EZH2-triggered methylation of SMAD3 promotes its activation and tumor metastasis
Changsheng Huang, Fuqing Hu, Da Song, Xuling Sun, Anyi Liu, Qi Wu, Xiaowei She, Yaqi Chen, Lisheng Chen, Fayong Hu, Feng Xu, Xuelai Luo, Yongdong Feng, Xiangping Yang, Junbo Hu, Guihua Wang
Changsheng Huang, Fuqing Hu, Da Song, Xuling Sun, Anyi Liu, Qi Wu, Xiaowei She, Yaqi Chen, Lisheng Chen, Fayong Hu, Feng Xu, Xuelai Luo, Yongdong Feng, Xiangping Yang, Junbo Hu, Guihua Wang
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Research Article Oncology

EZH2-triggered methylation of SMAD3 promotes its activation and tumor metastasis

Abstract

SMAD3 plays a central role in cancer metastasis, and its hyperactivation is linked to poor cancer outcomes. Thus, it is critical to understand the upstream signaling pathways that govern SMAD3 activation. Here, we report that SMAD3 underwent methylation at K53 and K333 (K53/K333) by EZH2, a process crucial for cell membrane recruitment, phosphorylation, and activation of SMAD3 upon TGFB1 stimulation. Mechanistically, EZH2-triggered SMAD3 methylation facilitated SMAD3 interaction with its cellular membrane localization molecule (SARA), which in turn sustained SMAD3 phosphorylation by the TGFB receptor. Pathologically, increased expression of EZH2 expression resulted in the accumulation of SMAD3 methylation to facilitate SMAD3 activation. EZH2-mediated SMAD3 K53/K333 methylation was upregulated and correlated with SMAD3 hyperactivation in breast cancer, promoted tumor metastasis, and was predictive of poor survival outcomes. We used 2 TAT peptides to abrogate SMAD3 methylation and therapeutically inhibit cancer metastasis. Collectively, these findings reveal the complicated layers involved in the regulation of SMAD3 activation coordinated by EZH2-mediated SMAD3 K53/K333 methylation to drive cancer metastasis.

Authors

Changsheng Huang, Fuqing Hu, Da Song, Xuling Sun, Anyi Liu, Qi Wu, Xiaowei She, Yaqi Chen, Lisheng Chen, Fayong Hu, Feng Xu, Xuelai Luo, Yongdong Feng, Xiangping Yang, Junbo Hu, Guihua Wang

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Figure 3

SMAD3 methylation is triggered by EZH2.

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SMAD3 methylation is triggered by EZH2. (A) WCEs of MDA-MB-231 and MCF-...
(A) WCEs of MDA-MB-231 and MCF-7 cells were collected and subjected to co-IP and IB assays. (B) HEK293T and MCF-7 cells were serum starved and treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. (C) MDA-MB-231 and MCF-7 cells were treated with TGFB1 (5 ng/mL) and the EZH2 inhibitors GSK126 or GSK503, and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. (D) HEK293T cells were transfected with WT HA-SMAD3 or mutant plasmids and a Flag-EZH2 plasmid as indicated/WCEs were then collected for IP with anti-HA antibody, followed by IB analysis. (E) Immunoprecipitated EZH2 from HEK293 cells was incubated with SAM along with SMAD3 protein for in vitro methylation of SMAD3. The methylated proteins were separated by SDS-PAGE, and SMAD3 methylation was analyzed by IB using anti–SMAD3 K53/K333 trimethylation–specific antibodies. (F) MDA-MB-231 cells silenced with control (shNC) or EZH2 shRNA (nos. 1 and 2) were treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. (G) HEK293T cells were transfected with vector, EZH2WT, or EZH2H689A and then treated with TGFB1 (5 ng/mL). WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. (H) HEK293T cells were transfected with vector, EZH2WT, or EZH2Y641H, and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. All immunoblotting was performed 3 times, independently, with similar results.