TWIST represses estrogen receptor-alpha expression by recruiting the NuRD protein complex in breast cancer cells

Abstract

Loss of estrogen receptor α (ERα) expression and gain of TWIST (TWIST1) expression in breast tumors correlate with increased disease recurrence and metastasis and poor disease-free survival. However, the molecular and functional regulatory relationship between TWIST and ERα are unclear. In this study, we found TWIST was associated with a chromatin region in intron 7 of the human ESR1 gene coding for ERα. This association of TWIST efficiently recruited the nucleosome remodeling and deacetylase (NuRD) repressor complex to this region, which subsequently decreased histone H3K9 acetylation, increased histone H3K9 methylation and repressed ESR1 expression in breast cancer cells. In agreement with these molecular events, TWIST expression was inversely correlated with ERα expression in both breast cancer cell lines and human breast ductal carcinomas. Forced expression of TWIST in TWIST-negative and ERα-positive breast cancer cells such as T47D and MCF-7 cells reduced ERα expression, while knockdown of TWIST in TWIST-positive and ERα-negative breast cancer cells such as MDA-MB-435 and 4T1 cells increased ERα expression. Furthermore, inhibition of histone deacetylase (HDAC) activity including the one in NuRD complex significantly increased ERα expression in MDA-MB-435 and 4T1 cells. HDAC inhibition together with TWIST knockdown did not further increase ERα expression in 4T1 and MDA-MB-435 cells. These results demonstrate that TWIST/NuRD represses ERα expression in breast cancer cells. Therefore, TWIST may serve as a potential molecular target for converting ERα-negative breast cancers to ERα-positive breast cancers, allowing these cancers to restore their sensitivity to endocrine therapy with selective ERα antagonists such as tamoxifen and raloxifene.

Keywords: ERα; NuRD complex; TWIST; breast cancer; gene repression.

Fig 1

TWIST is associated with a chromatin region in intron 7 of the ESR1 gene. A. The ChIP-Seq map showed one TWIST binding site located in intron 7 of the ESR1 gene in Flag-Twist HEK293 cells. The peak value was obtained by normalizing to the background, if any, in Flag HEK293 control cells. The locations of the ESR1 gene in chromosome 6 and the TWIST binding sites are indicated. The locations of primer pairs used for ChIP assays are also indicated. B. ChIP assays were performed using Flag-TWIST (FT) and Flag (F) HEK293 cells and Flag antibody beads. Precipitated DNA was assayed by PCR using three pairs of primers indicated in panel A. Data obtained from Flag-TWIST cells were normalized with that from Flag control cells. C. ChIP assay in MDA-MB-435 cells with endogenously expressed TWIST. ChIP assay was performed using TWIST antibody or using non-immune IgG as a negative control. The precipitated DNA was assayed by PCR using primers P3 and P4 shown in panel A.

Fig 2

TWIST recruits NuRD complex to the TWIST-binding site of the ESR1 gene. A. ChIP-re-ChIP assays were performed with Flag HEK293 (F) and Flag-TWIST HEK293 (FT) cells using the Flag antibody beads in the first step ChIP and the antibodies against Mi2, MTA2 or HDAC2 in the second step ChIP. The precipitated DNA from the second step ChIP was assayed by real time PCR using primers P3 and P4 indicated in Fig. 1A. B. MDA-MB-435 cells expressing non-targeting shRNA (shCtrl) or shRNA targeting TWIST mRNA (shT) were used in ChIP assays with antibodies against TWIST, MTA2, HDAC2 or RbAp46 as indicated. Real-time PCR was performed as panel A. C. MDA-MB-435 cells with shCtrl or shT were used in ChIP assays with antibodies against acetylated or methylated histone H3K9 as indicated. Real-time PCR was performed as panel A.

Fig 3

TWIST expression is inversely correlated with ERα expression in human breast cancer cell lines. A. Real-time RT-PCR analysis for the mRNA levels of TWIST (T) and ERα (E) in SUM1315, MDA-MB-435 (MDA435), MDA-MB-231 (MDA231), T47D and MCF-7 human breast cancer cell lines. The data were normalized to the mRNA levels of GAPDH and presented as average ± standard deviation. B. Western blot analysis for TWIST and ERα in the above human breast cancer cell lines. β-actin was assayed as a control for the loaded total cellular proteins.

Fig 4

TWIST represses ERα expression. A. 293T cells were transiently co-transfected with 120 ng of HA-TWIST expression plasmid or parent control plasmid DNA and 50 ng of pGL3-332ESR1-Pro-Luc or pGL-Pro-Luc reporter plasmid DNA as indicated. Western blot analysis using antibodies against HA and β-actin (lower panel) and luciferase activity assay (upper panel) were performed in triplicates. **, p < 0.001. B. T47D cell lines with control parent vector (T47D/V) and TWIST expression vector (T47D/T) were established. The levels of TWIST and ERα mRNAs were measured by real time RT-PCR. **, p < 0.001. C. Western blot analysis for ERα, E-cadherin, TWIST and β-actin in MCF-7 cells transfected with a control parent vector or a TWIST expression vector. D. Measurement of ERα mRNA levels by real-time RT-PCR in 4T1 and MDA-MB-435 (MDA435) cells expressing a non-targeting shRNA (shCtrl) or an shRNA targeting TWIST mRNA (shT). **, p < 0.001. E. The 4T1 and MDA-MB-435 cells with TWIST knockdown (shTWIST) or control shRNA (shCtrl) were treated with vehicle (-) or 330 nM of TSA (+) as indicated. Western blot analysis was performed using antibodies against ERα and β-actin.

Fig 5

TWIST expression inversely correlates with the levels of ERα protein in human breast invasive ductal carcinomas. A. Semi-quantitative analysis of ERα, TWIST and GAPDH mRNA expression by RT-PCR in human breast invasive ductal carcinomas. A total of 28 independent tumor mRNA samples were assayed with three repeat experiments and a part of the representative results is shown. B. Immunohistochemistry analysis (brown color) for TWIST and ERα in human breast invasive ductal carcinomas. Tissue sections prepared from the 28 human invasive breast ductal carcinomas were immunostained with TWIST and ERα antibodies. Representative results obtained from two tumors are shown. Photos were taken under a microscope set at 400×magnification. C. A summary of the data according to TWIST and ERα immunohistochemistry in the 28 human invasive breast ductal carcinomas.

Fig 6

A model for the repression of ERα expression by TWIST. TWIST protein (T) binds to E-boxes in the 7^th intron of ESR1 gene as homodimer or heterodimer with E12 or TWIST2, which recruits NuRD complex to decrease H3K9 acetylation (Ac) and increase H3K9 methylation (Me1) levels, resulting in transcriptional repression of ERα expression.