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.

  • Original Article
  • Published:

Induction of epithelial–mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent

Oncogene volume 33, pages 2307–2316 (2014)Cite this article

Subjects

Abstract

Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial–mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Kalluri R, Weinberg RA . The basics of epithelial-mesenchymal transition. J Clin Invest 2009; 119: 1420–1428.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Zeisberg M, Neilson EG . Biomarkers for epithelial-mesenchymal transitions. J Clin Invest 2009; 119: 1429–1437.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Nieto MA . The ins and outs of the epithelial to mesenchymal transition in health and disease. Annu Rev Cell Dev Biol 2011; 27: 347–376.

    Article  CAS  PubMed  Google Scholar 

  4. Polyak K, Weinberg RA . Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer 2009; 9: 265–273.

    Article  CAS  PubMed  Google Scholar 

  5. Prat A, Parker JS, Karginova O, Fan C, Livasy C, Herschkowitz JI et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 2010; 12: R68.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Sarrio D, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, Palacios J . Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype. Cancer Res 2008; 68: 989–997.

    Article  CAS  PubMed  Google Scholar 

  7. Proia TA, Keller PJ, Gupta PB, Klebba I, Jones AD, Sedic M et al. Genetic predisposition directs breast cancer phenotype by dictating progenitor cell fate. Cell Stem Cell 2011; 8: 149–163.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. DiMeo TA, Anderson K, Phadke P, Fan C, Perou CM, Naber S et al. A novel lung metastasis signature links Wnt signaling with cancer cell self-renewal and epithelial-mesenchymal transition in basal-like breast cancer. Cancer Res 2009; 69: 5364–5373.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Mani SA, Guo W, Liao M-J, Eaton EN, Ayyanan A, Zhou AY et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008; 133: 704–715.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, Sjolund A et al. Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci USA 2009; 106: 13820–13825.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Thiery JP, Acloque H, Huang RYJ, Angela Nieto M . Epithelial-mesenchymal transitions in development and disease. Cell 2009; 139: 871–890.

    Article  CAS  PubMed  Google Scholar 

  12. Hugo H, Ackland ML, Blick T, Lawrence MG, Clements JA, Williams ED et al. Epithelial-mesenchymal and mesenchymal-epithelial transitions in carcinoma progression. J Cell Physiol 2007; 213: 374–383.

    Article  CAS  PubMed  Google Scholar 

  13. Lo H-W, Hsu S-C, Xia W, Cao X, Shih J-Y, Wei Y et al. Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial-mesenchymal transition in cancer cells via up-regulation of TWIST gene expression. Cancer Res 2007; 67: 9066–9076.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Berg T . Inhibition of transcription factors with small organic molecules. Curr Opin Chem Biol 2008; 12: 464–471.

    Article  CAS  PubMed  Google Scholar 

  15. Berridge MJ, Bootman MD, Roderick HL . Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 2003; 4: 517–529.

    Article  CAS  PubMed  Google Scholar 

  16. Ronnov-Jessen L, Bissell MJ . Breast cancer by proxy: can the microenvironment be both the cause and consequence? Trends Mol Med 2009; 15: 5–13.

    Article  PubMed  Google Scholar 

  17. Monteith GR, Davis FM, Roberts-Thompson SJ . Calcium channels and pumps in cancer: changes and consequences. J Biol Chem 2012; 287: 31666–31673.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yang SL, Cao Q, Zhou KC, Feng YJ, Wang YZ . Transient receptor potential channel C3 contributes to the progression of human ovarian cancer. Oncogene 2009; 28: 1320–1328.

    Article  CAS  PubMed  Google Scholar 

  19. Prevarskaya N, Skryma R, Shuba Y . Calcium in tumour metastasis: new roles for known actors. Nat Rev Cancer 2011; 11: 609–618.

    Article  CAS  PubMed  Google Scholar 

  20. Monet M, Lehen'kyi Vy, Gackiere F, Firlej V, Vandenberghe M, Roudbaraki M et al. Role of cationic channel TRPV2 in promoting prostate cancer migration and progression to androgen resistance. Cancer Res 2010; 70: 1225–1235.

    Article  CAS  PubMed  Google Scholar 

  21. Yang S, Zhang JJ, Huang X-Y . Orai1 and STIM1 are critical for breast tumor cell migration and metastasis. Cancer Cell 2009; 15: 124–134.

    Article  CAS  PubMed  Google Scholar 

  22. Davis FM, Peters AA, Grice DM, Cabot PJ, Parat MO, Roberts-Thomson SJ et al. Non-stimulated, agonist-stimulated and store-operated Ca2+ influx in MDA-MB-468 breast cancer cells and the effect of EGF-induced EMT on calcium entry. PLoS ONE 2012; 7: e36923.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hu JJ, Qin KH, Zhang Y, Gong JB, Li N, Lv D et al. Downregulation of transcription factor Oct4 induces an epithelial-to-mesenchymal transition via enhancement of Ca(2+) influx in breast cancer cells. Biochem Biophys Res Commun 2011; 411: 786–791.

    Article  CAS  PubMed  Google Scholar 

  24. Gilles C, Polette M, Zahm JM, Tournier JM, Volders L, Foidart JM et al. Vimentin contributes to human mammary epithelial cell migration. J Cell Sci 1999; 112: 4615–4625.

    CAS  PubMed  Google Scholar 

  25. Bonnomet A, Syne L, Brysse A, Feyereisen E, Thompson EW, Noel A et al. A dynamic in vivo model of epithelial-to-mesenchymal transitions in circulating tumor cells and metastases of breast cancer. Oncogene 2011; 31: 3741–3753.

    Article  PubMed  Google Scholar 

  26. Wyckoff J, Wang WG, Lin EY, Wang YR, Pixley F, Stanley ER et al. A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res 2004; 64: 7022–7029.

    Article  CAS  PubMed  Google Scholar 

  27. Berridge MJ, Lipp P, Bootman MD . The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 2000; 1: 11–21.

    Article  CAS  PubMed  Google Scholar 

  28. Ji QS, Carpenter G . Role of basal calcium in the EGF activation of MAP kinases. Oncogene 2000; 19: 1853–1856.

    Article  CAS  PubMed  Google Scholar 

  29. Cook SJ, Beltman J, Cadwallader KA, McMahon M, McCormick F . Regulation of mitogen-activated protein kinase phosphatase-1 expression by extracellular signal-related kinase-dependent and Ca2+-dependent signal pathways in Rat-1 cells. J Biol Chem 1997; 272: 13309–13319.

    Article  CAS  PubMed  Google Scholar 

  30. Colomiere M, Ward AC, Riley C, Trenerry MK, Cameron-Smith D, Findlay J et al. Cross talk of signals between EGFR and IL-6R through JAK2/STAT3 mediate epithelial-mesenchymal transition in ovarian carcinomas. Br J Cancer 2009; 100: 134–144.

    Article  CAS  PubMed  Google Scholar 

  31. Wei C, Wang X, Chen M, Ouyang K, Song L-S, Cheng H . Calcium flickers steer cell migration. Nature 2009; 457: 901–905.

    Article  CAS  PubMed  Google Scholar 

  32. Cheng X, Jin J, Hu L, Shen D, Dong X-P, Samie MA et al. TRP channel regulates EGFR signaling in hair morphogenesis and skin barrier formation. Cell 2010; 141: 331–343.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Gao H, Chen X, Du X, Guan B, Liu Y, Zhang H . EGF enhances the migration of cancer cells by up-regulation of TRPM7. Cell Calcium 2011; 50: 559–568.

    Article  CAS  PubMed  Google Scholar 

  34. Middelbeek J, Kuipers AJ, Henneman L, Visser D, Eidhof I, van Horssen R et al. TRPM7 is required for breast tumor cell metastasis. Cancer Res 2012; 72: 4250–4261.

    Article  CAS  PubMed  Google Scholar 

  35. Chubanov V, Mederos y Schnitzler M, Meissner M, Schafer S, Abstiens K, Hofmann T et al. Natural and synthetic modulators of SK (K(ca)2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7. Br J Pharmacol 2012; 166: 1357–1376.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Langeslag M, Clark K, Moolenaar WH, van Leeuwen FN, Jalink K . Activation of TRPM7 channels by phospholipase C-coupled receptor agonists. J Biol Chem 2007; 282: 232–239.

    Article  CAS  PubMed  Google Scholar 

  37. Ramsey IS, Delling M, Clapham DE . An introduction to TRP channels. Annu Rev Physiol 2006; 68: 619–647.

    Article  CAS  PubMed  Google Scholar 

  38. Shabir S, Southgate J . Calcium signalling in wound-responsive normal human urothelial cell monolayers. Cell Calcium 2008; 44: 453–464.

    Article  CAS  PubMed  Google Scholar 

  39. Chang HY, Nuyten DSA, Sneddon JB, Hastie T, Tibshirani R, Sorlie T et al. Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival. Proc Natl Acad Sci USA 2005; 102: 3738–3743.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Grande M, Franzen A, Karlsson JO, Ericson LE, Heldin NE, Nilsson M . Transforming growth factor-beta and epidermal growth factor synergistically stimulate epithelial to mesenchymal transition (EMT) through a MEK-dependent mechanism in primary cultured pig thyrocytes. J Cell Sci 2002; 115: 4227–4236.

    Article  CAS  PubMed  Google Scholar 

  41. Su LT, Liu W, Chen HC, Gonzalez-Pagan O, Habas R, Runnels LW . TRPM7 regulates polarized cell movements. Biochem J 2011; 434: 513–521.

    Article  CAS  PubMed  Google Scholar 

  42. Takahashi T, Fukuda K, Pan J, Kodama H, Sano M, Makino S et al. Characterization of insulin-like growth factor-1-induced activation of the JAK/STAT pathway in rat cardiomyocytes. Circ Res 1999; 85: 884–891.

    Article  CAS  PubMed  Google Scholar 

  43. Vultur A, Cao J, Arulanandam R, Turkson J, Jove R, Greer P et al. Cell-to-cell adhesion modulates Stat3 activity in normal and breast carcinoma cells. Oncogene 2004; 23: 2600–2616.

    Article  CAS  PubMed  Google Scholar 

  44. Mellstrom B, Savignac M, Gomez-Villafuertes R, Naranjo JR . Ca2+-operated transcriptional networks: molecular mechanisms and in vivo models. Physiol Rev 2008; 88: 421–449.

    Article  CAS  PubMed  Google Scholar 

  45. Mancini M, Toker A . NFAT proteins: emerging roles in cancer progression. Nat Rev Cancer 2009; 9: 810–820.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Davis FM, Kenny PA, Soo ETL, van Denderen BJW, Thompson EW, Cabot PJ et al. Remodeling of purinergic receptor-mediated Ca(2+) signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells. PLoS ONE 2011; 6: e23464.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Clark K, Langeslag M, van Leeuwen B, Ran L, Ryazanov AG, Figdor CG et al. TRPM7, a novel regulator of actomyosin contractility and cell adhesion. EMBO J 2006; 25: 290–301.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Lundgren K, Nordenskjold B, Landberg G . Hypoxia, Snail and incomplete epithelial-mesenchymal transition in breast cancer. Br J Cancer 2009; 101: 1769–1781.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lester RD, Jo M, Montel V, Takimoto S, Gonias SL . uPAR induces epithelial-mesenchymal transition in hypoxic breast cancer cells. J Cell Biol 2007; 178: 425–436.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Liang C-C, Park AY, Guan J-L . In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007; 2: 329–333.

    Article  CAS  PubMed  Google Scholar 

  51. Elgoyhen AB, Vetter DE, Katz E, Rothlin CV, Heinemann SF, Boulter J . alpha 10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells. Proc Natl Acad Sci USA 2001; 98: 3501–3506.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Grice DM, Vetter I, Faddy HM, Kenny PA, Roberts-Thomson SJ, Monteith GR . Golgi calcium pump secretory pathway calcium ATPase 1 (SPCA1) is a key regulator of insulin-like growth factor receptor (IGF1R) processing in the basal-like breast cancer cell line MDA-MB-231. J Biol Chem 2010; 285: 37458–37466.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Suchanek KM, May FJ, Robinson JA, Lee WJ, Holman NA, Monteith GR et al. Peroxisome proliferator-activated receptor alpha in the human breast cancer cell lines MCF-7 and MDA-MB-231. Mol Carcinog 2002; 34: 165–171.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The research was partially supported by the National Health and Medical Research Council (NHMRC; project grants 569645 and 1022263) and the Intramural Research Program of the US National Institutes of Health, National Institute of Environmental Health Sciences (NIEHS). FD was funded by an NHMRC Biomedical Postgraduate Scholarship (511262), RF was funded by a UQ Postdoctoral Fellowship and EWT was funded in part by the National Breast Cancer Foundation.

Author information

Author notes

  1. F M Davis

    Present address: 8Current address: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.,

Authors and Affiliations

  1. School of Pharmacy, The University of Queensland, Brisbane, Queensland, Australia

    F M Davis, I Azimi, A A Peters, S J Roberts-Thomson & G R Monteith

  2. Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia

    R A Faville & G J Goodhill

  3. Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    K Jalink

  4. Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA

    J W Putney Jr

  5. School of Mathematics and Physics, The University of Queensland, Brisbane, Queensland, Australia

    G J Goodhill

  6. St Vincent’s Institute, Fitzroy, Victoria, Australia

    E W Thompson

  7. Department of Surgery, University of Melbourne, St Vincent’s Hospital, Fitzroy, Victoria, Australia

    E W Thompson

Authors
  1. F M Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. I Azimi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R A Faville
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A A Peters
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J W Putney Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. G J Goodhill
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. E W Thompson
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. S J Roberts-Thomson
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. G R Monteith
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G R Monteith.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Davis, F., Azimi, I., Faville, R. et al. Induction of epithelial–mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent. Oncogene 33, 2307–2316 (2014). https://doi.org/10.1038/onc.2013.187

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2013.187

Keywords

This article is cited by

Search

Advanced search

Quick links