1. Volume 51, Issue 5, Pages 559-572 (September 2013)
TRAF4 Promotes TGF-β Receptor Signaling and Drives Breast Cancer Metastasis 
Long Zhang, Fangfang Zhou, Amaya García de Vinuesa, Esther M. de Kruijf, Wilma E. Mesker, Li Hui, Yvette Drabsch, Yihao Li, Andreas Bauer, Adrien Rousseau, Kelly-Ann Sheppard, Craig Mickanin, Peter J.K. Kuppen, Chris X. Lu, Peter ten Dijke 
Molecular Cell 
Volume 51, Issue 5, Pages (September 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Figure 1 TRAF4 Is an E3 Ligase Required for TGF-β Signaling
(A) Diagram of genome-wide cDNA screening data in HEK293T cells; TRAF4 (green square), which activates the TGF-β-induced SMAD3-SMAD4-dependent CAGA12-Luc transcriptional reporter, is indicated. SMAD3 (red square) and SMAD7 (yellow square) are indicated as positive/negative control. Grey squares refer to all other cDNA clones tested. The x and y axes represent the relative luciferase activity in two replicates, p < 0.01.
(B) Effect of TRAF4 wild-type (TRAF4 fl) and E3 ligase-inactive TRAF4 mutants (TRAF4-CA, TRAF4-dR) on the CAGA12-Luc transcriptional response induced by TGF-β (1 ng/ml) in HEK293T cells. The data are presented as the mean ± SD. Co.vec, empty vector.
(C) (Left panel) Effect of TRAF4 knockdown (shTRAF4 #1 and #2) on the CAGA12-Luc transcriptional response induced by TGF-β (5 ng/ml) in HEK293T cells. Co.sh, non-targeting shRNA. (Right panel) qRT-PCR (qPCR) analysis of TRAF4 expression in control and stably TRAF4-depleted (shTRAF4 #1 and #2) HEK293T cells. Co.sh, non-targeting shRNA. The data are presented as the mean ± SD.
(D) qRT-PCR analysis of the TGF-β target genes PAI-1, Smad7, and CTGF in wild-type and TRAF4−/− MEFs treated with TGF-β (5 ng/ml) for the indicated lengths of time. Values and error bars represent the mean ± SD of triplicates and are representative of at least two independent experiments.
(E) Analysis of TGF-β-induced SMAD2-SMAD4 heteromeric complex formation in wild-type and TRAF4−/− MEFs (1 ng/ml) by immunoprecipitation (IP) followed by immunoblotting (IB). Treatment times are indicated. Cell lysates were also immunoblotted for phosphorylated SMAD2 (p-SMAD2), phosphorylated p38 (p-p38) MAP kinase, total p38 MAP kinase (p38), total SMAD4, and total SMAD2.
(F) Oncomine box plots of TRAF4 expression levels in multiple human advanced cancers.
(G) Kaplan-Meier curves showing that the overall survival of individuals with ovarian carcinoma and higher TRAF4 gene expression (with overexpression defined as greater than 1.8 SDs above the mean) is significantly lower than that of individuals with lower TRAF4 expression (p = 0.003) by a log rank test.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 TRAF4 Is an E3 Ubiquitin Ligase for SMURF2
(A and B) Proteomic screening for proteins that interact with SMURF2. Red or gray ovals in (B) indicate the known or unknown interacting proteins of SMURF2 (also listed in Table S1). TRAF4 is highlighted in green. The TRAF4 running position is indicated (A). The graph was created using Cytoscape’s Edge-Weighted Spring Embedded layout, with the Fisher’s exact p values as weights (Smoot et al., 2011).
(C) Yeast two-hybrid system data verifying TRAF4 as a SMURF2-associating protein. p53 and SV40T served as positive controls.
(D) Immunoblots (IB) of the total cell lysate (TCL) and immunoprecipitates from HEK293T cells transfected with the indicated plasmids.
(E) LUMIER analysis of HEK293T cells transfected with Renilla-tagged SMURF2 and Flag-tagged TRAF4 (Barrios-Rodiles et al., 2005). The interactions were determined by measuring Renilla activity in anti-Flag immunoprecipitates. The data represent the mean ± SD (n = 3). Flag-SMAD3 and Flag-SMAD7 served as positive controls.
(F) Endogenous interaction between SMURF2 and TRAF4 in MDA-MB-231 cells as analyzed by immunoprecipitation (IP) followed by IB with indicated antibodies.
(G) IB analysis of TCL and immunoprecipitates derived from ubiquitin (Ub)-expressing HEK293T cells transfected with the indicated plasmids and treated with MG132 (5 μM) for 5 hr.
(H) In vitro ubiquitination assays were performed with His-SMURF2 and GST-TRAF4, E1, E2 (UbcH6), and ubiquitin. The assay mixtures were incubated for 3 hr at 37°C and subjected to IP with a SMURF2 antibody and IB with an anti-ubiquitin antibody.
(I and J) IB analysis of the TCL and Ni-NTA pull-down products from HEK293T cells transfected with the indicated plasmids. SMURF2 polyubiquitination is visible as a ladder pattern.
(K) SMURF2 expression by IB analysis in wild-type and TRAF4−/− MEFs treated with CHX (20 μg/ml) for the indicated lengths of time.
(L) IB analysis of TCL and immunoprecipitates from HEK293T cells transfected with the indicated plasmids and treated with MG132 (5 μM) for 5 hr. TRAF4 polyubiquitination is visible as a ladder.
See also Figures S1 and S2 and Table S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

4. Figure 3
TRAF4 Is Associated with Activated TGF-β Receptors and Sustains the Level of TβRI on the Plasma Membrane
(A) Immunoblots (IB) of whole-cell lysates and immunoprecipitates from HEK293T cells transfected with TRAF4 and treated with TGF-β (5 ng/ml) as indicated.
(B) IB analysis of whole-cell lysates and immunoprecipitates from MDA-MB-231 cells treated with TGF-β (5 ng/ml) for 1 hr.
(C) IB analysis of total cell lysates (TCL) and immunoprecipitates from HEK293T cells transfected with the indicated plasmids and treated with TGF-β (5 ng/ml) for 1 hr.
(D) IB analysis of TCL and immunoprecipitates from wild-type and TRAF4−/− MEFs transfected with Flag-tagged TβRI and treated with or without TGF-β (5 ng/ml) for 1 hr.
(E) IB analysis of TCL and immunoprecipitates from HEK293T cells transfected with or without HA-tagged TβRII and Flag-tagged TβRI and treated with TGF-β (5 ng/ml) for the indicated lengths of time.
(F) IB analysis of TCL and immunoprecipitates (two rounds of immunoprecipitation: first with an anti-HA antibody and then with an anti-Flag antibody) from HEK293T cells transfected with the indicated plasmids.
(G) IB analysis of TCl and immunoprecipitates (two rounds of immunoprecipitation: first with an anti-HA antibody and then with an anti-Myc antibody) from HEK293T cells transfected with the indicated plasmids.
(H) Immunoblots (IB) of biotinylated TβRI in HeLa cells transfected with the indicated plasmids.
(I) Immunoblots of biotinylated TβRI in MDA-MB-231 cells infected with TRAF4 shRNA lentivirus and treated with TGF-β (5 ng/ml) for the indicated lengths of time. The data are presented in the right panel as the mean ± SD.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 4
TGF-β Promotes K63 Ubiquitination of TRAF4 and Its Interaction with TAK1
(A) Immunoblot (IB) analysis of whole-cell lysates and immunoprecipitates from MDA-MB-231 cells treated with TGF-β (5 ng/ml) for the indicated lengths of time.
(B) IB analysis of total cell lysates (TCL) and immunoprecipitates from HEK293T cells transfected with wild-type TRAF4 (WT) or the TRAF4 ΔRING mutant and treated with TGF-β (5 ng/ml) as indicated.
(C) IB analysis of TCL and immunoprecipitates from HEK293T cells transfected with wild-type, K63 only, or K48 only ubiquitin (Ub) plasmids and treated with TGF-β (5 ng/ml) as indicated.
(D) IB analysis of the TCL and two rounds of immunoprecipitation (first with an anti-Flag antibody and then with an anti-Myc antibody, as indicated) from HEK293T cells transfected with plasmids and treated with TGF-β (5 ng/ml) as indicated.
(E) IB analysis of the TCL and immunoprecipitates from HEK293T cells transfected with Flag-TRAF4 and treated with or without TGF-β (5 ng/ml) for 30 min.
(F) IB analysis of TCL and immunoprecipitates from HEK293T cells treated with or without TGF-β (5 ng/ml) for 30 min.
(G) IB analysis of TCL and immunoprecipitates from HEK293T cells transfected with the indicated plasmids and treated with or without TGF-β (5 ng/ml) for 15 min.
(H) IB analysis of TCL and TUBE1 pull-down products from wild-type and TRAF4−/− MEFs treated with or without TGF-β (5 ng/ml) for 30 min.
(I) NFκB reporter activity in HEK293T cells transfected with TRAF4 and treated with or without TGF-β (5 ng/ml) overnight as indicated. The data are presented as the mean ± SD.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

6. Figure 5
TRAF4 Is Critical for TGF-β-Induced EMT, Cell Migration, and Invasion
(A) qRT-PCR analysis of TRAF4 and E-cadherin levels in different breast cancer cell lines. The data are presented as the mean ± SD.
(B and C) qRT-PCR (B) and immunoblot (IB) (C) analyses of E-cadherin expression in wild-type and TRAF4−/− MEFs treated with or without TGF-β (5 ng/ml) overnight. The data are presented as the mean ± SD.
(D) Transwell migration assay of wild-type and TRAF4−/− MEFs treated with or without TGF-β (5 ng/ml) for 8 hr. Migrated cells were counted in four random fields, and the mean ± SD was calculated (p < 0.05).
(E and F) IB analysis of MCF10A-Ras cells stably expressing TRAF4 WT/CA (F) or depleted of TRAF4 by two independent shRNAs (E) and treated with SB or TGF-β (5 ng/ml) for 48 hr, as indicated.
(G) Transwell migration assay of control and MDA-MB-231 cells stably depleted of TRAF4 by two independent shRNAs and treated with or without TGF-β (5 ng/ml) for 8 hr. Representative images are shown. Migrated cells were counted from four random fields, and the mean ± SD was calculated (p < 0.05). Representative images are shown in Figure S4B.
(H) TRAF4 depletion inhibits TGF-β-induced invasion of MDA-MB-231 breast cancer spheroid cell cultures in collagen. The data are presented as the mean ± SD. Co.sh, non-targeting shRNA. Representative images are shown in Figure S4C.
(I) Scatterplot showing the positive correlation between TRAF4 and Slug/Fibronectin/CTGF/CXCR4 expression in patients of the NKI295 clinical data set. Pearson’s coefficient tests were performed to assess statistical significance.
(J) Kaplan-Meier plots of distant metastasis-free survival of patients stratified by TRAF4 expression in the NKI295 clinical data set. The p value was calculated by a log rank test.
See also Figures S4 and S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

7. Figure 6 TRAF4 Is Required for Efficient Metastasis In Vivo
(A–C) Control and MDA-MB-231 cells stably depleted of TRAF4 by two independent shRNAs were injected into the blood circulation of 48 hpf zebrafish. Representative images of the zebrafish at 6 dpi are shown (A). Experimental micrometastasis (B) was detected in the posterior tail fin after 6 days. The final percentage of embryos displaying metastasis (C) at 6 dpi is shown. The results of two independent experiments are shown (p < 0.05). The numbers (n) of each experimental group are indicated in the figure.
(D–J) Bioluminescent imaging (BLI) of three representative mice from each group at week 6 injected with control or MDA-MB-231 cells stably depleted of TRAF4 by two independent shRNAs. Both ventral and dorsal images are shown (D). Bioluminescent imaging (BLI) signals of each mouse (E) and the percentage of bone metastasis-free mice (F) in each experimental group, followed over time. The number of bone metastasis in each mouse in each experimental group (G).
(H–J) Ex vivo analysis of bone metastatic cells. qRT-PCR analysis (I) and protein levels (J) of TRAF4 expression in control and TRAF4-depleted MDA-MB-231 cells harvested before injection and after bone metastasis. #1(∗) is the mouse in the TRAF4-depleted group (shRNA #1) that developed metastasis. The data are presented as the mean ± SD (I).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

8. Figure 7
Amplification of the TRAF4 Gene Confers Poor Prognosis in Breast Cancer Patients
(A) Graph showing the number of TRAF4 amplifications (%) in different tumor types. Data were obtained from the TCGA database. Detailed values are listed in Table S2.
(B) Graph showing the correlation between TRAF4 gene copy numbers and mRNA expression in human breast cancer patients (n = 825). Data were obtained from the TCGA database.
(C) Scatterplot showing the positive correlation between TRAF4 and ERBB2 expression in patients of the NKI295 clinical data set. Pearson’s coefficient tests were performed to assess statistical significance.
(D) Kaplan-Meier plots of distant metastasis-free survival of patients, stratified by expression of TRAF4. Data obtained from the Kaplan-Meier plotter database. The p value was calculated by a log rank test.
(E) Immunohistochemical analysis of TRAF4, p-SMAD2, and p-TAK1 in breast cancer tissue microarrays.
(F) Percentage of specimens displaying low or high TRAF4 expression compared to the expression levels of p-SMAD2 and p-TAK1.
(G) Percentage of specimens displaying low or high p-TAK1 expression compared to the expression level of p-SMAD2.
(H and I) Kaplan-Meier curves showing that the relapse free period of individuals is inversely correlated with TRAF4 (H) or p-TAK1 (I) expression by a log rank test.
(J) Working model of TRAF4-mediated regulation of TGF-β-induced cancer cell migration and metastasis.
See also Figures S6 and S7 and Tables S2, S3, S4, and S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions