Breast Cancer Research and Treatment

, Volume 138, Issue 3, pp 741–752 | Cite as

EZH2 inhibition decreases p38 signaling and suppresses breast cancer motility and metastasis

  • Heather M. Moore
  • Maria E. Gonzalez
  • Kathy A. Toy
  • Ashley Cimino-Mathews
  • Pedram Argani
  • Celina G. Kleer
Preclinical study


EZH2 is a Polycomb group protein that exerts oncogenic functions in breast cancer, where its overexpression is associated with metastatic disease. While it reportedly acts a transcriptional repressor through trimethylation of histone H3 at lysine 27, EZH2 may exhibit context-dependent activating functions. Despite associations with worse outcome and metastasis in breast cancer, a functional role of EZH2 in breast cancer metastasis in vivo has not been demonstrated. Furthermore, whether EZH2 regulates cancer cell phenotype and motility are unknown. In this study, we discovered that knockdown of EZH2 induces a phenotypic reprogramming from mesenchymal to epithelial, reduces motility, and blocks invasion in breast cancer cell lines. In vivo, EZH2 downregulation in MDA-MB-231 cells decreases spontaneous metastasis to the lungs. We uncover an unexpected role of EZH2 in inducing the p38 mitogen-activated protein kinase signaling pathway, an important regulator of breast cancer invasion and metastasis. In breast cancer cells, EZH2 binds to phosphorylated p38 (p-p38) in association with other core members of the Polycomb repressive complex 2, EED, and SUZ12, and EZH2 overexpression leads to increased levels of p-p38 and of activated, downstream pathway proteins. The effect on p-p38 was confirmed in vivo, where it correlated with decreased spontaneous metastasis. In clinical specimens of matched primary and invasive breast carcinomas, we found that EZH2 expression was upregulated in 100 % of the metastases, and that EZH2 and p-p38 were coexpressed in 63 % of cases, consistent with the functional results. Together our findings reveal a new mechanism by which EZH2 functions in breast cancer, and provide direct evidence that EZH2 inhibition reduces breast cancer metastasis in vivo.


Breast cancer EZH2 p38 Cell motility Metastasis Snail 



Epithelial-to-mesenchymal transition


Enhancer of zeste homolog 2


Mesenchymal-to-epithelial transition

p-p38 MAPK

Phosphorylated p38 mitogen-activated protein kinase


Polycomb repressive complex 2



We thank Xin Li for help in mouse colony maintenance and executing the spontaneous metastasis assay. We thank Wei Huang and Paul Moore for helpful experimental suggestions and support. We thank Yali Dou and Bo Zhou for assistance with the in vitro methylation assay. This study was supported by the Department of Defense Breast Cancer Research Program through a Predoctoral Traineeship Award (BC093828; to H. M. M) and the National Institutes of Health Grants R01 CA107469, R01 CA125577 and U01 CA154224 (to C. G. K.), and the University of Michigan’s Cancer Center Support Grant (5 P30 CA46592).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10549_2013_2498_MOESM1_ESM.pdf (931 kb)
Supplementary material 1 (PDF 931 kb)


  1. 1.
    Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ (2003) Cancer statistics, 2003. CA Cancer J Clin 53(1):5–26PubMedCrossRefGoogle Scholar
  2. 2.
    American Cancer Society (2012) Breast cancer facts and figures 2011–2012. American Cancer Society, AtlantaGoogle Scholar
  3. 3.
    Hayes DF, Isaacs C, Stearns V (2001) Prognostic factors in breast cancer: current and new predictors of metastasis. J Mammary Gland Biol Neoplasia 6(4):375–392PubMedCrossRefGoogle Scholar
  4. 4.
    Laible G, Wolf A, Dorn R, Reuter G, Nislow C, Lebersorger A, Popkin D, Pillus L, Jenuwein T (1997) Mammalian homologues of the Polycomb-group gene enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres. EMBO J 16(11):3219–3232. doi: 10.1093/emboj/16.11.3219 PubMedCrossRefGoogle Scholar
  5. 5.
    Ringrose L, Paro R (2004) Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu Rev Genet 38:413–443. doi: 10.1146/annurev.genet.38.072902.091907 PubMedCrossRefGoogle Scholar
  6. 6.
    Satijn DP, Otte AP (1999) Polycomb group protein complexes: do different complexes regulate distinct target genes? Biochim Biophys Acta 1447(1):1–16 S0167-4781(99)00130-XPubMedCrossRefGoogle Scholar
  7. 7.
    Tonini T, Bagella L, D’Andrilli G, Claudio PP, Giordano A (2004) Ezh2 reduces the ability of HDAC1-dependent pRb2/p130 transcriptional repression of cyclin A. Oncogene 23(28):4930–4937. doi: 10.1038/sj.onc.1207608 PubMedCrossRefGoogle Scholar
  8. 8.
    Shi B, Liang J, Yang X, Wang Y, Zhao Y, Wu H, Sun L, Zhang Y, Chen Y, Li R, Hong M, Shang Y (2007) Integration of estrogen and Wnt signaling circuits by the Polycomb group protein EZH2 in breast cancer cells. Mol Cell Biol 27(14):5105–5119. doi: 10.1128/MCB.00162-07 PubMedCrossRefGoogle Scholar
  9. 9.
    Su IH, Dobenecker MW, Dickinson E, Oser M, Basavaraj A, Marqueron R, Viale A, Reinberg D, Wulfing C, Tarakhovsky A (2005) Polycomb group protein ezh2 controls actin polymerization and cell signaling. Cell 121(3):425–436. doi: 10.1016/j.cell.2005.02.029 PubMedCrossRefGoogle Scholar
  10. 10.
    Asangani IA, Ateeq B, Cao Q, Dodson L, Pandhi M, Kunju LP, Mehra R, Lonigro RJ, Siddiqui J, Palanisamy N, Wu YM, Cao X, Kim JH, Zhao M, Qin ZS, Iyer MK, Maher CA, Kumar-Sinha C, Varambally S, Chinnaiyan AM (2012) Characterization of the EZH2-MMSET histone methyltransferase regulatory axis in cancer. Mol Cell. doi: 10.1016/j.molcel.2012.10.008 PubMedGoogle Scholar
  11. 11.
    Chang CJ, Hung MC (2012) The role of EZH2 in tumour progression. Br J Cancer 106(2):243–247. doi: 10.1038/bjc.2011.551 PubMedCrossRefGoogle Scholar
  12. 12.
    Sauvageau M, Sauvageau G (2010) Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer. Cell Stem Cell 7(3):299–313. doi: 10.1016/j.stem.2010.08.002 PubMedCrossRefGoogle Scholar
  13. 13.
    Chase A, Cross NC (2011) Aberrations of EZH2 in cancer. Clin Cancer Res 17(9):2613–2618. doi: 10.1158/1078-0432.CCR-10-2156 PubMedCrossRefGoogle Scholar
  14. 14.
    Kleer CG, Cao Q, Varambally S, Shen R, Ota I, Tomlins SA, Ghosh D, Sewalt RG, Otte AP, Hayes DF, Sabel MS, Livant D, Weiss SJ, Rubin MA, Chinnaiyan AM (2003) EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc Natl Acad Sci USA 100(20):11606–11611. doi: 10.1073/pnas.1933744100 PubMedCrossRefGoogle Scholar
  15. 15.
    Bachmann IM, Halvorsen OJ, Collett K, Stefansson IM, Straume O, Haukaas SA, Salvesen HB, Otte AP, Akslen LA (2006) EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast. J Clin Oncol 24(2):268–273. doi: 10.1200/JCO.2005.01.5180 PubMedCrossRefGoogle Scholar
  16. 16.
    Collett K, Eide GE, Arnes J, Stefansson IM, Eide J, Braaten A, Aas T, Otte AP, Akslen LA (2006) Expression of enhancer of zeste homologue 2 is significantly associated with increased tumor cell proliferation and is a marker of aggressive breast cancer. Clin Cancer Res 12(4):1168–1174. doi: 10.1158/1078-0432.CCR-05-1533 PubMedCrossRefGoogle Scholar
  17. 17.
    Gonzalez ME, Li X, Toy K, DuPrie M, Ventura AC, Banerjee M, Ljungman M, Merajver SD, Kleer CG (2009) Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1. Oncogene 28(6):843–853. doi: 10.1038/onc.2008.433 PubMedCrossRefGoogle Scholar
  18. 18.
    Ding L, Erdmann C, Chinnaiyan AM, Merajver SD, Kleer CG (2006) Identification of EZH2 as a molecular marker for a precancerous state in morphologically normal breast tissues. Cancer Res 66(8):4095–4099. doi: 10.1158/0008-5472.CAN-05-4300 PubMedCrossRefGoogle Scholar
  19. 19.
    Wagner EF, Nebreda AR (2009) Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer 9(8):537–549. doi: 10.1038/nrc2694 PubMedCrossRefGoogle Scholar
  20. 20.
    del Barco Barrantes I, Nebreda AR (2012) Roles of p38 MAPKs in invasion and metastasis. Biochem Soc Trans 40(1):79–84. doi: 10.1042/BST20110676 CrossRefGoogle Scholar
  21. 21.
    Rosenthal DT, Iyer H, Escudero S, Bao L, Wu Z, Ventura AC, Kleer CG, Arruda EM, Garikipati K, Merajver SD (2011) p38gamma promotes breast cancer cell motility and metastasis through regulation of RhoC GTPase, cytoskeletal architecture, and a novel leading edge behavior. Cancer Res 71(20):6338–6349. doi: 10.1158/0008-5472.CAN-11-1291 PubMedCrossRefGoogle Scholar
  22. 22.
    Kleer CG, Zhang Y, Pan Q, Merajver SD (2004) WISP3 (CCN6) is a secreted tumor-suppressor protein that modulates IGF signaling in inflammatory breast cancer. Neoplasia 6(2):179–185. doi: 10.1593/neo.03316 PubMedCrossRefGoogle Scholar
  23. 23.
    Cao Q, Yu J, Dhanasekaran SM, Kim JH, Mani RS, Tomlins SA, Mehra R, Laxman B, Cao X, Kleer CG, Varambally S, Chinnaiyan AM (2008) Repression of E-cadherin by the Polycomb group protein EZH2 in cancer. Oncogene 27(58):7274–7284. doi: 10.1038/onc.2008.333 PubMedCrossRefGoogle Scholar
  24. 24.
    Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, Ghosh D, Pienta KJ, Sewalt RG, Otte AP, Rubin MA, Chinnaiyan AM (2002) The Polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 419(6907):624–629. doi: 10.1038/nature01075 PubMedCrossRefGoogle Scholar
  25. 25.
    Cimino-Mathews A, Hicks JL, Illei PB, Halushka MK, Fetting JH, De Marzo AM, Park BH, Argani P (2012) Androgen receptor expression is usually maintained in initial surgically resected breast cancer metastases but is often lost in end-stage metastases found at autopsy. Hum Pathol 43(7):1003–1011. doi: 10.1016/j.humpath.2011.08.007 PubMedCrossRefGoogle Scholar
  26. 26.
    Singhi AD, Cimino-Mathews A, Jenkins RB, Lan F, Fink SR, Nassar H, Vang R, Fetting JH, Hicks J, Sukumar S, De Marzo AM, Argani P (2012) MYC gene amplification is often acquired in lethal distant breast cancer metastases of unamplified primary tumors. Mod Pathol 25(3):378–387. doi: 10.1038/modpathol.2011.171 PubMedCrossRefGoogle Scholar
  27. 27.
    Pal A, Huang W, Li X, Toy KA, Nikolovska-Coleska Z, Kleer CG (2012) CCN6 modulates BMP signaling via the Smad-independent TAK1/p38 pathway, acting to suppress metastasis of breast cancer. Cancer Res 72(18):4818–4828. doi: 10.1158/0008-5472.CAN-12-0154 PubMedCrossRefGoogle Scholar
  28. 28.
    Cuadrado A, Nebreda AR (2010) Mechanisms and functions of p38 MAPK signalling. Biochem J 429(3):403–417. doi: 10.1042/BJ20100323 PubMedCrossRefGoogle Scholar
  29. 29.
    Drasin DJ, Robin TP, Ford HL (2011) Breast cancer epithelial-to-mesenchymal transition: examining the functional consequences of plasticity. Breast Cancer Res 13(6):226. doi: 10.1186/bcr3037 PubMedCrossRefGoogle Scholar
  30. 30.
    Tiwari N, Gheldof A, Tatari M, Christofori G (2012) EMT as the ultimate survival mechanism of cancer cells. Semin Cancer Biol 22(3):194–207. doi: 10.1016/j.semcancer.2012.02.013 PubMedCrossRefGoogle Scholar
  31. 31.
    Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial-mesenchymal transitions in development and disease. Cell 139(5):871–890. doi: 10.1016/j.cell.2009.11.007 PubMedCrossRefGoogle Scholar
  32. 32.
    Hong J, Zhou J, Fu J, He T, Qin J, Wang L, Liao L, Xu J (2011) Phosphorylation of serine 68 of Twist1 by MAPKs stabilizes Twist1 protein and promotes breast cancer cell invasiveness. Cancer Res 71(11):3980–3990. doi: 10.1158/0008-5472.CAN-10-2914 PubMedCrossRefGoogle Scholar
  33. 33.
    Hipp S, Berg D, Ergin B, Schuster T, Hapfelmeier A, Walch A, Avril S, Schmalfeldt B, Hofler H, Becker KF (2010) Interaction of Snail and p38 mitogen-activated protein kinase results in shorter overall survival of ovarian cancer patients. Virchows Arch 457(6):705–713. doi: 10.1007/s00428-010-0986-5 PubMedCrossRefGoogle Scholar
  34. 34.
    Zohn IE, Li Y, Skolnik EY, Anderson KV, Han J, Niswander L (2006) p38 and a p38-interacting protein are critical for downregulation of E-cadherin during mouse gastrulation. Cell 125(5):957–969. doi: 10.1016/j.cell.2006.03.048 PubMedCrossRefGoogle Scholar
  35. 35.
    Parvani JG, Taylor MA, Schiemann WP (2011) Noncanonical TGF-beta signaling during mammary tumorigenesis. J Mammary Gland Biol Neoplasia 16(2):127–146. doi: 10.1007/s10911-011-9207-3 PubMedCrossRefGoogle Scholar
  36. 36.
    Hsieh YH, Wu TT, Huang CY, Hsieh YS, Hwang JM, Liu JY (2007) p38 mitogen-activated protein kinase pathway is involved in protein kinase Calpha-regulated invasion in human hepatocellular carcinoma cells. Cancer Res 67(9):4320–4327. doi: 10.1158/0008-5472.CAN-06-2486 PubMedCrossRefGoogle Scholar
  37. 37.
    Junttila MR, Ala-Aho R, Jokilehto T, Peltonen J, Kallajoki M, Grenman R, Jaakkola P, Westermarck J, Kahari VM (2007) p38alpha and p38delta mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells. Oncogene 26(36):5267–5279. doi: 10.1038/sj.onc.1210332 PubMedCrossRefGoogle Scholar
  38. 38.
    Demuth T, Reavie LB, Rennert JL, Nakada M, Nakada S, Hoelzinger DB, Beaudry CE, Henrichs AN, Anderson EM, Berens ME (2007) MAP-ing glioma invasion: mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival. Mol Cancer Ther 6(4):1212–1222. doi: 10.1158/1535-7163.MCT-06-0711 PubMedCrossRefGoogle Scholar
  39. 39.
    Hsieh MJ, Chen KS, Chiou HL, Hsieh YS (2010) Carbonic anhydrase XII promotes invasion and migration ability of MDA-MB-231 breast cancer cells through the p38 MAPK signaling pathway. Eur J Cell Biol 89(8):598–606. doi: 10.1016/j.ejcb.2010.03.004 PubMedCrossRefGoogle Scholar
  40. 40.
    Johansson N, Ala-aho R, Uitto V, Grenman R, Fusenig NE, Lopez-Otin C, Kahari VM (2000) Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase. J Cell Sci 2:227–235Google Scholar
  41. 41.
    Kumar P, Yadav A, Patel SN, Islam M, Pan Q, Merajver SD, Teknos TN (2010) Tetrathiomolybdate inhibits head and neck cancer metastasis by decreasing tumor cell motility, invasiveness and by promoting tumor cell anoikis. Mol Cancer 9:206. doi: 10.1186/1476-4598-9-206 PubMedCrossRefGoogle Scholar
  42. 42.
    Xu L, Chen S, Bergan RC (2006) MAPKAPK2 and HSP27 are downstream effectors of p38 MAP kinase-mediated matrix metalloproteinase type 2 activation and cell invasion in human prostate cancer. Oncogene 25(21):2987–2998. doi: 10.1038/sj.onc.1209337 PubMedCrossRefGoogle Scholar
  43. 43.
    Park SY, Jeong KJ, Panupinthu N, Yu S, Lee J, Han JW, Kim JM, Lee JS, Kang J, Park CG, Mills GB, Lee HY (2011) Lysophosphatidic acid augments human hepatocellular carcinoma cell invasion through LPA1 receptor and MMP-9 expression. Oncogene 30(11):1351–1359. doi: 10.1038/onc.2010.517 PubMedCrossRefGoogle Scholar
  44. 44.
    Dreissigacker U, Mueller MS, Unger M, Siegert P, Genze F, Gierschik P, Giehl K (2006) Oncogenic K-Ras down-regulates Rac1 and RhoA activity and enhances migration and invasion of pancreatic carcinoma cells through activation of p38. Cell Signal 18(8):1156–1168. doi: 10.1016/j.cellsig.2005.09.004 PubMedCrossRefGoogle Scholar
  45. 45.
    Loesch M, Chen G (2008) The p38 MAPK stress pathway as a tumor suppressor or more? Front Biosci 13:3581–3593PubMedCrossRefGoogle Scholar
  46. 46.
    Lee ST, Li Z, Wu Z, Aau M, Guan P, Murthy Karuturi RK, Liou YC, Yu Q (2011) Context-specific regulation of NF-kB target gene expression by EZH2 in breast cancers. Mol Cell 43:789–810Google Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Heather M. Moore
    • 1
    • 2
  • Maria E. Gonzalez
    • 1
    • 3
  • Kathy A. Toy
    • 1
    • 3
  • Ashley Cimino-Mathews
    • 4
  • Pedram Argani
    • 4
  • Celina G. Kleer
    • 1
    • 2
    • 3
    • 5
  1. 1.Department of PathologyUniversity of MichiganAnn ArborUSA
  2. 2.Cellular and Molecular Biology ProgramUniversity of MichiganAnn ArborUSA
  3. 3.Comprehensive Cancer CenterUniversity of MichiganAnn ArborUSA
  4. 4.Department of PathologyThe Johns Hopkins HospitalBaltimoreUSA
  5. 5.Department of PathologyUniversity of Michigan Medical SchoolAnn ArborUSA

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