(A) MSigDB-based pathway enrichment analysis shows that CD73 expression in TCGA TNBC samples is positively correlated with TGF-β signaling hallmark. (B) MDA-MB468-ShTRIM21 cells were treated with IFN-γ (100 μg/mL), and OTUD4 and CD73 protein levels were determined. (C) Spearman’s rank correlation analysis using CPTAC data set showing the CD73 protein expression is highly positively correlated with TGF-β signaling pathway–related proteins. (D) The MSigDB-based pathway enrichment analysis showing that OTUD4 expression in TCGA TNBC samples is positively correlated with TGF-β signaling hallmark. (E) Spearman’s correlation analysis showing the positive correlation between OTUD4 expression and TGF-β signaling pathway. (F) MDA-MB468 were treated with 3 ng/mL TGF-β, OTUD4, and CD73 protein levels were determined by immunoblotting. (G) MDA-MB468-CD73-V5 were treated with 3 ng/mL TGF-β, CD73 immune complexes were immunoprecipitated and ubiquitylation levels were determined. (H) MDA-MB468 cells were treated with 3 ng/mL TGF-β, adenosine production levels were determined. (I) MDA-MB468 cells were treated with CHX and MG-132. Cell lysates were collected at indicated time points and followed by measuring CD73 protein expression. (J and K) MDA-MB468-ShCon and MDA-MB468-ShOTUD4 were treated with 3 ng/mL TGF-β, OTUD4 and CD73 protein levels (J) and adenosine production (K) were determined. (L) MDA-MB468-CD73^WT and MDA-MB468-CD73^V300P/I301Q cells were treated with 3 ng/mL TGF-β, V5-tagged CD73 and CD73^V300P/I301Q were immunoprecipitated and ubiquitylation level was determined. (M and N) CD73 protein turnover rate (M) and adenosine production level (N) were determined in MDA-MB468-CD73^V300P/I301Q and TGF-β–treated MDA-MB468-CD73^V300P/I301Q cells. Data (mean ± SEM) are representative of at least 3 independent experiments. *P < 0.05 and ****P < 0.0001, by unpaired t test, 1-way ANOVA with Tukey’s multiple comparisons test.