EZH2-triggered methylation of SMAD3 promotes its activation and tumor metastasis
Changsheng Huang, … , Junbo Hu, Guihua Wang
Changsheng Huang, … , Junbo Hu, Guihua Wang
Published January 27, 2022
Citation Information: J Clin Invest. 2022;132(5):e152394. https://doi.org/10.1172/JCI152394.
View: Text | PDF
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

×

Figure 1

SMAD3 K53/K333 methylation is critical for SMAD3 activation.

Options: View larger image (or click on image) Download as PowerPoint
SMAD3 K53/K333 methylation is critical for SMAD3 activation. (A and B) ...
(A and B) HEK293T cells and MDA-MB-231 cells were serum starved and treated with TGFB1 (5 ng/mL) for the indicated durations, and whole-cell extracts (WCEs) were collected for IP with anti-SMAD3 antibody, followed by immunoblot (IB) analysis. (C) HEK293T cells were transfected with WT HA-SMAD3 or mutant plasmids as indicated and treated with TGFB1 (5 ng/mL). WCEs were then collected for IP with anti-HA antibody, followed by IB analysis. (D) SMAD3 K53/K333 site aa in different species. (E) HEK293T cells were transfected with WT HA-SMAD3 or K53/333R-mutant plasmids and then treated with TGFB1 (5 ng/mL). WCEs were collected for IP with anti-HA antibody, followed by IB analysis. (F) ddH2O (10 μL) containing different peptides (0.1–0.75 μg) was added onto the PVDF membranes, followed by IB analysis using a K53-specific trimethylation antibody (anti–SMAD3 K53me3) and a K333-specific trimethylation antibody (anti–SMAD3 K333me3). (G) MDA-MB-231 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 with SMAD3 K53/K333 trimethylation–specific antibodies. (H) MDA-MB-231SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL). WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. All immunoblotting was performed 3 times, independently, with similar results.