Breast Cancer Research and Treatment

, Volume 131, Issue 1, pp 65–73 | Cite as

FOXC1, a target of polycomb, inhibits metastasis of breast cancer cells

  • Juan Du
  • Lin Li
  • Zhouluo Ou
  • Chenfei Kong
  • Yu Zhang
  • Zhixiong Dong
  • Shan Zhu
  • Hao Jiang
  • Zhimin Shao
  • Baiqu HuangEmail author
  • Jun LuEmail author
Preclinical study


Polycomb group (PcG) proteins have recently been shown related to cancer development. The PcG protein EZH2 is involved in progression of prostate and breast cancers, and has been identified as a molecular marker in breast cancer. Nevertheless, the molecular mechanism by which PcG proteins regulate cancer progression and malignant metastasis is still unclear. PcG proteins methylate H3K27 in undifferentiated epithelial cells, resulting in the repression of differentiation genes such as HOX. FOXC1 is a member of the Forkhead box transcription factor family, which plays an important role in differentiation, and is involved in eye development. We discovered in this study that the expression of FOXC1 gene was negatively correlated to that of PcG genes, i.e., Bmi1, EZH2, and SUZ12, in MCF-7 and MDA-MB-231 cells. To investigate the regulatory effects of PcG proteins on FOXC1 gene, the two cell lines were transfected with either expression plasmids or siRNA plasmids of Bmi1, EZH2, and SUZ12, and we found that PcGs, especially EZH2, could repress the transcription of FOXC1 gene. Chromatin immunoprecipitation (ChIP) assay showed that histone methylation and acetylation modifications played critical roles in this regulatory process. When FOXC1 was stably transfected into MDA-MB-231 cells, the migration and invasion of the cells were repressed. Moreover, the tumorigenicity and the spontaneous metastatic capability regulated by FOXC1 were determined by using an orthotropic xenograft tumor model of athymic mice with the FOXC1-MDA-MB-231HM and the GFP-MDA-MB-231HM cells, and the results showed that FOXC1 in MDA-MB-231HM cells inhibited migration and invasion in vitro and reduced the pulmonary metastasis in vivo. Data presented in this report contribute to the understanding of the mechanisms by which EZH2 participates in tumor development.


FOXC1 Polycomb EZH2 Metastasis Breast cancer 



Polycomb group


The enhancer of zeste homologue 2


Lysine 27 of histone H3


Chromatin immunoprecipitation




Epithelial-mesenchymal transition



This study was supported by grants from The National Natural Science Foundation of China (30671184 and 30971613), The Program for Changjiang Scholars and Innovative Research Team (PCSIRT) in Universities (IRT0519), and The Fundamental Research Funds for the Central Universities.

Conflicts of interest


Supplementary material

10549_2011_1396_MOESM1_ESM.docx (122 kb)
Supplement Fig. 1. Generation of stable MDA-MB-231 cell lines with constitutive FOXC1 overexpression. MDA-MB-231 cells were transfected with FOXC1-pEGFP-N1 and pEGFP-N1 control plasmids using Lipofectamine-2000. After 48 h of incubation, cells were selected for 8 weeks using G418 (800 μg/ml). Western blots detecting the constitutive expression of FOXC1 and GFP in FOXC1-MDA-MB-231 and GFP-MDA-MB-231 cells with specific GFP antibody. (DOCX 121 kb)
10549_2011_1396_MOESM2_ESM.docx (99 kb)
Supplement Figs. 2, 3. Generation of stable MDA-MB-231HM cell line with FOXC1 overexpression. MDA-MB-231HM cells were transfected with FOXC1-pEGFP-N1 and pEGFP-N1 control plasmids using Lipofectamine-2000. After 48 h of incubation, cells were selected for 8 weeks using G418 (800 μg/ml). RT-PCR (S2) and western blotting (S3) detecting the constitutive expression of FOXC1 and GFP in FOXC1-MDA-MB-231HM and GFP-MDA-MB-231HM cells, were performed. (DOCX 99 kb)
10549_2011_1396_MOESM3_ESM.docx (122 kb)
Supplement Fig. 4. Knockdown of FOXC1 inhibited E-cadherin expression in MCF-7 cells. Cells were transfected with FOXC1siRNA1# and FOXC1siRNA2# vectors, and after 48 h, the whole cell lysates were extracted for western blotting. E-cadherin protein level was determined by western blotting (middle). The interfering efficiency of FOXC1siRNAs in MCF-7 cells was verified by western blotting (top). (DOCX 122 kb)
10549_2011_1396_MOESM4_ESM.docx (113 kb)
Supplement Fig. 5. Expression of EZH2, Bmi1, and FOXC1 in ten breast cells. Real-time RT-PCR assessments of EZH2 (A), Bmi1 (B) and FOXC1 (C) mRNAs in ten breast cancer cells are shown. The ten breast cell lines were as follows: MCF10A, MCF7, T47D, MDA-MB-468, MDA-MB435, MDA-MB-435H, MDA-MB-231, MD-MB-231H, BT-474, SKBR3. (DOCX 112 kb)


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Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Juan Du
    • 1
  • Lin Li
    • 1
  • Zhouluo Ou
    • 2
  • Chenfei Kong
    • 3
  • Yu Zhang
    • 1
  • Zhixiong Dong
    • 1
  • Shan Zhu
    • 3
  • Hao Jiang
    • 1
  • Zhimin Shao
    • 2
  • Baiqu Huang
    • 3
    Email author
  • Jun Lu
    • 1
    Email author
  1. 1.The Institute of Genetics and CytologyNortheast Normal UniversityChangchunChina
  2. 2.Department of OncologyBreast Cancer Institute, Cancer Hospital, Shanghai Medical College, Fudan UniversityShanghaiChina
  3. 3.The Key Laboratory of Molecular Epigenetics of the Ministry of EducationNortheast Normal UniversityChangchunChina

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