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 3

FOXM1 increases nuclear retention of SMAD3.

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FOXM1 increases nuclear retention of SMAD3. (A) FOXM1 increased nuclear ...
(A) FOXM1 increased nuclear retention of SMAD3. HaCaT cells transfected with Flag-FOXM1 were treated with TGF-β1 (5 ng/ml) for 30 minutes; cells were washed 3 times to remove TGF-β1 and treated with SB431542 for up to 4 hours. Cells were harvested at indicated times, and both the nuclear and cytoplasmic fractions were collected. Values are mean ± SD from 2 experiments. (B) FOXM1 increased nuclear retention of SMAD3. 4T07 control and FOXM1 overexpression cells were treated with TGF-β1 (5 ng/ml) for 30 minutes; cells were washed 3 times to remove TGF-β1 and treated with SB431542 for 4 hours. Intensity of nuclear SMAD3 among these cells was quantified using NIH ImageJ software. The percentages of nuclear SMAD3 level shown at the right represent the mean of 3 independent experiments, and error bars indicate the SEM. Scale bars: 20 μm. (C) SMAD3 nuclear export as determined by a heterokaryon assay. HaCaT stable cells expressing FOXM1 were transfected with Flag-SMAD3 plasmids, and the cells were fused with murine NIH 3T3 cells. NIH 3T3 nuclei that are easily distinguished by the presence of brightly stained blocks in heterorkaryons are marked by arrows. Scale bars: 10 μm. Relocalization of SMAD3 to the mouse nucleus was rarely seen in heterokaryons expressing FOXM1, as shown with the percentages of heterokaryons in right panel. (D) Knockdown of FOXM1 reduced the level of nuclear SMAD3. MDA-MB-231 cells stably expressing shControl or shFOXM1 were treated with TGF-β1 (5 ng/ml) for 1 hour.