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

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

doi:10.1172/jci152394

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.

Highlights

Tumor metastasis is highly responsible for tumor-related death[1,2,3,4]
We proved that K53 and K333 methylation of SMAD3 is crucial for SMAD3 cell membrane recruitment, phosphorylation and biological function stimulated by TGFB1
Inhibiting SMAD3 methylation with designed peptides blocks cancer cell metastasis Based on the fact that EZH2-mediated SMAD3 K53 and K333 methylation is necessary for TGFB/SMAD signaling pathway activation and cancer metastasis, we investigated whether SMAD3 methylation could be therapeutically targeted

Summary

Introduction

Tumor metastasis is highly responsible for tumor-related death[1,2,3,4]. During metastasis, epithelial cells gradually discard their differentiated characteristics, including losing cellular polarity, cell-cell adhesion capacity, whereby gaining mesenchymal characteristics such as invasion, migration, and motility, which called Epithelial-mesenchymal transition (EMT) [5, 6]. Overexpression of wild-type EZH2, but not the EZH2 H689A mutant deficient in methyltransferase activity[38, 39], enhanced basal and TGFB1-induced SMAD3 at K53 and K333 tri-methylation and SMAD3S423/S425 phosphorylation We found that gain of wild-type EZH2, but not the EZH2 T372A mutant, enhanced basal TGFB1-induced SMAD3 at K53 and K333 tri-methylation and SMAD3S423/S425 phosphorylation, cells ectopicly expressing EZH2 T372A had higher Histone 3 at K27 tri-methylation compared with cells ectopicly expressing wild-type EZH2(Figure. Inhibiting SMAD3 methylation with designed peptides blocks cancer cell metastasis Based on the fact that EZH2-mediated SMAD3 K53 and K333 methylation is necessary for TGFB/SMAD signaling pathway activation and cancer metastasis, we investigated whether SMAD3 methylation could be therapeutically targeted. Combining TAT-peptides 1 and TAT-peptide 2 significantly inhibited EZH2-mediated SMAD3 K53 and K333 methylation, as well as cancer cell epithelial-mesenchymal transition (EMT) Our data underscore the importance of EZH2-mediated SMAD3 K53 and K333 methylation in metastasis; SMAD3 methylation might function as a marked factor to predict breast cancer patients’ overall survival time

Discussion

Findings

Methods