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
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
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.
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.
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;67:7–30.
Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871–90.
Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Investig. 2009;119:1420–8.
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.
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.
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.
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.
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.
Nieto MA, Huang RY, Jackson RA, Thiery JP. Emt: 2016. Cell. 2016;166:21–45.
Puisieux A, Brabletz T, Caramel J. Oncogenic roles of EMT-inducing transcription factors. Nat Cell Biol. 2014;16:488–94.
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.
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.
Nieto MA. Epithelial-Mesenchymal Transitions in development and disease: old views and new perspectives. Int J Dev Biol. 2009;53:1541–7.
Tam WL, Weinberg RA. The epigenetics of epithelial-mesenchymal plasticity in cancer. Nat Med. 2013;19:1438–49.
McGranahan N, Swanton C. Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell. 2015;27:15–26.
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.
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.
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.
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.
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
Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36:1461–73.
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.
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.
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.
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.
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.
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.
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.
Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature. 2003;423:349–55.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Driessens G, Beck B, Caauwe A, Simons BD, Blanpain C. Defining the mode of tumour growth by clonal analysis. Nature. 2012;488:527–30.
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.
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.
Bedard PL, Hansen AR, Ratain MJ, Siu LL. Tumour heterogeneity in the clinic. Nature. 2013;501:355–64.
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.
Derynck R, Muthusamy BP, Saeteurn KY. Signaling pathway cooperation in TGF-beta-induced epithelial-mesenchymal transition. Curr Opin Cell Biol. 2014;31:56–66.
Gonzalez DM, Medici D. Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal. 2014;7:re8.
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.
Magnani L, Eeckhoute J, Lupien M. Pioneer factors: directing transcriptional regulators within the chromatin environment. Trends Genet. 2011;27:465–74.
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.
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.
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.
Komori T. Regulation of bone development and maintenance by Runx2. Front Biosci. 2008;13:898–903.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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
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.
Rights and permissions
About this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41388-020-1351-z
This article is cited by
-
RUNX transcription factors: biological functions and implications in cancer
Clinical and Experimental Medicine (2024)
-
Single cell multi-omics reveal intra-cell-line heterogeneity across human cancer cell lines
Nature Communications (2023)
-
Wnt signaling in colorectal cancer: pathogenic role and therapeutic target
Molecular Cancer (2022)
-
Intestinal cellular heterogeneity and disease development revealed by single-cell technology
Cell Regeneration (2022)
-
The SWI/SNF chromatin remodeling factor DPF3 regulates metastasis of ccRCC by modulating TGF-β signaling
Nature Communications (2022)