G9a interacts with Snail and is critical for Snail-mediated E-cadherin repression in human breast cancer
Chenfang Dong, … , B. Mark Evers, Binhua P. Zhou
Chenfang Dong, … , B. Mark Evers, Binhua P. Zhou
Published March 12, 2012
Citation Information: J Clin Invest. 2012;122(4):1469-1486. https://doi.org/10.1172/JCI57349.
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Research Article Oncology

G9a interacts with Snail and is critical for Snail-mediated E-cadherin repression in human breast cancer

Abstract

Breast cancers are highly heterogeneous but can be grouped into subtypes based on several criteria, including level of expression of certain markers. Claudin-low breast cancer (CLBC) is associated with early metastasis and resistance to chemotherapy, while gene profiling indicates it is characterized by the expression of markers of epithelial-mesenchymal transition (EMT) — a phenotypic conversion linked with metastasis. Although the epigenetic program controlling the phenotypic and cellular plasticity of EMT remains unclear, one contributor may be methylation of the E-cadherin promoter, resulting in decreased E-cadherin expression, a hallmark of EMT. Indeed, reduced E-cadherin often occurs in CLBC and may contribute to the early metastasis and poor patient survival associated with this disease. Here, we have determined that methylation of histone H3 on lysine 9 (H3K9me2) is critical for promoter DNA methylation of E-cadherin in three TGF-β–induced EMT model cell lines, as well as in CLBC cell lines. Further, Snail interacted with G9a, a major euchromatin methyltransferase responsible for H3K9me2, and recruited G9a and DNA methyltransferases to the E-cadherin promoter for DNA methylation. Knockdown of G9a restored E-cadherin expression by suppressing H3K9me2 and blocking DNA methylation. This resulted in inhibition of cell migration and invasion in vitro and suppression of tumor growth and lung colonization in in vivo models of CLBC metastasis. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT but also paves a way for the development of new treatment strategies for CLBC.

Authors

Chenfang Dong, Yadi Wu, Jun Yao, Yifan Wang, Yinhua Yu, Piotr G. Rychahou, B. Mark Evers, Binhua P. Zhou

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Figure 3

G9a is required for H3K9me2 and DNA methylation at the E-cadherin promoter.

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G9a is required for H3K9me2 and DNA methylation at the E-cadherin promot...
(A) G9a, Snail, or NTC siRNA was expressed in, or BIX01294 (BIX; 2.5 μM) was added to NMuMG cells followed by treatment with or without TGF-β1 (5 ng/ml) for 3 days. H3K9me2 and H3K9 acetylation at the E-cadherin promoter was analyzed by ChIP. ChIP samples were also analyzed by quantitative real-time PCR (mean ± SD from 3 separate experiments; bottom panel). (B) NMuMG cells were treated as described in A. DNA methylation at the E-cadherin promoter was analyzed by MSP. Samples from MSP analyses were also analyzed by quantitative real-time PCR, and the ratio of methylated to unmethylated DNA was plotted (mean ± SD from 3 separate experiments; bottom panel). (C) NMuMG cells were treated as described in B. The expression of E-cadherin mRNA was analyzed by either semi-quantitative RT-PCR (bottom panel) or quantitative real-time PCR (top panel) (mean ± SD from 3 separate experiments). (D) NMuMG and MCF10A cells were treated with TGF-β1 for 3 and 12 days, respectively. After immunoprecipitation of endogenous Snail, associated endogenous G9a and DNMT1 were analyzed by Western blotting.