Advertisement

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

Abstract

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.

Keywords

FOXC1 Polycomb EZH2 Metastasis Breast cancer 

Abbreviations

PcG

Polycomb group

EZH2

The enhancer of zeste homologue 2

H3K27

Lysine 27 of histone H3

ChIP

Chromatin immunoprecipitation

YY1

Yin-Yang-1

EMT

Epithelial-mesenchymal transition

Notes

Acknowledgments

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

None.

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)

References

  1. 1.
    Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ (2003) Cancer statistics. CA Cancer J Clin 53(1):5–26PubMedCrossRefGoogle Scholar
  2. 2.
    Ellis M, Heyas D, Lippman M (2000) Treatment of metastatic disease. Dis Breast 2000:749–798Google Scholar
  3. 3.
    Hayes DF (2000) Do we need prognostic factors in nodal-negative breast cancer? Arbiter. Eur J Cancer 36(3):302–306PubMedCrossRefGoogle Scholar
  4. 4.
    Yamashita J, Ogawa M, Shirakusa T (1995) Free-form neutrophil elastase is an independent marker predicting recurrence in primary breast cancer. J Leukoc Biol 57(3):375–378PubMedGoogle Scholar
  5. 5.
    Orlando V (2003) Polycomb, epigenomes, and control of cell identity. Cell 112(5):599–606PubMedCrossRefGoogle Scholar
  6. 6.
    Shao Z, Raible F, Mollaaghababa R, Guyon JR, Wu CT, Bender W, Kingston RE (1999) Stabilization of chromatin structure by PRC1, a polycomb complex. Cell 98(1):37–46PubMedCrossRefGoogle Scholar
  7. 7.
    Sparmann A, van Lohuizen M (2006) Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer 6(11):846–856PubMedCrossRefGoogle Scholar
  8. 8.
    Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K (2003) EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J 22(20):5323–5335PubMedCrossRefGoogle Scholar
  9. 9.
    Raaphorst FM, Meijer CJ, Fieret E, Blokzijl T, Mommers E, Buerger H, Packeisen J, Sewalt RA, Otte AP, van Diest PJ (2003) Poorly differentiated breast carcinoma is associated with increased expression of the human polycomb group EZH2 gene. Neoplasia 5(6):481–488PubMedGoogle Scholar
  10. 10.
    Kleer CG, Cao Q, Varambally S, Shen R, Ota I, Tomlins SA, Ghosh D, Sewalt RG, Otte AP, Hayes DF et al (2003) EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc Natl Acad Sci USA 100(20):11606–11611PubMedCrossRefGoogle Scholar
  11. 11.
    Zeidler M, Kleer CG (2006) The polycomb group protein Enhancer of Zeste 2: its links to DNA repair and breast cancer. J Mol Histol 37(5–7):219–223PubMedCrossRefGoogle Scholar
  12. 12.
    Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, Jones RS, Zhang Y (2002) Role of histone H3 lysine 27 methylation in polycomb-group silencing. Science 298(5595):1039–1043PubMedCrossRefGoogle Scholar
  13. 13.
    Sen GL, Webster DE, Barragan DI, Chang HY, Khavari PA (2008) Control of differentiation in a self-renewing mammalian tissue by the histone demethylase JMJD3. Genes Dev 22(14):1865–1870PubMedCrossRefGoogle Scholar
  14. 14.
    Makarevich G, Leroy O, Akinci U, Schubert D, Clarenz O, Goodrich J, Grossniklaus U, Kohler C (2006) Different polycomb group complexes regulate common target genes in Arabidopsis. EMBO Rep 7(9):947–952PubMedCrossRefGoogle Scholar
  15. 15.
    Mortemousque B, Amati-Bonneau P, Couture F, Graffan R, Dubois S, Colin J, Bonneau D, Morissette J, Lacombe D, Raymond V (2004) Axenfeld-Rieger anomaly: a novel mutation in the forkhead box C1 (FOXC1) gene in a 4-generation family. Arch Ophthalmol 122(10):1527–1533PubMedCrossRefGoogle Scholar
  16. 16.
    Aldinger KA, Lehmann OJ, Hudgins L, Chizhikov VV, Bassuk AG, Ades LC, Krantz ID, Dobyns WB, Millen KJ (2009) FOXC1 is required for normal cerebellar development and is a major contributor to chromosome 6p25.3 Dandy-Walker malformation. Nat Genet 41(9):1037–1042PubMedCrossRefGoogle Scholar
  17. 17.
    Lehmann OJ, Sowden JC, Carlsson P, Jordan T, Bhattacharya SS (2003) Fox’s in development and disease. Trends Genet 19(6):339–344PubMedCrossRefGoogle Scholar
  18. 18.
    Wang X, Pan L, Feng Y, Wang Y, Han Q, Han L, Han S, Guo J, Huang B, Lu J (2008) P300 plays a role in p16(INK4a) expression and cell cycle arrest. Oncogene 27(13):1894–1904PubMedCrossRefGoogle Scholar
  19. 19.
    Hou YF, Yuan ST, Li HC, Wu J, Lu JS, Liu G, Lu LJ, Shen ZZ, Ding J, Shao ZM (2004) ERbeta exerts multiple stimulative effects on human breast carcinoma cells. Oncogene 23(34):5799–5806PubMedCrossRefGoogle Scholar
  20. 20.
    Bloushtain-Qimron N, Yao J, Snyder EL, Shipitsin M, Campbell LL, Mani SA, Hu M, Chen H, Ustyansky V, Antosiewicz JE et al (2008) Cell type-specific DNA methylation patterns in the human breast. Proc Natl Acad Sci USA 105(37):14076–14081PubMedCrossRefGoogle Scholar
  21. 21.
    Kim JH, Yoon SY, Jeong SH, Kim SY, Moon SK, Joo JH, Lee Y, Choe IS, Kim JW (2004) Overexpression of Bmi-1 oncoprotein correlates with axillary lymph node metastases in invasive ductal breast cancer. Breast 13(5):383–388PubMedCrossRefGoogle Scholar
  22. 22.
    Pirrotta V, Poux S, Melfi R, Pilyugin M (2003) Assembly of Polycomb complexes and silencing mechanisms. Genetica 117(2–3):191–197PubMedCrossRefGoogle Scholar
  23. 23.
    Liu ZB, Hou YF, Di GH, Wu J, Shen ZZ, Shao ZM (2009) PA-MSHA inhibits proliferation and induces apoptosis through the up-regulation and activation of caspases in the human breast cancer cell lines. J Cell Biochem 108(1):195–206PubMedCrossRefGoogle Scholar
  24. 24.
    Chang XZ, Li DQ, Hou YF, Wu J, Lu JS, Di GH, Jin W, Ou ZL, Shen ZZ, Shao ZM (2008) Identification of the functional role of AF1Q in the progression of breast cancer. Breast Cancer Res Treat 111(1):65–78PubMedCrossRefGoogle Scholar
  25. 25.
    Schwartz YB, Kahn TG, Nix DA, Li XY, Bourgon R, Biggin M, Pirrotta V (2006) Genome-wide analysis of polycomb targets in Drosophila melanogaster. Nat Genet 38(6):700–705PubMedCrossRefGoogle Scholar
  26. 26.
    Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K (2006) Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev 20(9):1123–1136PubMedCrossRefGoogle Scholar
  27. 27.
    Grimaud C, Negre N, Cavalli G (2006) From genetics to epigenetics: the tale of polycomb group and trithorax group genes. Chromosome Res 14(4):363–375PubMedCrossRefGoogle Scholar
  28. 28.
    Chang YL, Peng YH, Pan IC, Sun DS, King B, Huang DH (2001) Essential role of Drosophila Hdac1 in homeotic gene silencing. Proc Natl Acad Sci USA 98(17):9730–9735PubMedCrossRefGoogle Scholar
  29. 29.
    Muggerud AA, Ronneberg JA, Warnberg F, Botling J, Busato F, Jovanovic J, Solvang H, Bukholm I, Borresen-Dale AL, Kristensen VN et al (2010) Frequent aberrant DNA methylation of ABCB1, FOXC1, PPP2R2B and PTEN in ductal carcinoma in situ and early invasive breast cancer. Breast Cancer Res 12(1):R3PubMedCrossRefGoogle Scholar
  30. 30.
    Srinivasan L, Atchison ML (2004) YY1 DNA binding and PcG recruitment requires CtBP. Genes Dev 18(21):2596–2601PubMedCrossRefGoogle Scholar
  31. 31.
    Dellino GI, Schwartz YB, Farkas G, McCabe D, Elgin SC, Pirrotta V (2004) Polycomb silencing blocks transcription initiation. Mol Cell 13(6):887–893PubMedCrossRefGoogle Scholar
  32. 32.
    Mani SA, Yang J, Brooks M, Schwaninger G, Zhou A, Miura N, Kutok JL, Hartwell K, Richardson AL, Weinberg RA (2007) Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proc Natl Acad Sci USA 104(24):10069–10074PubMedCrossRefGoogle Scholar
  33. 33.
    Hannenhalli S, Kaestner KH (2009) The evolution of Fox genes and their role in development and disease. Nat Rev Genet 10(4):233–240PubMedCrossRefGoogle Scholar
  34. 34.
    Thiery JP (2002) Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2(6):442–454PubMedCrossRefGoogle Scholar
  35. 35.
    Zhou Y, Kato H, Asanoma K, Kondo H, Arima T, Kato K, Matsuda T, Wake N (2002) Identification of FOXC1 as a TGF-beta1 responsive gene and its involvement in negative regulation of cell growth. Genomics 80(5):465–472PubMedCrossRefGoogle Scholar

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

Personalised recommendations