cells.In addition,SUMOylation level of ?-catenin was enhanced by Ubc9 and deSUMOylated by SENP1.And we found that endougenous ?-catenin interacted with I?B?.So,we believe that ?-catenin is modified by SUMO.We also found ?-catenin SUMOylation site was its lysine 870 using site mutation assay.2.SUMOylation did not affect subcellular distribution between WT ?-catenin and mutant.However,expected ?-catenin-SUMO1 band was mainly evident in the cytoplasm.We also observed that both WT ?-catenin and the K870 R ?-catenin mutant were degraded at a similar rate.3.?-catenin SUMOylation mutant showed significantly reduced inhibition of NF-?B signaling using gene reportor assay.In addition,we found that SUMOylation enhanced interaction between ?-catenin and I?B? using immunoprecipitation assay.4.Cell growth assay,Transwell assay and colony formation assay demonstrated that SUMOylation deficient ?-catenin showed reduced inhibiton of cell growth and migration.Additionally,?-catenin SUMOylation mutant showed impared tumor suppression activity in 4T1/Luc cells in BALB/c mice.Collectively,our data identified a new mechanistic pathway for the regulation of ?-catenin SUMOylation and NF-?B signaling,one that is based on the regulation of interaction between ?-catenin and I?B? by direct SUMOylation of ?-catenin.