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.

Integrative multi-omics analysis of a colon cancer cell line with heterogeneous Wnt activity revealed RUNX2 as an epigenetic regulator of EMT

Abstract

Epithelial–mesenchymal transition (EMT) program, which facilitates tumor metastasis, stemness and therapy resistance, is a reversible biological process that is largely orchestrated at the epigenetic level under the regulation of different cell signaling pathways. EMT state is often heterogeneous within individual tumors, though the epigenetic drivers underlying such heterogeneity remain elusive. In colon cancer, hyperactivation of the Wnt/β-catenin signaling not only drives tumor initiation, but also promotes metastasis in late stage by promoting EMT program. However, it is unknown whether the intratumorally heterogeneous Wnt activity could directly drive EMT heterogeneity, and, if so, what are the underlying epigenetic driver(s). Here, by analyzing a phenotypically and molecularly heterogeneous colon cancer cell line using single-cell RNA sequencing, we identified two distinct cell populations with positively correlated Wnt activity and EMT state. Integrative multi-omics analysis of these two cell populations revealed RUNX2 as a critical transcription factor epigenetically driving the EMT heterogeneity. Both in vitro and in vivo genetic perturbation assays validated the EMT-enhancing effect of RUNX2, which remodeled chromatin landscape and activated a panel of EMT-associated genes through binding to their promoters and/or potential enhancers. Finally, by exploring the clinical data, we showed that RUNX2 expression is positively correlated with metastasis development and poor survival of colon cancer patients, as well as patients afflicted with other types of cancer. Taken together, our work revealed RUNX2 as a new EMT-promoting epigenetic regulator in colon cancer, which may potentially serve as a prognostic marker for tumor metastasis.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Identification and characterization of two distinct cell populations within SW480 cell line.
Fig. 2: Transcriptomic analysis of Wnt-low and Wnt-high cells.
Fig. 3: Analysis of chromatin accessibility revealed RUNX2 as a master TF in Wnt-high cells.
Fig. 4: RUNX2 promotes epithelial–mesenchymal transition in vitro.
Fig. 5: RUNX2 promotes metastasis formation of colon cancer cells in vivo.
Fig. 6: RUNX2 modulates chromatin landscape and regulates the expression of EMT-related genes.
Fig. 7: High expression of RUNX2 is associated with metastasis development and poor survival of cancer patients.

References

  1. Arnold M, Sierra MS, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global patterns and trends in colorectal cancer incidence and mortality. Gut. 2017;66:683–91.

    PubMed  Article  Google Scholar 

  2. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424.

    PubMed  Article  Google Scholar 

  3. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;67:7–30.

    Article  PubMed  Google Scholar 

  4. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871–90.

    CAS  PubMed  Article  Google Scholar 

  5. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Investig. 2009;119:1420–8.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 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.

    CAS  PubMed  Article  Google Scholar 

  7. Guo W, Keckesova Z, Donaher JL, Shibue T, Tischler V, Reinhardt F, et al. Slug and Sox9 cooperatively determine the mammary stem cell state. Cell. 2012;148:1015–28.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Krebs AM, Mitschke J, Lasierra Losada M, Schmalhofer O, Boerries M, Busch H, et al. The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer. Nat Cell Biol. 2017;19:518–29.

    CAS  PubMed  Article  Google Scholar 

  9. Rhim AD, Mirek ET, Aiello NM, Maitra A, Bailey JM, McAllister F, et al. EMT and dissemination precede pancreatic tumor formation. Cell. 2012;148:349–61.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133:704–15.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Nieto MA, Huang RY, Jackson RA, Thiery JP. Emt: 2016. Cell. 2016;166:21–45.

    CAS  PubMed  Article  Google Scholar 

  12. Puisieux A, Brabletz T, Caramel J. Oncogenic roles of EMT-inducing transcription factors. Nat Cell Biol. 2014;16:488–94.

    CAS  PubMed  Article  Google Scholar 

  13. Singh A, Settleman J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene. 2010;29:4741–51.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Ye X, Tam WL, Shibue T, Kaygusuz Y, Reinhardt F, Ng Eaton E, et al. Distinct EMT programs control normal mammary stem cells and tumour-initiating cells. Nature. 2015;525:256–60.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Nieto MA. Epithelial-Mesenchymal Transitions in development and disease: old views and new perspectives. Int J Dev Biol. 2009;53:1541–7.

    PubMed  Article  Google Scholar 

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. McGranahan N, Swanton C. Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell. 2015;27:15–26.

    CAS  PubMed  Article  Google Scholar 

  18. Bian S, Hou Y, Zhou X, Li X, Yong J, Wang Y, et al. Single-cell multiomics sequencing and analyses of human colorectal cancer. Science. 2018;362:1060–3.

    CAS  PubMed  Article  Google Scholar 

  19. Li H, Courtois ET, Sengupta D, Tan Y, Chen KH, Goh JJL, et al. Reference component analysis of single-cell transcriptomes elucidates cellular heterogeneity in human colorectal tumors. Nat Genet. 2017;49:708–18.

    CAS  Article  PubMed  Google Scholar 

  20. Ligorio M, Sil S, Malagon-Lopez J, Nieman LT, Misale S, Di Pilato M, et al. Stromal microenvironment shapes the intratumoral architecture of pancreatic cancer. Cell. 2019;178:160–75.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 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.

    CAS  PubMed  Article  Google Scholar 

  22. Puram SV, Tirosh I, Parikh AS, Patel AP, Yizhak K, Gillespie S, et al. Single-cell transcriptomic analysis of primary and metastatic tumor ecosystems in head and neck Cancer. Cell. 2017;171:1611–24. e1624

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36:1461–73.

    CAS  PubMed  Article  Google Scholar 

  24. Sanchez-Tillo E, de Barrios O, Siles L, Cuatrecasas M, Castells A, Postigo A. beta-catenin/TCF4 complex induces the epithelial-to-mesenchymal transition (EMT)-activator ZEB1 to regulate tumor invasiveness. Proc Natl Acad Sci USA. 2011;108:19204–9.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. Yook JI, Li XY, Ota I, Hu C, Kim HS, Kim NH, et al. A Wnt-Axin2-GSK3beta cascade regulates Snail1 activity in breast cancer cells. Nat Cell Biol. 2006;8:1398–406.

    CAS  PubMed  Article  Google Scholar 

  26. Vermeulen L, De Sousa EMF, van der Heijden M, Cameron K, de Jong JH, Borovski T, et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat Cell Biol. 2010;12:468–76.

    CAS  PubMed  Article  Google Scholar 

  27. Guinney J, Dienstmann R, Wang X, de Reynies A, Schlicker A, Soneson C, et al. The consensus molecular subtypes of colorectal cancer. Nat Med. 2015;21:1350–6.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Sveen A, Bruun J, Eide PW, Eilertsen IA, Ramirez L, Murumagi A, et al. Colorectal cancer consensus molecular subtypes translated to preclinical models uncover potentially targetable cancer cell dependencies. Clin Cancer Res. 2018;24:794–806.

    CAS  PubMed  Article  Google Scholar 

  29. Fang L, Zhu Q, Neuenschwander M, Specker E, Wulf-Goldenberg A, Weis WI, et al. A small-molecule antagonist of the beta-Catenin/TCF4 interaction blocks the self-renewal of cancer stem cells and suppresses tumorigenesis. Cancer Res. 2016;76:891–901.

    CAS  PubMed  Article  Google Scholar 

  30. Buenrostro JD, Wu B, Chang HY, Greenleaf WJ. ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr Protoc Mol Biol. 2015;109:21–29.

    PubMed  PubMed Central  Article  Google Scholar 

  31. Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature. 2003;423:349–55.

    CAS  PubMed  Article  Google Scholar 

  32. Rahman MS, Akhtar N, Jamil HM, Banik RS, Asaduzzaman SM. TGF-beta/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation. Bone Res. 2015;3:15005.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Wu M, Chen G, Li YP. TGF-beta and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res. 2016;4:16009.

    PubMed  PubMed Central  Article  Google Scholar 

  34. Jagan I, Fatehullah A, Deevi RK, Bingham V, Campbell FC. Rescue of glandular dysmorphogenesis in PTEN-deficient colorectal cancer epithelium by PPARgamma-targeted therapy. Oncogene. 2013;32:1305–15.

    CAS  PubMed  Article  Google Scholar 

  35. Soares KC, Foley K, Olino K, Leubner A, Mayo SC, Jain A, et al. A preclinical murine model of hepatic metastases. J Vis Exp. 2014;91:51677.

    Google Scholar 

  36. Blois SM, Sulkowski G, Tirado-Gonzalez I, Warren J, Freitag N, Klapp BF, et al. Pregnancy-specific glycoprotein 1 (PSG1) activates TGF-beta and prevents dextran sodium sulfate (DSS)-induced colitis in mice. Mucosal Immunol. 2014;7:348–58.

    CAS  PubMed  Article  Google Scholar 

  37. Lin CW, Wang LK, Wang SP, Chang YL, Wu YY, Chen HY, et al. Daxx inhibits hypoxia-induced lung cancer cell metastasis by suppressing the HIF-1alpha/HDAC1/Slug axis. Nat Commun. 2016;7:13867.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Pistore C, Giannoni E, Colangelo T, Rizzo F, Magnani E, Muccillo L, et al. DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells. Oncogene. 2017;36:5551–66.

    CAS  PubMed  Article  Google Scholar 

  39. van Staalduinen J, Baker D, Ten Dijke P, van Dam H. Epithelial-mesenchymal-transition-inducing transcription factors: new targets for tackling chemoresistance in cancer? Oncogene. 2018;37:6195–211.

    PubMed  Article  CAS  Google Scholar 

  40. Hu Y, Gaedcke J, Emons G, Beissbarth T, Grade M, Jo P, et al. Colorectal cancer susceptibility loci as predictive markers of rectal cancer prognosis after surgery. Genes Chromosom Cancer. 2018;57:140–9.

    CAS  PubMed  Article  Google Scholar 

  41. Driessens G, Beck B, Caauwe A, Simons BD, Blanpain C. Defining the mode of tumour growth by clonal analysis. Nature. 2012;488:527–30.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Gerlinger M, Rowan AJ, Horswell S, Math M, Larkin J, Endesfelder D, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl J Med. 2012;366:883–92.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Navin N, Kendall J, Troge J, Andrews P, Rodgers L, McIndoo J, et al. Tumour evolution inferred by single-cell sequencing. Nature. 2011;472:90–94.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. Bedard PL, Hansen AR, Ratain MJ, Siu LL. Tumour heterogeneity in the clinic. Nature. 2013;501:355–64.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. de Sousa e Melo F, Kurtova AV, Harnoss JM, Kljavin N, Hoeck JD, Hung J, et al. A distinct role for Lgr5(+) stem cells in primary and metastatic colon cancer. Nature. 2017;543:676–80.

    Article  CAS  PubMed  Google Scholar 

  46. Derynck R, Muthusamy BP, Saeteurn KY. Signaling pathway cooperation in TGF-beta-induced epithelial-mesenchymal transition. Curr Opin Cell Biol. 2014;31:56–66.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. Gonzalez DM, Medici D. Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal. 2014;7:re8.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  48. Gaur T, Lengner CJ, Hovhannisyan H, Bhat RA, Bodine PV, Komm BS, et al. Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression. J Biol Chem. 2005;280:33132–40.

    CAS  PubMed  Article  Google Scholar 

  49. Magnani L, Eeckhoute J, Lupien M. Pioneer factors: directing transcriptional regulators within the chromatin environment. Trends Genet. 2011;27:465–74.

    CAS  PubMed  Article  Google Scholar 

  50. Lee JW, Kim DM, Jang JW, Park TG, Song SH, Lee YS, et al. RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point. Nat Commun. 2019;10:1897.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  51. Barnes GL, Javed A, Waller SM, Kamal MH, Hebert KE, Hassan MQ, et al. Osteoblast-related transcription factors Runx2 (Cbfa1/AML3) and MSX2 mediate the expression of bone sialoprotein in human metastatic breast cancer cells. Cancer Res. 2003;63:2631–7.

    CAS  PubMed  Google Scholar 

  52. Barnes GL, Hebert KE, Kamal M, Javed A, Einhorn TA, Lian JB, et al. Fidelity of Runx2 activity in breast cancer cells is required for the generation of metastases-associated osteolytic disease. Cancer Res. 2004;64:4506–13.

    CAS  PubMed  Article  Google Scholar 

  53. Komori T. Regulation of bone development and maintenance by Runx2. Front Biosci. 2008;13:898–903.

    CAS  PubMed  Article  Google Scholar 

  54. Yang J, Fizazi K, Peleg S, Sikes CR, Raymond AK, Jamal N, et al. Prostate cancer cells induce osteoblast differentiation through a Cbfa1-dependent pathway. Cancer Res. 2001;61:5652–9.

    CAS  PubMed  Google Scholar 

  55. Yeung F, Law WK, Yeh CH, Westendorf JJ, Zhang Y, Wang R, et al. Regulation of human osteocalcin promoter in hormone-independent human prostate cancer cells. J Biol Chem. 2002;277:2468–76.

    CAS  PubMed  Article  Google Scholar 

  56. Baniwal SK, Khalid O, Gabet Y, Shah RR, Purcell DJ, Mav D, et al. Runx2 transcriptome of prostate cancer cells: insights into invasiveness and bone metastasis. Mol Cancer. 2010;9:258.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  57. Niu DF, Kondo T, Nakazawa T, Oishi N, Kawasaki T, Mochizuki K, et al. Transcription factor Runx2 is a regulator of epithelial-mesenchymal transition and invasion in thyroid carcinomas. Lab Investig. 2012;92:1181–90.

    CAS  PubMed  Article  Google Scholar 

  58. Chimge NO, Baniwal SK, Little GH, Chen YB, Kahn M, Tripathy D, et al. Regulation of breast cancer metastasis by Runx2 and estrogen signaling: the role of SNAI2. Breast Cancer Res. 2011;13:R127.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. Inman CK, Shore P. The osteoblast transcription factor Runx2 is expressed in mammary epithelial cells and mediates osteopontin expression. J Biol Chem. 2003;278:48684–9.

    CAS  PubMed  Article  Google Scholar 

  60. Pratap J, Javed A, Languino LR, van Wijnen AJ, Stein JL, Stein GS, et al. The Runx2 osteogenic transcription factor regulates matrix metalloproteinase 9 in bone metastatic cancer cells and controls cell invasion. Mol Cell Biol. 2005;25:8581–91.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  61. Selvamurugan N, Kwok S, Partridge NC. Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-beta1-stimulated collagenase-3 expression in human breast cancer cells. J Biol Chem. 2004;279:27764–73.

    CAS  PubMed  Article  Google Scholar 

  62. Sanchez-Vega F, Mina M, Armenia J, Chatila WK, Luna A, La KC, et al. Oncogenic signaling pathways in the cancer genome Atlas. Cell. 2018;173:321–37. e310.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  63. Lee MH, Kim YJ, Kim HJ, Park HD, Kang AR, Kyung HM, et al. BMP-2-induced Runx2 expression is mediated by Dlx5, and TGF-beta 1 opposes the BMP-2-induced osteoblast differentiation by suppression of Dlx5 expression. J Biol Chem. 2003;278:34387–94.

    CAS  PubMed  Article  Google Scholar 

  64. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012;483:603–7.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  65. Ghandi M, Huang FW, Jane-Valbuena J, Kryukov GV, Lo CC, McDonald ER 3rd, et al. Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature. 2019;569:503–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  66. Hinohara K, Wu HJ, Vigneau S, McDonald TO, Igarashi KJ, Yamamoto KN, et al. KDM5 histone demethylase activity links cellular transcriptomic heterogeneity to therapeutic resistance. Cancer Cell. 2018;34:939–53. e939.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  67. Stark AL, Zhang W, Zhou T, O’Donnell PH, Beiswanger CM, Huang RS, et al. Population differences in the rate of proliferation of international HapMap cell lines. Am J Hum Genet. 2010;87:829–33.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 31701237) and the Shenzhen Science and Technology Program (Grant No. JCYJ20170817110925887, KQTD20180411143432337, and GJHZ20170310161947503). Bioinformatic analysis was supported by the Center for Computational Science and Engineering of Southern University of Science and Technology. Animal experiments were supported by the Laboratory Animal Center of Southern University of Science and Technology.

Author information

Authors and Affiliations

Authors

Contributions

WC, LF, HY, and GL developed the concept of the project and wrote the paper. LF and HY designed and performed experiments. GL, YL, and BZ performed bioinformatic analysis. WL, HC, and YT prepared NGS samples for ATAC-, ChIP- and mRNA-sequencing, respectively. YL, LS, DD, YT, CM, ST, YC, and QZ assisted in performing experiments. QZ and YH reviewed and discussed results and contributed to the paper preparation. YH, WC, and LF supervised the project.

Corresponding authors

Correspondence to Yuhui Hu, Wei Chen or Liang Fang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

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

Yi, H., Li, G., Long, Y. et al. Integrative multi-omics analysis of a colon cancer cell line with heterogeneous Wnt activity revealed RUNX2 as an epigenetic regulator of EMT. Oncogene 39, 5152–5164 (2020). https://doi.org/10.1038/s41388-020-1351-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-020-1351-z

This article is cited by

Search

Quick links