Advertisement

Clinical & Experimental Metastasis

, Volume 31, Issue 4, pp 423–433 | Cite as

Contribution of acidic melanoma cells undergoing epithelial-to-mesenchymal transition to aggressiveness of non-acidic melanoma cells

  • Silvia Peppicelli
  • Francesca Bianchini
  • Eugenio Torre
  • Lido CaloriniEmail author
Research Paper

Abstract

Tumor cell plasticity largely depends on epithelial-to-mesenchymal transition (EMT) and its reversion. It was ascertained that EMT characterizes disease progression, including melanoma malignancy. As most solid tumors, melanoma shows extracellular acidosis, we analyse the impact of acidic environment on the EMT development in human melanoma cells. Melanoma cells were exposed to an acidic extracellular environment (pH 6.7) and tested for EMT markers. We found that acidic cells express a significant up-regulation of mesenchymal markers (N-cadherin, Vimentin), transcription factors (Twist, NF-κB) and a significant, although modest, reduction of E-cadherin expression. Acidic cell also express an increased invasiveness through Matrigel associated with an up-regulation of MMP-9 activity. When we injected acidic cells intravenously into immunodeficient animals, we found a number of lung micrometastases not different from non-acidic cells. Indeed, they show a partial G1 cell cycle arrest, which might interfere with the growth of lung colonies. When we investigated the in vitro invasiveness and lung colonization of a mixed population of acidic and non acidic melanoma cells, we found that acidic cells promote in vitro invasiveness of non-acidic cells and this cooperation leads to an higher migration rate than acidic cells. Moreover, acidic cells cooperate for a better lung colonization of non-acidic cells, that represent the greater part of cells participating to lung micrometastases. We found evidence that acidity triggers in melanoma cells an EMT program, which although “incomplete”, potentiates migration rate and development of lung colonies into immunodeficient host of cells grown in standard pH.

Keywords

Extracellular acidity Epithelial-to-mesenchymal transition NF-κB Invasiveness Tumor cell dissemination Human melanoma 

Notes

Acknowledgments

This study was financially supported by grants from Istituto Toscano Tumori and Ente Cassa di Risparmio di Firenze.

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Alonso SR, Tracey L, Ortiz P, Pérez-Gómez B, Palacios J, Pollán M, Linares J, Serrano S, Sáez-Castillo AI, Sánchez L, Pajares R, Sánchez-Aguilera A, Artiga MJ, Piris MA, Rodríguez-Peralto JL (2007) A high-throughput study in melanoma identifies epithelial–mesenchymal transition as a major determinant of metastasis. Cancer Res 67:3450–3460PubMedCrossRefGoogle Scholar
  2. 2.
    Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA (2009) Epithelial–mesenchymal transitions: the importance of changing cell state in development and disease. J Clin Invest 119:1438–1449PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Kalluri R, Weinberg RA (2009) The basics of epithelial–mesenchymal transition. J Clin Invest 119:1420–1428PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA (2008) The epithelial–mesenchymal transition generates cells with properties of stem cells. Cell 133:704–715PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial–mesenchymal transitions. Nat Rev Mol Cell Biol 7:131–142PubMedCrossRefGoogle Scholar
  6. 6.
    Polyak K, Weinberg RA (2009) Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer 9:265–273PubMedCrossRefGoogle Scholar
  7. 7.
    Scheel C, Weinberg RA (2012) Cancer stem cells and epithelial–mesenchymal transition: concepts and molecular links. Semin Cancer Biol 22:396–403PubMedCrossRefGoogle Scholar
  8. 8.
    Giannoni E, Bianchini F, Masieri L, Serni S, Torre E, Calorini L, Chiarugi P (2010) Reciprocal activation of prostate cancer cells and cancer-associated fibroblasts stimulates epithelial–mesenchymal transition and cancer stemness. Cancer Res 70:6945–6956PubMedCrossRefGoogle Scholar
  9. 9.
    Thiery JP (2003) Epithelial–mesenchymal transitions in development and pathologies. Curr Opin Cell Biol 15(6):740–746PubMedCrossRefGoogle Scholar
  10. 10.
    Yang J, Weinberg RA (2008) Epithelial–mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell 14:818–829PubMedCrossRefGoogle Scholar
  11. 11.
    Massague J (2008) TGF-β in cancer. Cell 134:215–230PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Shi Y, Massagué J (2003) Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113:685–700PubMedCrossRefGoogle Scholar
  13. 13.
    Radisky DC, Levy DD, Littlepage LE, Liu H, Nelson CM, Fata JE, Leake D, Godden EL, Albertson DG, Nieto MA, Werb Z, Bissell MJ (2005) Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature 436:123–127PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Gort EH, Groot AJ, van der Wall E, van Diest PJ, Vooijs MA (2008) Hypoxic regulation of metastasis via hypoxia-inducible factors. Curr Mol Med 8:60–67PubMedCrossRefGoogle Scholar
  15. 15.
    Webb BA, Chimenti M, Jacobson MP, Barber DL (2011) Dysregulated pH: a perfect storm for cancer progression. Nat Rev Cancer 11:671–677PubMedCrossRefGoogle Scholar
  16. 16.
    Gatenby RA, Gillies RJ (2004) Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4:891–899PubMedCrossRefGoogle Scholar
  17. 17.
    Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324:1029–1033PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674PubMedCrossRefGoogle Scholar
  19. 19.
    Calorini L, Peppicelli S, Bianchini F (2012) Extracellular acidity as favouring factor of tumor progression and metastatic dissemination. Exp Oncol 34:79–84PubMedGoogle Scholar
  20. 20.
    Walenta S, Wetterling M, Lehrke M, Schwickert G, Sundfør K, Rofstad EK, Mueller-Klieser W (2000) High lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical cancers. Cancer Res 60:916–921PubMedGoogle Scholar
  21. 21.
    Morita T, Nagaki T, Fukuda I, Okumura K (1992) Clastogenicity of low pH to various cultured mammalian cells. Mutat Res 268:297–305PubMedCrossRefGoogle Scholar
  22. 22.
    Rottinger EM, Mendonca M (1982) Radioresistance secon-dary to low pH in human glial cells and Chinese hamster ovary cells. Int J Radiat Oncol Biol Phys 8:1309–1314PubMedCrossRefGoogle Scholar
  23. 23.
    Fischer K, Hoffmann P, Voelkl S, Meidenbauer N, Ammer J, Edinger M, Gottfried E, Schwarz S, Rothe G, Hoves S, Renner K, Timischl B, Mackensen A, Kunz-Schughart L, Andreesen R, Krause SW, Kreutz M (2007) Inhibitory effect of tumor cell-derived lactic acid on human T cells. Blood 109:3812–3819PubMedCrossRefGoogle Scholar
  24. 24.
    Thews O, Gassner B, Kelleher DK, Schwerdt G, Gekle M (2006) Impact of extracellular acidity on the activity of P-glycoprotein and the cytotoxicity of chemotherapeutic drugs. Neoplasia 8:143–152PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Chen TR (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res 104:255–262PubMedCrossRefGoogle Scholar
  26. 26.
    Vaupel P, Kallinowski F, Okunieff P (1989) Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res 49:6449–6465PubMedGoogle Scholar
  27. 27.
    Wike-Hooley JL, Haveman J, Reinhold HS (1984) The relevance of tumour pH to the treatment of malignant disease. Radiother Oncol 2:343–366PubMedCrossRefGoogle Scholar
  28. 28.
    Hsu MY, Wheelock MJ, Johnson KR, Herlyn M (1996) Shifts in cadherin profiles between human normal melanocytes and melanomas. J Investig Dermatol Symp Proc 1:188–194PubMedGoogle Scholar
  29. 29.
    Hazan RB, Phillips GR, Qiao RF, Norton L, Aaronson SA (2000) Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis. J Cell Biol 148:779–790PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Krengel S, Grotelüschen F, Bartsch S, Tronnier M (2004) Cadherin expression pattern in melanocytic tumors more likely depends on the melanocyte environment than on tumor cell progression. J Cutan Pathol 31:1–7PubMedCrossRefGoogle Scholar
  31. 31.
    De Wever O, Pauwels P, De Craene B, Sabbah M, Emami S, Redeuilh G, Gespach C, Bracke M, Berx G (2008) Molecular and pathological signatures of epithelial–mesenchymal transitions at the cancer invasion front. Histochem Cell Biol 130:481–494PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Yang MH, Hsu DS, Wang HW, Wang HJ, Lan HY, Yang WH, Huang CH, Kao SY, Tzeng CH, Tai SK, Chang SY, Lee OK, Wu KJ (2010) Bmi1 is essential in Twist1-induced epithelial–mesenchymal transition. Nat Cell Biol 12:982–992PubMedCrossRefGoogle Scholar
  33. 33.
    Peppicelli S, Bianchini F, Contena C, Tombaccini D, Calorini L (2013) Acidic pH via NF-κB favours VEGF-C expression in human melanoma cells. Clin Exp Metastasis 30(8):957–967PubMedCrossRefGoogle Scholar
  34. 34.
    Amiri KI, Richmond A (2005) Role of nuclear factor-kappa B in melanoma. Cancer Metastasis Rev 24:301–313PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Kato Y, Lambert CA, Colige AC, Mineur P, Noël A, Frankenne F, Foidart JM, Baba M, Hata R, Miyazaki K, Tsukuda M (2005) Acidic extracellular pH induces matrix metalloproteinase-9 expression in mouse metastatic melanoma cells through the phospholipase D-mitogen-activated protein kinase signaling. J Biol Chem 280:10938–10944PubMedCrossRefGoogle Scholar
  36. 36.
    Moellering RE, Black KC, Krishnamurty C, Baggett BK, Stafford P, Rain M, Gatenby RA, Gillies RJ (2008) Acid treatment of melanoma cells selects for invasive phenotypes. Clin Exp Metastasis 25:411–425PubMedCrossRefGoogle Scholar
  37. 37.
    Friedl P, Wolf K (2003) Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 3:362–374PubMedCrossRefGoogle Scholar
  38. 38.
    Celià-Terrassa T, Meca-Cortés O, Mateo F, de Paz AM, Rubio N, Arnal-Estapé A, Ell BJ, Bermudo R, Díaz A, Guerra-Rebollo M, Lozano JJ, Estarás C, Ulloa C, Álvarez-Simón D, Milà J, Vilella R, Paciucci R, Martínez-Balbás M, de Herreros AG, Gomis RR, Kang Y, Blanco J, Fernández PL, Thomson TM (2012) Epithelial–mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells. J Clin Invest 122:1849–1868PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Tsuji T, Ibaragi S, Hu GF (2009) Epithelial–mesenchymal transition and cell cooperativity in metastasis. Cancer Res 69:7135–7139PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Rofstad EK, Mathiesen B, Kindem K, Galappathi K (2006) Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice. Cancer Res 66:6699–6707PubMedCrossRefGoogle Scholar
  41. 41.
    Jang A, Hill RP (1997) An examination of the effects of hypoxia, acidosis, and glucose starvation on the expression of metastasis-associated genes in murine tumor cells. Clin Exp Metastasis 15:469–483PubMedCrossRefGoogle Scholar
  42. 42.
    Casey JR, Grinstein S, Orlowski J (2010) Sensors and regulators of intracellular pH. Nat Rev Mol Cell Biol 11:50–61PubMedCrossRefGoogle Scholar
  43. 43.
    Robey IF, Baggett BK, Kirkpatrick ND, Roe DJ, Dosescu J, Sloane BF, Hashim AI, Morse DL, Raghunand N, Gatenby RA, Gillies RJ (2009) Bicarbonate increases tumor pH and inhibits spontaneous metastases. Cancer Res 69:2260–2268PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Silvia Peppicelli
    • 1
    • 2
  • Francesca Bianchini
    • 1
    • 2
  • Eugenio Torre
    • 1
    • 2
  • Lido Calorini
    • 1
    • 2
    Email author
  1. 1.Sezione di Patologia e Oncologia Sperimentali, Dipartimento di Scienze Biomediche Sperimentali e ClinicheUniversità di FirenzeFlorenceItaly
  2. 2.Istituto Toscano TumoriFlorenceItaly

Personalised recommendations