Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

EZH2 endorses cell plasticity to non-small cell lung cancer cells facilitating mesenchymal to epithelial transition and tumour colonization

Abstract

Reversible transition between the epithelial and mesenchymal states are key aspects of carcinoma cell dissemination and the metastatic disease, and thus, characterizing the molecular basis of the epithelial to mesenchymal transition (EMT) is crucial to find druggable targets and more effective therapeutic approaches in cancer. Emerging studies suggest that epigenetic regulators might endorse cancer cells with the cell plasticity required to conduct dynamic changes in cell state during EMT. However, epigenetic mechanisms involved remain mostly unknown. Polycomb Repressive Complexes (PRCs) proteins are well-established epigenetic regulators of development and stem cell differentiation, but their role in different cancer systems is inconsistent and sometimes paradoxical. In this study, we have analysed the role of the PRC2 protein EZH2 in lung carcinoma cells. We found that besides its described role in CDKN2A-dependent cell proliferation, EZH2 upholds the epithelial state of cancer cells by repressing the transcription of hundreds of mesenchymal genes. Chemical inhibition or genetic removal of EZH2 promotes the residence of cancer cells in the mesenchymal state during reversible epithelial–mesenchymal transition. In fitting, analysis of human patient samples and tumour xenograft models indicate that EZH2 is required to efficiently repress mesenchymal genes and facilitate tumour colonization in vivo. Overall, this study discloses a novel role of PRC2 as a master regulator of EMT in carcinoma cells. This finding has important implications for the design of therapies based on EZH2 inhibitors in human cancer patients.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: EZH2 binds and represses mesenchymal genes in lung carcinoma cells.
Fig. 2: TGF-β-induced EMT is reversible in A549 cells.
Fig. 3: Binding of EZH2 to target promoters remains constant during EMT in A549 cells.
Fig. 4: EZH2 represses a large set of mesenchymal genes during EMT–MET in A549 cells.
Fig. 5: EZH2-null A549 cells acquire mesenchymal features but remain responsive to TGF-β stimulation.
Fig. 6: EZH2-null A549 cells display reduced tumor colonization capacity in xenograft assays.

Data availability

Datasets are available at GEO-NCBI with accession number GSE180067.

References

  1. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Investig. 2009;119:1420–8. https://doi.org/10.1172/jci39104

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871–90. https://doi.org/10.1016/j.cell.2009.11.007

    CAS  Article  PubMed  Google Scholar 

  3. Dongre A, Weinberg RA. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol cell Biol. 2019;20:69–84. https://doi.org/10.1038/s41580-018-0080-4

    CAS  Article  PubMed  Google Scholar 

  4. Tam WL, Weinberg RA. The epigenetics of epithelial-mesenchymal plasticity in cancer. Nat Med. 2013;19:1438–49. https://doi.org/10.1038/nm.3336

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol cell Biol. 2014;15:178–96. https://doi.org/10.1038/nrm3758

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Derynck R, Muthusamy BP, Saeteurn KY. Signaling pathway cooperation in TGF-β-induced epithelial-mesenchymal transition. Curr Opin cell Biol. 2014;31:56–66. https://doi.org/10.1016/j.ceb.2014.09.001

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Lambert AW, Pattabiraman DR, Weinberg RA. Emerging biological principles of metastasis. Cell. 2017;168:670–91. https://doi.org/10.1016/j.cell.2016.11.037

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Shibue T, Weinberg RA. EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol. 2017;14:611–29. https://doi.org/10.1038/nrclinonc.2017.44

    Article  PubMed  PubMed Central  Google Scholar 

  9. Schuettengruber B, Bourbon HM, Di Croce L, Cavalli G. Genome regulation by Polycomb and Trithorax: 70 years and counting. Cell. 2017;171:34–57. https://doi.org/10.1016/j.cell.2017.08.002

    CAS  Article  PubMed  Google Scholar 

  10. Piunti A & Shilatifard A. The roles of Polycomb repressive complexes in mammalian development and cancer. Nat rev Mol cell biol. 2021. https://doi.org/10.1038/s41580-021-00341-1

  11. Comet I, Riising EM, Leblanc B, Helin K. Maintaining cell identity: PRC2-mediated regulation of transcription and cancer. Nat Rev Cancer. 2016;16:803–10. https://doi.org/10.1038/nrc.2016.83.

    CAS  Article  PubMed  Google Scholar 

  12. Jacobs JJ, Kieboom K, Marino S, DePinho RA, van Lohuizen M. The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature. 1999;397:164–8. https://doi.org/10.1038/16476

    CAS  Article  PubMed  Google Scholar 

  13. Bracken AP, Kleine-Kohlbrecher D, Dietrich N, Pasini D, Gargiulo G, Beekman C, et al. The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes Dev. 2007;21:525–30. https://doi.org/10.1101/gad.415507

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Koppens M & van Lohuizen M. Context-dependent actions of Polycomb repressors in cancer. Oncogene. 2015. https://doi.org/10.1038/onc.2015.195

  15. Schwartzentruber J, Korshunov A, Liu XY, Jones DTW, Pfaff E, Jacob K, et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature. 2012;482:226–31. https://doi.org/10.1038/nature10833

    CAS  Article  PubMed  Google Scholar 

  16. De Raedt T, Beert E, Pasmant E, Luscan A, Brems H, Ortonne N, et al. PRC2 loss amplifies Ras-driven transcription and confers sensitivity to BRD4-based therapies. Nature. 2014;514:247–51. https://doi.org/10.1038/nature13561

    CAS  Article  PubMed  Google Scholar 

  17. Zhang M, Wang Y, Jones S, Sausen M, McMahon K, Sharma R, et al. Somatic mutations of SUZ12 in malignant peripheral nerve sheath tumors. Nat Genet. 2014;46:1170–2. https://doi.org/10.1038/ng.3116

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Serresi M, Gargiulo G, Proost N, Siteur B, Cesaroni M, Koppens M, et al. Polycomb repressive complex 2 is a barrier to KRAS-driven inflammation and epithelial-mesenchymal transition in non-small-cell lung cancer. Cancer cell. 2016;29:17–31. https://doi.org/10.1016/j.ccell.2015.12.006

    CAS  Article  PubMed  Google Scholar 

  19. Kim KH, Roberts CW. Targeting EZH2 in cancer. Nat Med. 2016;22:128–34. https://doi.org/10.1038/nm.4036

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Flavahan WA, Gaskell E & Bernstein BE. Epigenetic plasticity and the hallmarks of cancer. Science. 2017;357. https://doi.org/10.1126/science.aal2380

  21. Cao Q, Yu J, Dhanasekaran SM, Kim JH, Mani RS, Tomlins SA, et al. Repression of E-cadherin by the polycomb group protein EZH2 in cancer. Oncogene. 2008;27:7274–84. https://doi.org/10.1038/onc.2008.333

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Herranz N, Pasini D, Díaz VM, Francí C, Gutierrez A, Dave N, et al. Polycomb complex 2 is required for E-cadherin repression by the Snail1 transcription factor. Mol Cell Biol. 2008;28:4772–81. https://doi.org/10.1128/MCB.00323-08

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Azuara V, Perry P, Sauer S, Spivakov M, Jørgensen HF, John RM, et al. Chromatin signatures of pluripotent cell lines. Nat cell Biol. 2006;8:532–8. https://doi.org/10.1038/ncb1403

    CAS  Article  PubMed  Google Scholar 

  24. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell. 2006;125:315–26. https://doi.org/10.1016/j.cell.2006.02.041

    CAS  Article  PubMed  Google Scholar 

  25. McCabe MT, Ott HM, Ganji G, Korenchuk S, Thompson C, Van Aller GS, et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature. 2012;492:108–12. https://doi.org/10.1038/nature11606

    CAS  Article  PubMed  Google Scholar 

  26. Knutson SK, Warholic NM, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, et al. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proc Natl Acad Sci USA. 2013;110:7922–7. https://doi.org/10.1073/pnas.1303800110

    Article  PubMed  PubMed Central  Google Scholar 

  27. Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development. 2005;132:3151–61. https://doi.org/10.1242/dev.01907

    CAS  Article  PubMed  Google Scholar 

  28. Takawa M, Masuda K, Kunizaki M, Daigo Y, Takagi K, Iwai Y, et al. Validation of the histone methyltransferase EZH2 as a therapeutic target for various types of human cancer and as a prognostic marker. Cancer Sci. 2011;102:1298–305. https://doi.org/10.1111/j.1349-7006.2011.01958.x

    CAS  Article  PubMed  Google Scholar 

  29. Hardavella G, George R, Sethi T. Lung cancer stem cells-characteristics, phenotype. Transl Lung Cancer Res. 2016;5:272–9. https://doi.org/10.21037/tlcr.2016.02.01

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Serresi M, Siteur B, Hulsman D, Company C, Schmitt MJ, Lieftink C, et al. Ezh2 inhibition in Kras-driven lung cancer amplifies inflammation and associated vulnerabilities. J Exp Med. 2018;215:3115–35. https://doi.org/10.1084/jem.20180801

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Iliopoulos D, Lindahl-Allen M, Polytarchou C, Hirsch HA, Tsichlis PN, Struhl K. Loss of miR-200 inhibition of Suz12 leads to polycomb-mediated repression required for the formation and maintenance of cancer stem cells. Mol cell. 2010;39:761–72. https://doi.org/10.1016/j.molcel.2010.08.013

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Tiwari N, Tiwari VK, Waldmeier L, Balwierz PJ, 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. https://doi.org/10.1016/j.ccr.2013.04.020

    CAS  Article  PubMed  Google Scholar 

  33. Fillmore CM, Xu C, Desai PT, Berry JM, Rowbotham SP, Lin YJ, et al. EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors. Nature. 2015;520:239–42. https://doi.org/10.1038/nature14122

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer. 2011;11:761–74. https://doi.org/10.1038/nrc3106

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Pattabiraman DR, Bierie B, Kober KI, Thiru P, Krall JA, Zill C, et al. Activation of PKA leads to mesenchymal-to-epithelial transition and loss of tumor-initiating ability. Science. 2016;351:aad3680 https://doi.org/10.1126/science.aad3680

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Mohn F, Weber M, Rebhan M, Roloff TC, Richter J, Stadler MB, et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol cell. 2008;30:755–66. https://doi.org/10.1016/j.molcel.2008.05.007

    CAS  Article  PubMed  Google Scholar 

  37. Marasca F, Bodega B, Orlando V. How Polycomb-mediated cell memory deals with a changing environment: variations in PcG complexes and proteins assortment convey plasticity to epigenetic regulation as a response to environment. BioEssays: N. Rev Mol, Cell developmental Biol. 2018;40:e1700137 https://doi.org/10.1002/bies.201700137

    Article  Google Scholar 

  38. Horibata S, Vo TV, Subramanian V, Thompson PR & Coonrod SA. Utilization of the soft agar colony formation assay to identify inhibitors of tumorigenicity in breast cancer cells. J of vis exp: JoVE. 2015:e52727. https://doi.org/10.3791/52727.

  39. Hernández-Camarero P, López-Ruiz E, Griñán-Lisón C, García MÁ, Chocarro-Wrona C, Marchal JA, et al. Pancreatic (pro)enzymes treatment suppresses BXPC-3 pancreatic Cancer Stem Cell subpopulation and impairs tumour engrafting. Sci Rep. 2019;9:11359 https://doi.org/10.1038/s41598-019-47837-7

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Gonzalez-Gonzalez A, Munoz-Muela E, Marchal JA, Cara FE, Molina MP, Cruz-Lozano M, et al. Activating transcription factor 4 modulates TGFbeta-induced aggressiveness in triple-negative breast cancer via SMAD2/3/4 and mTORC2 signaling. Clin Cancer Res. 2018;24:5697–709. https://doi.org/10.1158/1078-0432.ccr-17-3125

    CAS  Article  PubMed  Google Scholar 

  41. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8:2281–308. https://doi.org/10.1038/nprot.2013.143

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Landeira D, Bagci H, Malinowski AR, Brown KE, Soza-Ried J, Feytout A, et al. Jarid2 coordinates Nanog Expression and PCP/Wnt signaling required for efficient ESC differentiation and early embryo development. Cell Rep. 2015;12:573–86. https://doi.org/10.1016/j.celrep.2015.06.060

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Gallardo A, Molina A, Asenjo HG, Martorell-Marugán J, Montes R, Ramos-Mejia V et al. The molecular clock protein Bmal1 regulates cell differentiation in mouse embryonic stem cells. Life Sci Alliance. 2020;3. https://doi.org/10.26508/lsa.201900535.

  44. Asenjo HG, Gallardo A, López-Onieva L, Tejada I, Martorell-Marugán J, Carmona-Sáez P et al. Polycomb regulation is coupled to cell cycle transition in pluripotent stem cells. Sci adv. 2020;6. https://doi.org/10.1126/sciadv.aay4768.

  45. Fursova NA, Blackledge NP, Nakayama M, Ito S, Koseki Y, Farcas AM et al. Synergy between variant PRC1 complexes defines polycomb-mediated gene repression. Mol cell. 2019. https://doi.org/10.1016/j.molcel.2019.03.024.

  46. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat methods. 2012;9:357–9. https://doi.org/10.1038/nmeth.1923

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–9. https://doi.org/10.1093/bioinformatics/btp352

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. Ramirez F, Ryan DP, Gruning B, Bhardwaj V, Kilpert F, Richter AS, et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic acids Res. 2016;44:W160–W165. https://doi.org/10.1093/nar/gkw257

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008;9:R137 https://doi.org/10.1186/gb-2008-9-9-r137

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol cell. 2010;38:576–89. https://doi.org/10.1016/j.molcel.2010.05.004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Lawrence M, Huber W, Pages H, Aboyoun P, Carlson M, Gentleman R, et al. Software for computing and annotating genomic ranges. PLoS computational Biol. 2013;9:e1003118 https://doi.org/10.1371/journal.pcbi.1003118

    CAS  Article  Google Scholar 

  52. Dobin A, Gingeras TR. Mapping RNA-seq Reads with STAR. Curr Protoc Bioinforma. 2015;51:11.14.11–11.14.19. https://doi.org/10.1002/0471250953.bi1114s51

    Article  Google Scholar 

  53. Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010;11:R25 https://doi.org/10.1186/gb-2010-11-3-r25

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Tarazona S, Furió-Tarí P, Turrà D, Pietro AD, Nueda MJ, Ferrer A, et al. Data quality aware analysis of differential expression in RNA-seq with NOISeq R/Bioc package. Nucleic acids Res. 2015;43:e140–e140. https://doi.org/10.1093/nar/gkv711

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550 https://doi.org/10.1186/s13059-014-0550-8

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. McCarthy DJ, Chen Y, Smyth GK. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic acids Res. 2012;40:4288–97. https://doi.org/10.1093/nar/gks042

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. Győrffy B, Surowiak P, Budczies J, Lánczky A. Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer. PloS one. 2013;8:e82241 https://doi.org/10.1371/journal.pone.0082241

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are very grateful to Diego de Miguel Perez and Dra. Maria Jose Serrano for providing cell lines and technical advice. We thank Dra. Aurora Serrano, Natasa Vukovic, core facilities at GENYO and the Animal Experimentation Unit at University of Granada for excellent technical support. We also thank the genomics unit at the CRG for assistance with RNA-seq and ChIP-seq experiments.

Funding

CGL was funded by the Consejería de Salud y Familias, Junta de Andalucía (RH-0139-2020) and SG-P is funded by Instituto de Salud Carlos III (CP19/00029, PI15/00336, PI19/01533). JAM is supported by RTI2018.101309B-C22 funded by MCIN/AEI/10.13039/501100011033/FEDER “Una manera de hacer Europa” and by the Chair “Doctors Galera-Requena in cancer stem cell research”. PCS is funded by Ministerio de Ciencia e Innovación (grant PID2020-119032RB-I00) and FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades (grants P20_00335 and B‐CTS‐40‐UGR20). The Landeira lab is supported by the Spanish ministry of science and innovation (PID2019-108108-100, EUR2021-122005), the Andalusian regional government (PC-0246-2017, PIER-0211-2019, PY20_00681) and the University of Granada (A-BIO-6-UGR20) grants.

Author information

Authors and Affiliations

Authors

Contributions

DL designed and conceptualized the study. DL and AG designed experiments. AG, AM, HGA, LLO, FEC and MEM performed and analysed experiments. CGL, JCAV and SANM performed xenograft assays. JMM performed bioinformatic analyses. PCS, SGP, LLO, JAM, PPM, ASP and DL provided scientific advice and resources. DL, LLO and ASP obtained funding and supervised research.

Corresponding author

Correspondence to David Landeira.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gallardo, A., Molina, A., Asenjo, H.G. et al. EZH2 endorses cell plasticity to non-small cell lung cancer cells facilitating mesenchymal to epithelial transition and tumour colonization. Oncogene (2022). https://doi.org/10.1038/s41388-022-02375-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41388-022-02375-x

Search

Quick links