Sustained activation of SMAD3/SMAD4 by FOXM1 promotes TGF-β–dependent cancer metastasis
Jianfei Xue, … , Mien-Chie Hung, Suyun Huang
Jianfei Xue, … , Mien-Chie Hung, Suyun Huang
Published January 2, 2014
Citation Information: J Clin Invest. 2014;124(2):564-579. https://doi.org/10.1172/JCI71104.
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Research Article Oncology

Sustained activation of SMAD3/SMAD4 by FOXM1 promotes TGF-β–dependent cancer metastasis

Abstract

A key feature of TGF-β signaling activation in cancer cells is the sustained activation of SMAD complexes in the nucleus; however, the drivers of SMAD activation are poorly defined. Here, using human and mouse breast cancer cell lines, we found that oncogene forkhead box M1 (FOXM1) interacts with SMAD3 to sustain activation of the SMAD3/SMAD4 complex in the nucleus. FOXM1 prevented the E3 ubiquitin-protein ligase transcriptional intermediary factor 1 γ (TIF1γ) from binding SMAD3 and monoubiquitinating SMAD4, which stabilized the SMAD3/SMAD4 complex. Loss of FOXM1 abolished TGF-β–induced SMAD3/SMAD4 formation. Moreover, the interaction of FOXM1 and SMAD3 promoted TGF-β/SMAD3–mediated transcriptional activity and target gene expression. We found that FOXM1/SMAD3 interaction was required for TGF-β–induced breast cancer invasion, which was the result of SMAD3/SMAD4-dependent upregulation of the transcription factor SLUG. Importantly, the function of FOXM1 in TGF-β–induced invasion was not dependent on FOXM1’s transcriptional activity. Knockdown of SMAD3 diminished FOXM1-induced metastasis. Furthermore, FOXM1 levels correlated with activated TGF-β signaling and metastasis in human breast cancer specimens. Together, our data indicate that FOXM1 promotes breast cancer metastasis by increasing nuclear retention of SMAD3 and identify crosstalk between FOXM1 and TGF-β/SMAD3 pathways. This study highlights the critical interaction of FOXM1 and SMAD3 for controlling TGF-β signaling during metastasis.

Authors

Jianfei Xue, Xia Lin, Wen-Tai Chiu, Yao-Hui Chen, Guanzhen Yu, Mingguang Liu, Xin-Hua Feng, Raymond Sawaya, René H. Medema, Mien-Chie Hung, Suyun Huang

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Figure 5

FOXM1 is required for formation of the nuclear SMAD3/SMAD4 complex.

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FOXM1 is required for formation of the nuclear SMAD3/SMAD4 complex. (A) ...
(A) FOXM1 knockdown blocked formation of the SMAD3/SMAD4 complex. MDA-MB-231 shControl or shFOXM1 cells were treated with TGF-β1 (5 ng/ml) for 1 hour before immunoprecipitation of SMAD3. The amount of SMAD4 bound to SMAD3 was detected by immunoblot using an anti-SMAD4 antibody. (B) MDA-MB-231 shControl or shFOXM1 cells were treated with TGF-β1 for 1 hour. The cells were stained with an anti-SMAD3 or anti-SMAD4 antibody and then by a rhodamine-conjugated anti-mouse secondary antibody (red, for SMAD4) or a FITC-conjugated anti-rabbit secondary antibody (green, for SMAD3), and the nuclei were visualized with DAPI (blue). Scale bar: 20 μm. Results are indicated at bottom as the percentage of cells showing mostly cytoplasmic or mostly nuclear fluorescence or both. (C) FOXM1 deficiency inhibited formation of the SMAD3/SMAD4 complex. Foxm1fl/fl or Foxm1–/– MEFs were treated with TGF-β1 (5 ng/ml) for 1 hour. Then the immunoprecipitation of SMAD3 and the immunoblots of SMAD3 and SMAD4 were conducted as described for panel A. (D) Foxm1fl/fl or Foxm1–/– MEFs were treated with TGF-β1 for 1 hour. Scale bar: 20 μm.