Histone deacetylases, Mbd3/NuRD, and Tet2 hydroxylase are crucial regulators of epithelial–mesenchymal plasticity and tumor metastasis

Abstract

An epithelial–mesenchymal transition (EMT) represents a basic morphogenetic process of high cell plasticity underlying embryogenesis, wound healing, cancer metastasis and drug resistance. It involves a profound transcriptional and epigenetic reprogramming of cells. A critical role of epigenetic modifiers and their specific chromatin modifications has been demonstrated during EMT. However, it has remained elusive whether epigenetic control differs between the dynamic cell state transitions of reversible EMT and the fixed differentiation status of irreversible EMT. We have employed varying EMT models of murine breast cancer cells to identify the key players establishing epithelial–mesenchymal cell plasticity during reversible and irreversible EMT. We demonstrate that the Mbd3/NuRD complex and the activities of histone deacetylases (HDACs), and Tet2 hydroxylase play a critical role in keeping cancer cells in a highly metastatic mesenchymal state. Combinatorial interference with their functions leads to mesenchymal–epithelial transition (MET) and efficiently represses metastasis formation by invasive murine and human breast cancer cells in vivo.

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Data availability

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Gerhard Christofori (gerhard.christofori@unibas.ch). The RNA sequencing data are deposited at Gene Expression Omnibus (GEO accession number: GSE100553).

References

  1. 1.

    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.

  2. 2.

    Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer. 2002;2:442–54.

  3. 3.

    Yang J, Weinberg RA. Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell 2008;14:818–29.

  4. 4.

    Nieto MA. The ins and outs of the epithelial to mesenchymal transition in health and disease. Annu Rev Cell Dev Biol. 2011;27:347–76.

  5. 5.

    Diepenbruck M, Christofori G. Epithelial-mesenchymal transition (EMT) and metastasis: yes, no, maybe? Curr Opin Cell Biol. 2016;43:7–13.

  6. 6.

    Fantozzi A, Gruber DC, Pisarsky L, Heck C, Kunita A, Yilmaz M, et al. VEGF-mediated angiogenesis links EMT-induced cancer stemness to tumor initiation. Cancer Res. 2014;74:1566–75.

  7. 7.

    McDonald OG, Wu H, Timp W, Doi A, Feinberg AP. Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition. Nat Struct Mol Biol. 2011;18:867–74.

  8. 8.

    Tam WL, Weinberg RA. The epigenetics of epithelial-mesenchymal plasticity in cancer. Nat Med. 2013;19:1438–49.

  9. 9.

    Glozak MA, Seto E. Histone deacetylases and cancer. Oncogene. 2007;26:5420–32.

  10. 10.

    Tate CR, Rhodes LV, Segar HC, Driver JL, Pounder FN, Burow ME, et al. Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat. Breast Cancer Res. 2012;14:R79.

  11. 11.

    Srivastava RK, Kurzrock R, Shankar S. MS-275 sensitizes TRAIL-resistant breast cancer cells, inhibits angiogenesis and metastasis, and reverses epithelial-mesenchymal transition in vivo. Mol Cancer Ther. 2010;9:3254–66.

  12. 12.

    Tang HM, Kuay KT, Koh PF, Asad M, Tan TZ, Chung VY, et al. An epithelial marker promoter induction screen identifies histone deacetylase inhibitors to restore epithelial differentiation and abolishes anchorage independence growth in cancers. Cell Death Discov. 2016;2:16041.

  13. 13.

    Grozinger CM, Schreiber SL. Deacetylase enzymes: biological functions and the use of small-molecule inhibitors. Chem Biol. 2002;9:3–16.

  14. 14.

    Denslow SA, Wade PA. The human Mi-2/NuRD complex and gene regulation. Oncogene. 2007;26:5433–8.

  15. 15.

    Hayakawa T, Nakayama J. Physiological roles of class I HDAC complex and histone demethylase. J Biomed Biotechnol. 2011;2011:129383.

  16. 16.

    Yang Y, Huang W, Qiu R, Liu R, Zeng Y, Gao J, et al. LSD1 coordinates with the SIN3A/HDAC complex and maintains sensitivity to chemotherapy in breast cancer. J Mol Cell Biol. 2018;10:285–301.

  17. 17.

    Wang Y, Zhang H, Chen Y, Sun Y, Yang F, Yu W, et al. LSD1 is a subunit of the NuRD complex and targets the metastasis programs in breast. Cancer Cell. 2009;138:660–72.

  18. 18.

    Peinado H, Ballestar E, Esteller M, Cano A. Snail mediates E-cadherin repression by the recruitment of the Sin3A/Histone deacetylase 1 (HDAC1)/HDAC2 complex. Mol Cell Biol. 2004;24:306.

  19. 19.

    Fujita N, Jaye DL, Kajita M, Geigerman C, Moreno CS, Wade PA. MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast. Cancer Cell. 2003;113:207–19.

  20. 20.

    Fu J, Qin L, He T, Qin J, Hong J, Wong J, et al. The TWIST/Mi2/NuRD protein complex and its essential role in cancer metastasis. Cell Res. 2011;21:275–89.

  21. 21.

    Verstappen G, van Grunsven LA, Michiels C, Van de Putte T, Souopgui J, Van Damme J, et al. A typical Mowat–Wilson patient confirms the importance of the novel association between ZFHX1B/SIP1 and NuRD corepressor complex. Hum Mol Genet. 2008;17:1175–83.

  22. 22.

    Lai AY, Wade PA. Cancer biology and NuRD: a multifaceted chromatin remodelling complex. Nat Rev Cancer. 2011;11:588–96.

  23. 23.

    Kaji K, Caballero IM, MacLeod R, Nichols J, Wilson VA, Hendrich B. The NuRD component Mbd3 is required for pluripotency of embryonic stem cells. Nat Cell Biol. 2006;8:285–92.

  24. 24.

    Kaji K, Nichols J, Hendrich B. Mbd3, a component of the NuRD co-repressor complex, is required for development of pluripotent cells. Development. 2007;134:1123–32.

  25. 25.

    Rais Y, Zviran A, Geula S, Gafni O, Chomsky E, Viukov S, et al. Deterministic direct reprogramming of somatic cells to pluripotency. Nature. 2013;502:65–70.

  26. 26.

    Saito M, Ishikawa F. The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2. J Biol Chem. 2002;277:35434–9.

  27. 27.

    Fraga MF, Ballestar E, Montoya G, Taysavang P, Wade PA, Esteller M. The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties. Nucleic Acids Res. 2003;31:1765–74.

  28. 28.

    Yildirim O, Li R, Hung J-H, Chen Poshen B, Dong X, Ee L-S, et al. Mbd3/NURD complex regulates expression of 5-hydroxymethylcytosine marked genes in embryonic stem cells. Cell. 2011;147:1498–510.

  29. 29.

    Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009;324:930–5.

  30. 30.

    Cimmino L, Abdel-Wahab O, Levine Ross L, Aifantis I. TET family proteins and their role in stem cell differentiation and transformation. Cell Stem Cell. 2011;9:193–204.

  31. 31.

    Baubec T, Ivánek R, Lienert F, Schübeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family. Cell. 2013;153:480–92.

  32. 32.

    Hu X, Zhang L, Mao S-Q, Li Z, Chen J, Zhang R-R, et al. Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming. Cell Stem Cell. 2014;14:512–22.

  33. 33.

    Waldmeier L, Meyer-Schaller N, Diepenbruck M, Christofori G. Py2T murine breast cancer cells, a versatile model of TGFβ-induced EMT in vitro and in vivo. PLoS ONE. 2012;7:e48651.

  34. 34.

    Dumont N, Wilson MB, Crawford YG, Reynolds PA, Sigaroudinia M, Tlsty TD. Sustained induction of epithelial to mesenchymal transition activates DNA methylation of genes silenced in basal-like breast cancers. Proc Natl Acad Sci. 2008;105:14867–72.

  35. 35.

    Crea F, Paolicchi E, Marquez VE, Danesi R. Polycomb genes and cancer: time for clinical application? Crit Rev Oncol/Hematol. 2012;83:184–93.

  36. 36.

    Tiwari N, Tiwari Vijay K, Waldmeier L, Balwierz Piotr J, Arnold P, Pachkov M, et al. Sox4 is a master regulator of epithelial-mesenchymal transition by controlling Ezh2 expression and epigenetic reprogramming. Cancer Cell. 2013;23:768–83.

  37. 37.

    Zhang N, Liu Y, Wang Y, Zhao M, Tu L, Luo F. Decitabine reverses TGF-β1-induced epithelial–mesenchymal transition in non-small-cell lung cancer by regulating miR-200/ZEB axis. Drug Des, Dev Ther. 2017;11:969–83.

  38. 38.

    Yoshida M, Kijima M, Akita M, Beppu T. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J Biol Chem. 1990;265:17174–9.

  39. 39.

    Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus CR, et al. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol. 2012;8:890–6.

  40. 40.

    Meidhof S, Brabletz S, Lehmann W, Preca BT, Mock K, Ruh M, et al. ZEB1‐associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat. EMBO Mol Med. 2015;7:831–47.

  41. 41.

    Beckers T, Burkhardt C, Wieland H, Gimmnich P, Ciossek T, Maier T, et al. Distinct pharmacological properties of second generation HDAC inhibitors with the benzamide or hydroxamate head group. Int J Cancer. 2007;121:1138–48.

  42. 42.

    Li R, Liang J, Ni S, Zhou T, Qing X, Li H, et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell. 2010;7:51–63.

  43. 43.

    Batlle E, Sancho E, Francí C, Domínguez D, Monfar M, Baulida J, et al. The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol. 2000;2:84–9.

  44. 44.

    Cano A, Pérez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, et al. The transcription factor Snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol. 2000;2:76–83.

  45. 45.

    Eger A, Aigner K, Sonderegger S, Dampier B, Oehler S, Schreiber M, et al. DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells. Oncogene. 2005;24:2375–85.

  46. 46.

    Aigner K, Dampier B, Descovich L, Mikula M, Sultan A, Schreiber M, et al. The transcription factor ZEB1 (deltaEF1) promotes tumour cell dedifferentiation by repressing master regulators of epithelial polarity. Oncogene. 2007;26:6979–88.

  47. 47.

    Shirakihara T, Saitoh M, Miyazono K. Differential regulation of epithelial and mesenchymal markers by deltaEF1 proteins in epithelial mesenchymal transition induced by TGF-beta. Mol Biol Cell. 2007;18:3533–44.

  48. 48.

    Comijn J, Berx G, Vermassen P, Verschueren K, van Grunsven L, Bruyneel E, et al. The two-handed e box binding zinc finger protein SIP1 downregulates e-cadherin and induces invasion. Mol Cell. 2001;7:1267–78.

  49. 49.

    Vesuna F, van Diest P, Chen JH, Raman V. Twist is a transcriptional repressor of E-cadherin gene expression in breast cancer. Biochem Biophys Res Commun. 2008;367:235–41.

  50. 50.

    Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell. 2004;117:927–39.

  51. 51.

    Peng L, Li Y, Xi Y, Li W, Li J, Lv R, et al. MBD3L2 promotes Tet2 enzymatic activity for mediating 5-methylcytosine oxidation. J Cell Sci. 2016;129:1059–71.

  52. 52.

    Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, et al. Genes that mediate breast cancer metastasis to lung. Nature. 2005;436:518.

  53. 53.

    Kong D, Ahmad A, Bao B, Li Y, Banerjee S, Sarkar FH. Histone deacetylase inhibitors induce epithelial-to-mesenchymal transition in prostate cancer cells. PLoS ONE. 2012;7:e45045.

  54. 54.

    Jiang G-M, Wang H-S, Zhang F, Zhang K-S, Liu Z-C, Fang R, et al. Histone deacetylase inhibitor induction of epithelial–mesenchymal transitions via up-regulation of Snail facilitates cancer progression. Biochim Biophys Acta (BBA)—Mol Cell Res. 2013;1833:663–71.

  55. 55.

    George JT, Jolly MK, Xu S, Somarelli JA, Levine H. Survival outcomes in cancer patients predicted by a partial EMT gene expression scoring metric. Cancer Res. 2017;77:6415–28.

  56. 56.

    Jolly MK, Boareto M, Huang B, Jia D, Lu M, Ben-Jacob E, et al. Implications of the hybrid epithelial/mesenchymal phenotype in metastasis. Front Oncol. 2015;5:155.

  57. 57.

    Li W, Kang Y. Probing the fifty shades of EMT in metastasis. Trends Cancer. 2016;2:65–7.

  58. 58.

    Aiello NM, Maddipati R, Norgard RJ, Balli D, Li J, Yuan S, et al. EMT subtype influences epithelial plasticity and mode of cell migration. Dev Cell. 2018;45:681–95.e4.

  59. 59.

    Pastushenko I, Brisebarre A, Sifrim A, Fioramonti M, Revenco T, Boumahdi S, et al. Identification of the tumour transition states occurring during EMT. Nature. 2018;556:463–8.

  60. 60.

    Jolly MK, Tripathi SC, Jia D, Mooney SM, Celiktas M, Hanash SM, et al. Stability of the hybrid epithelial/mesenchymal phenotype. Oncotarget. 2016;7:27067–84.

  61. 61.

    Scourzic L, Mouly E, Bernard OA. TET proteins and the control of cytosine demethylation in cancer. Genome Med. 2015;7:9.

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Acknowledgements

We thank P. Schär and S. Weis (DBM Basel) for reagents and protocols, F. Noreen (DBM Basel) for pyrosequencing, C. Beisel (D-BSSE, ETH Zürich) for next generation RNA sequencing, P. Lorentz (DBM Basel) for excellent microscopy support, and Isabel Galm for technical support.

Funding

This work was supported by the SystemsX.ch RTD project Cellplasticity, the SystemsX.ch MTD project MetastasiX, the Swiss National Science Foundation, and the Swiss Cancer League. MKJ was also supported by a training fellowship from the Gulf Coast Consortia on the Computational Cancer Biology Training Program (CPRIT Grant No. RP170593).

Author information

ANK designed and performed the experiments, analyzed the data and wrote the paper; NS performed experiments for revision, RKRK performed bioinformatics analysis; HA performed animal experiments; HB analyzed animal experiments, DI-R performed Py2T MET Time-course, JTG, HL and MKJ performed EMT score analysis; and GC oversaw the project, designed experiments, analyzed the data and wrote the paper.

Correspondence to Gerhard Christofori.

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Nihan Kilinc, A., Sugiyama, N., Reddy Kalathur, R.K. et al. Histone deacetylases, Mbd3/NuRD, and Tet2 hydroxylase are crucial regulators of epithelial–mesenchymal plasticity and tumor metastasis. Oncogene 39, 1498–1513 (2020). https://doi.org/10.1038/s41388-019-1081-2

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