Supplementary Figure 8: A20 expression is correlated with relapse-free survival of human breast cancer patients.

(a) Using Kaplan-Meier (KM) Plotter Tool (http://kmplot.com/analysis)⁶⁶, the correlation between A20 expression and the relapse-free survival rates of breast cancer patients was analyzed in two independent public GEO datasets (left; GSE9195, right; GSE2603). P values were calculated using a log-rank test. HR = hazard ratio (b) Proposed model demonstrating Snail1 stabilization by A20-mediated multi-monoubiquitination. In the absence of A20, Snail1 is phosphorylated by GSK3β at one of serine 107, 111, 115 and 119 residues and exported from the nucleus to the cytoplasm. Additional phosphorylation occurs at one of the serine 96 and 100 residues by GSK3β in the cytoplasm. β-TrCP1 subsequently recognizes these Snail1 phosphorylations and builds a K48-linked polyubiquitin chain on Snail1, resulting in proteasomal degradation. In the presence of A20, Snail1 is monoubiquitinated by A20 at multiple sites of lysine 206, 234 and 235 residues in the nucleus. This multi-monoubiquitination inhibits the interaction between Snail1 and GSK3β. Thus, GSK3β-mediated Snail1 phosphorylation is decreased and Snail1 stability in the nucleus is increased, eventually promoting EMT and metastasis.