USP2a Supports Metastasis by Tuning TGF-β Signaling Yin Zhao, Xiaomeng Wang, Qingqing Wang, Yu Deng, Kang Li, Man Zhang, Qiang Zhang, Jin Zhou, Hong-Yan Wang, Peng Bai, Yujie Ren, Ni Zhang, Weina Li, Yongbo Cheng, Wuhan Xiao, Hai-Ning Du, Xiaoliang Cheng, Lei Yin, Xiangning Fu, Dandan Lin, Qianghui Zhou, Bo Zhong  Cell Reports  Volume 22, Issue 9, Pages 2442-2454 (February 2018) DOI: 10.1016/j.celrep.2018.02.007 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 22, 2442-2454DOI: (10.1016/j.celrep.2018.02.007) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Identification of USP2a as a Promoter of TGF-β-Triggered Signaling (A and B) Luciferase reporter assays analyzing SMAD2/3/4 activity in HEK293 cells transfected with empty vector or plasmids encoding FLAG-tagged DUBs (A) or with increasing amounts of plasmids encoding FLAG-USP2a (B) in the presence or absence of TGF-β (15 ng/mL) stimulation for 8 hr. (C) Immunoblot analysis of phosphorylated and total SMAD2/3, FLAG-USP2a, and β-actin of Hep3B cells (left) or HeLa cells (right) transfected with empty vector or FLAG-USP2a, followed by stimulation with TGF-β for 0–20 min. (D and E) qPCR analysis of p15 and p21 mRNA in HeLa cells (D) and immunoblot analysis of p21, FLAG-USP2a, and β-actin (E) of Hep3B or HeLa cells transfected with empty vector or FLAG-USP2a, followed by stimulation with TGF-β for 0–8 hr. (F) Immunoblot (left) and luciferase reporter assay of SMAD2/3/4 activity (right) of HEK293 cells transfected with a control shRNA vector (pLenti shControl) or a shRNA targeting USP2a (pLenti shUSP2a) in the presence or absence of TGF-β stimulation for 8 hr. (G) Immunoblot analysis of phosphorylated and total SMAD2/3, total USP2a, p21, and β-actin of Hep3B cells (left) or HeLa cells (right) transfected with a control shRNA vector (−) or a shRNA targeting USP2a (+), followed by stimulation with TGF-β for 0–20 min. (H and I) qPCR analysis of p15 and p21 mRNA (H) or immunoblot analysis (I) of p21, USP2a, and β-actin in HeLa or Hep3B cells transfected with a control shRNA vector (shCon) or a shRNA targeting USP2a (shUSP2a), followed by stimulation with TGF-β for 0–8 hr. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (analysis of two-way ANOVA, followed by Bonferroni post-test). Data are representative of three independent experiments (data are mean ± SD in A, B, D, F, and H; n = 3). See also Figures S1 and S2. Cell Reports 2018 22, 2442-2454DOI: (10.1016/j.celrep.2018.02.007) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 USP2a-Mediated Regulation of TGF-β Signaling Depends on Its DUB Activity (A) Luciferase reporter assays analyzing SMAD2/3/4 activity in HEK293 cells transfected with the empty vector or plasmids encoding FLAG-tagged USP2a or USP2a(D357A) in the presence or absence of TGF-β (15 ng/mL) stimulation for 8 hr. (B) qPCR analysis of p15 and p21 in HeLa cells transfected with an empty vector (Vec), FLAG-tagged USP2a, or USP2a(D357A), followed by TGF-β stimulation for 0–4 hr. (C and D) Immunoblot of phosphorylated and total SMAD2, FLAG-USP2a, FLAG-USP2a(D357A), or β-actin in HeLa cells (C) or USP2a−/− HCT116 cells (D) stably transfected with an empty vector (Vec), FLAG-tagged USP2a, or USP2a(D357A), followed by TGF-β stimulation for 0–20 min. ∗∗p < 0.01; ∗∗∗p < 0.001 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of at least three independent experiments (data are mean ± SD in A and B; n = 3). See also Figure S2. Cell Reports 2018 22, 2442-2454DOI: (10.1016/j.celrep.2018.02.007) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 TGFBR2 Phosphorylates USP2a at Ser207/Ser225 after TGF-β Treatment (A) Mass spectrometry analysis of the phosphorylation of USP2a in TGF-β-treated (15 ng/mL, 20 min) HEK293 cells transfected with FLAG-USP2a, followed by immunoprecipitation (with anti-FLAG agarose) and elution (with 3 × FLAG peptide). (B) Immunoblot analysis of phosphorylated and total USP2a in HEK293 cells transfected with FLAG-tagged USP2a, USP2a(S207A), or USP2a(D357A) in the presence or absence of TGF-β treatment for 20 min. (C) Luciferase reporter assay of SMAD2/3/4 activity in HEK293 cells transfected with immunoblot analysis of phosphorylated and total USP2a in HEK293 cells transfected with an empty vector (Vec), FLAG-tagged USP2a, USP2a(S207A), USP2a(S225A), or USP2a(Ser207/225A) in the presence or absence of TGF-β stimulation for 0–8 hr. (D) qPCR analysis of p15 and p21 in HeLa cells transfected with an empty vector (Vec), FLAG-tagged USP2a, or USP2a(Ser207/225A), followed by TGF-β stimulation for 0–4 hr. (E and F) Immunoblot of phosphorylated and total SMAD2, FLAG-USP2a, FLAG-USP2a(Ser207/225A), or β-actin in HeLa cells (E) or HCT116 USP2a−/− cells (F) stably transfected with an empty vector (Vec), FLAG-tagged USP2a, or USP2a(Ser207/225A), followed by TGF-β stimulation for 0–20 min. (G) Immunoblot of phosphorylated and total USP2a and SMAD2 and of total β-actin in Hep3B cells stimulated with TGF-β for 20 min in the presence of DMSO, LY2109761 (10 μM), MK22062HCL (5 μM), or LY294002 (10 μM). (H) Strategy for in vitro phosphorylation assay and immunoblot analysis of total and phosphorylated USP2a and SMAD2 and of total TGFBR1 and TGFBR2 in the presence of DMSO or LY2109761 (10 μM). (I) In vitro kinase assay to analyze the phosphorylation of GST, GST-USP2a(aa 193–231), or GST-USP2a(aa 193–231, Ser207/225A) by TGFBR2. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (analysis of two-way ANOVA, followed by Bonferroni post-test). Data are representative of three independent experiments (data are mean ± SD in C and D; n = 3). See also Figure S3. Cell Reports 2018 22, 2442-2454DOI: (10.1016/j.celrep.2018.02.007) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 USP2a Modulates SMAD2 Recruitment to and Disassociation from TGFBR1 (A) Immunoprecipitation (with a control immunoglobulin G [IgG] or anti-FLAG) and immunoblot analysis (with anti-pSMAD2, anti-SMAD2/3, anti-TGFBR1, anti-TGFBR2, anti-FLAG, or anti-β-actin) of USP2a−/− HCT116 cells reconstituted with FLAG-USP2a stimulated by TGF-β for 0–20 min. (B) Immunoblot of phosphorylated and total SMAD2 and tubulin or Na+-K+ ATPase in the membrane and cytoplasmic fractions of Hep3B cells stably transfected with a control shRNA (shCon) or shUSP2a stimulated with TGF-β for 0–20 min. (C) Proximity ligation assay (PLA) analysis of TGFBR1 and SMAD2 interaction in Hep3B cells transfected with a control shRNA (shCon) or shUSP2a stimulated with TGF-β for 0–40 min. (D) Immunoprecipitation (with anti-TGFBR1) and immunoblot analysis (with anti-SMAD2, anti-FLAG, anti-TGFBR1, or anti-β-actin) of USP2a−/− HCT116 cells reconstituted with an empty vector, FLAG-tagged USP2a, USP2a(D357A)(DA), or USP2a(Ser207/225A)(SA) stimulated by TGF-β for 20 min. (E) PLA analysis of TGFBR1 and SMAD2 or TGFBR1-FLAG-USP2a associations of USP2a−/− HCT116 cells reconstituted with an empty vector, FLAG-tagged USP2a, USP2a(D357A)(DA), or USP2a(Ser207/225A)(SA) stimulated by TGF-β for 0–40 min. DA, USP2a(D357A); SA, USP2a(Ser207/225A). Data are representative of three independent experiments. Cell Reports 2018 22, 2442-2454DOI: (10.1016/j.celrep.2018.02.007) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 USP2a Removes K33-Linked Polyubiquitin Chains from Lys502 of TGFBR1 after TGF-β Stimulation (A) Strategy and results of analysis on ubiquitinated sites of TGFBR1 after TGF-β treatment. (B) Immunoblot analysis of FLAG or ubiquitin in the GST-NZF1 pull-down fractions of Usp2a+/+ and Usp2a−/− MLFs transfected with FLAG-TGFBR1 or TGFBR1(K502R), followed by TGF-β treatment (10 ng/mL) for 0–20 min. (C) Luciferase reporter assay of SMAD2/3/4 activity and immunoblot analysis in HEK293 cells transfected with FLAG-tagged TGFBR1 or the KR mutants in the presence or absence of TGF-β treatment for 0–8 hr. (D and E) Immunoblot (with anti-FLAG, anti-SMAD2/3, anti-pSMAD2, or anti-β-actin) (D) or PLA analysis of FLAG-TGFBR1-SMAD2 associations (E) in R1B cells reconstituted with an empty vector, TGFBR1, or TGFBR1(K502R) stimulated with TGF-β (10 ng/mL) for 0–60 min. (F) Predicted structure of TGFBR1 cytoplasmic domain (aa 175–503) dimer. Ubiquitin (pink) modification on Lys502 (blue) of one TGFBR1 might interfere with the GS motif (red) of the other TGFBR1. Data are representative of three independent experiments (B–E) (data are mean ± SD in C). See also Figure S4. Cell Reports 2018 22, 2442-2454DOI: (10.1016/j.celrep.2018.02.007) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 USP2a Supports Metastasis (A and B) H&E staining (A, left), number and density (A, right), and size (B) of tumors metastasized in the lungs of nude mice at day 18 after intravenous injection with control or USP2aKO Hep3B cells (4 × 106). (C) Survival analysis of nude mice intravenously injected with control or USP2aKO Hep3B cells (4 × 106). (D and E) H&E staining (D, left), immunoblot analysis of FLAG and GAPDH (D, right), and number, density, or size of tumors metastasized in the lungs (E) of nude mice at day 18 after intravenous injection with USP2aKO Hep3B cells reconstituted with empty vector, USP2a, USP2a(D357A), or USP2a(Ser207/225A) (5 × 106). (F) Survival analysis of nude mice intravenously injected with USP2aKO Hep3B cells reconstituted with empty vector, USP2a, USP2a(D357A), or USP2a(Ser207/225A) (5 × 106). Scale bars represent 1 mm. Statistical analysis was performed with one-way ANOVA (A and E). Data are representative of two independent experiments (A–E). The survival in (F) was a combination of two independent experiments. See also Figures S5 and S6. Cell Reports 2018 22, 2442-2454DOI: (10.1016/j.celrep.2018.02.007) Copyright © 2018 The Author(s) Terms and Conditions

Figure 7 USP2a Serves as a Therapeutic Target for Metastasis (A) Immunoblot of pSMAD2, SMAD2/3, phosphorylated USP2a (pUSP2a), and β-actin in MLFs or Hep3B cells stimulated with TGF-β for 0–30 min in the presence of DMSO or ML364 (10 μM). (B) qPCR of Snai1, Snai2, or Angptl4 and immunoblot of vimentin (VIM) and SNAI1 in A549 or 4T1 cells stimulated with TGF-β for 0–8 hr in the presence of DMSO or ML364 (10 μM). (C) PLA analysis of TGFBR1-SMAD2 associations in MLFs stimulated with TGF-β for 0–30 min in the presence of DMSO or ML364 (10 μM). (D) Immunoblot analysis of FLAG or ubiquitin of the GST-NZF1 pull-down fractions in R1B cells reconstituted with TGFBR1 or TGFBR1(K502R) stimulated with TGF-β (5 ng/mL) for 20 min in the presence of DMSO or ML364 (10 μM). (E) Survival analysis of nude mice intravenously injected with Hep3B (4.5 × 106), followed by eight successive intraperitoneal injections of DMSO (n = 6) or ML364 (n = 6) (5 mg/kg body weight) every other day. (F) H&E staining, size, number, and density of tumors in lungs of nude mice at day 20 after intravenous injection with Hep3B (4 × 106), followed by eight successive intraperitoneal injections of DMSO (n = 6) or ML364 (n = 5) (5 mg/kg body weight) every other day. (G and H) Strategy (G, upper scheme), primary breast tumors and growth curve (G, lower image and graph), and H&E staining, size, number, and density of the metastatic tumors in lungs (H) of BALB/C mice orthotopically injected with 4T1 cells (1 × 105 per mouse). On day 10, these mice were intraperitoneally injected with DMSO (n = 5) or ML364 (n = 5) (5 mg/kg body weight) for 17 successive days. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (analysis of two-way ANOVA, followed by Bonferroni post-test in B and unpaired t test in F and H). Sale bars represent 1 mm. Data are representative of three (A–C) or two (D–H) independent experiments (data are mean ± SD in B and G). See also Figure S7. Cell Reports 2018 22, 2442-2454DOI: (10.1016/j.celrep.2018.02.007) Copyright © 2018 The Author(s) Terms and Conditions