Tumor Biology

, Volume 35, Issue 11, pp 11107–11120 | Cite as

EGFR signaling downstream of EGF regulates migration, invasion, and MMP secretion of immortalized cells derived from human ameloblastoma

  • Marina Rolo Pinheiro da Rosa
  • Aline Semblano Carreira Falcão
  • Hellen Thais Fuzii
  • Maria Sueli da Silva Kataoka
  • André L. R. Ribeiro
  • Enrique Boccardo
  • Adriane Sousa de Siqueira
  • Ruy G. Jaeger
  • João de Jesus Viana Pinheiro
  • Sérgio de Melo Alves Júnior
Research Article


Ameloblastoma is an odontogenic tumor characterized by local invasiveness and frequent recurrence. The surrounding stroma, composed of different cell types and extracellular matrix (ECM), may influence ameloblastoma invasive behavior. Furthermore, tumor and stromal cells secrete matrix metalloproteases (MMPs), which, in turn, can modulate the matrix and promote the release of ECM-bound growth factors. Among these growth factors, epidermal growth factor (EGF) and its receptor, EGFR, have already been shown to stimulate MMP synthesis, suggesting that an interdependent mechanism, involving MMP activity and growth factors release, may contribute to tumor invasiveness. The aim of this study was to evaluate the effects of the EGF/EGFR signaling pathway on migration, invasion, and MMP activity, in a primary cell line derived from human ameloblastoma. We established and characterized a primary cell line (AME-1) from a human ameloblastoma sample. This cell line was transduced with human papillomavirus type 16 (HPV16) E6/E7 oncogenes, generating the AME-HPV continuous cell line. EGF, MMP2, and MMP9 expression in ameloblastoma biopsies and in the AME-HPV cell line was analyzed by immunohistochemistry and immunofluorescence, respectively. Migratory activity of EGF-treated AME-HPV cells was investigated using monolayer wound assays and Transwell chambers. EGF-induced invasion was assessed in Boyden chambers coated with Matrigel. Conditioned medium from EGF-treated cells was subjected to zymography. EGFR expression in AME-HPV cells was silenced by small interfering RNA (siRNA), to verify the relationship between this receptor and MMP secretion. Ameloblastoma samples and AME-HPV cells expressed EGF, EGFR, MMP2, and MMP9. AME-HPV cells treated with EGF showed increased rates of migration and invasion, as well as enhanced MMP2 and MMP9 activity. EGFR knockdown decreased MMP2 and MMP9 levels in AME-HPV cells. EGFR signaling downstream of EGF probably regulates migration, invasion, and MMP secretion of ameloblastoma-derived cells.


Ameloblastoma ECM Matrix metalloproteases EGF EGFR 



This research was supported by the Brazilian National Council for Scientific and Technological Development (CNPq, grant 4815537/2010-4). Adriane S. Siqueira is recipient of a Graduate fellowship from The State of São Paulo Research Foundation (FAPESP, grant 2009/17336-6).

Conflict of interests



This study was approved by the Ethics Committee on Human Research, of the Institute of Health Sciences, Federal University of Pará (CEP-ICS/UFPA).


  1. 1.
    Hughes CA, Wilson WR, Olding M. Giant ameloblastoma: report of an extreme case and a description of its treatment. Ear Nose Throat J. 1999;78(568):570–62. 574.Google Scholar
  2. 2.
    Osterne RL, Brito RG, Alves AP, Cavalcante RB, Sousa FB. Odontogenic tumors: a 5-year retrospective study in a Brazilian population and analysis of 3,406 cases reported in the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;111:474–81.PubMedCrossRefGoogle Scholar
  3. 3.
    Siriwardena BS, Tennakoon TM, Tilakaratne WM. Relative frequency of odontogenic tumors in Sri Lanka: analysis of 1677 cases. Pathol Res Pract. 2012;208:225–30.PubMedCrossRefGoogle Scholar
  4. 4.
    Carlson ER, Marx RE. The ameloblastoma: primary, curative surgical management. J Oral Maxillofac Surg. 2006;64:484–94.PubMedCrossRefGoogle Scholar
  5. 5.
    Mendenhall WM, Werning JW, Fernandes R, Malyapa RS, Mendenhall NP. Ameloblastoma. Am J Clin Oncol. 2007;30:645–8.PubMedCrossRefGoogle Scholar
  6. 6.
    Mueller MM, Fusenig NE. Friends or foes—bipolar effects of the tumour stroma in cancer. Nat Rev Cancer. 2004;4:839–49.PubMedCrossRefGoogle Scholar
  7. 7.
    Beacham B, Hill C, McDermott F, O’Brien M, Turner J. Therapy with women with metastatic breast cancer. Australas Psychiatry. 2005;13:50–3.PubMedCrossRefGoogle Scholar
  8. 8.
    Hynes RO. The extracellular matrix: not just pretty fibrils. Science. 2009;326:1216–9.PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Harada H, Mitsuyasu T, Nakamura N, Higuchi Y, Toyoshima K, Taniguchi A, et al. Establishment of ameloblastoma cell line, am-1. J Oral Pathol Med. 1998;27:207–12.PubMedCrossRefGoogle Scholar
  10. 10.
    Tao Q, Huang H. Establishment of immortalized ameloblastoma cell line tam-1. Zhonghua Kou Qiang Yi Xue Za Zhi. 2002;37:167–9.PubMedGoogle Scholar
  11. 11.
    Thomas GT, Lewis MP, Speight PM. Matrix metalloproteinases and oral cancer. Oral Oncol. 1999;35:227–33.PubMedCrossRefGoogle Scholar
  12. 12.
    Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol. 2007;8:221–33.PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Pereira AL, Veras SS, Silveira EJ, Seabra FR, Pinto LP, Souza LB, et al. The role of matrix extracellular proteins and metalloproteinases in head and neck carcinomas: an updated review. Braz J Otorhinolaryngol. 2005;71:81–6.PubMedGoogle Scholar
  14. 14.
    Pinheiro JJ, Freitas VM, Moretti AI, Jorge AG, Jaeger RG. Local invasiveness of ameloblastoma. Role played by matrix metalloproteinases and proliferative activity. Histopathology. 2004;45:65–72.PubMedCrossRefGoogle Scholar
  15. 15.
    Baselga J. The EGFR as a target for anticancer therapy—focus on cetuximab. Eur J Cancer. 2001;37 Suppl 4:S16–22.PubMedCrossRefGoogle Scholar
  16. 16.
    Herbst RS. Review of epidermal growth factor receptor biology. Int J Radiat Oncol Biol Phys. 2004;59:21–6.PubMedCrossRefGoogle Scholar
  17. 17.
    Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res. 2003;284:31–53.PubMedCrossRefGoogle Scholar
  18. 18.
    Herr MJ, Mabry SE, Jameson JF, Jennings LK. Pro-mmp-9 upregulation in ht1080 cells expressing cd9 is regulated by epidermal growth factor receptor. Biochem Biophys Res Commun. 2013;442:99–104.PubMedCrossRefGoogle Scholar
  19. 19.
    Ribeiro AL, Nobre RM, Alves-Junior SM, Kataoka MS, Barroso RF, Jaeger RG, et al. Matrix metalloproteinases, tissue inhibitors of metalloproteinases, and growth factors regulate the aggressiveness and proliferative activity of keratocystic odontogenic tumors. Oral Surg Oral Med Oral Pathol Oral Radiol. 2012;114:487–96.PubMedCrossRefGoogle Scholar
  20. 20.
    Siqueira AS, Carvalho MR, Monteiro AC, Freitas VM, Jaeger RG, Pinheiro JJ. Matrix metalloproteinases, timps and growth factors regulating ameloblastoma behaviour. Histopathology. 2010;57:128–37.PubMedCrossRefGoogle Scholar
  21. 21.
    Howie HL, Katzenellenbogen RA, Galloway DA. Papillomavirus e6 proteins. Virology. 2009;384:324–34.PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Demers GW, Halbert CL, Galloway DA. Elevated wild-type p53 protein levels in human epithelial cell lines immortalized by the human papillomavirus type 16 e7 gene. Virology. 1994;198:169–74.PubMedCrossRefGoogle Scholar
  23. 23.
    Sok JC, Coppelli FM, Thomas SM, Lango MN, Xi S, Hunt JL, et al. Mutant epidermal growth factor receptor (EGFRvIII) contributes to head and neck cancer growth and resistance to EGFR targeting. Clin Cancer Res. 2006;12:5064–73.PubMedCrossRefGoogle Scholar
  24. 24.
    Wheeler SE, Suzuki S, Thomas SM, Sen M, Leeman-Neill RJ, Chiosea SI, et al. Epidermal growth factor receptor variant III mediates head and neck cancer cell invasion via stat3 activation. Oncogene. 2010;29:5135–45.PubMedCrossRefGoogle Scholar
  25. 25.
    Shrestha P, Yamada K, Higashiyama H, Takagi H, Mori M. Epidermal growth factor receptor in odontogenic cysts and tumors. J Oral Pathol Med. 1992;21:314–7.PubMedCrossRefGoogle Scholar
  26. 26.
    Harris RC, Chung E, Coffey RJ. EGF receptor ligands. Exp Cell Res. 2003;284:2–13.PubMedCrossRefGoogle Scholar
  27. 27.
    Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer. 2001;37 Suppl 4:S9–15.PubMedCrossRefGoogle Scholar
  28. 28.
    Ethier SP. Signal transduction pathways: the molecular basis for targeted therapies. Semin Radiat Oncol. 2002;12:3–10.PubMedCrossRefGoogle Scholar
  29. 29.
    Crivelini MM, de Araujo VC, de Sousa SO, de Araujo NS. Cytokeratins in epithelia of odontogenic neoplasms. Oral Dis. 2003;9:1–6.PubMedCrossRefGoogle Scholar
  30. 30.
    Pal SK, Sakamoto K, Aragaki T, Akashi T, Yamaguchi A. The expression profiles of acidic epithelial keratins in ameloblastoma. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;115:523–31.PubMedCrossRefGoogle Scholar
  31. 31.
    Wang A, Zhang B, Huang H, Zhang L, Zeng D, Tao Q, et al. Suppression of local invasion of ameloblastoma by inhibition of matrix metalloproteinase-2 in vitro. BMC Cancer. 2008;8:182.PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Hellner K, Mar J, Fang F, Quackenbush J, Munger K. Hpv16 e7 oncogene expression in normal human epithelial cells causes molecular changes indicative of an epithelial to mesenchymal transition. Virology. 2009;391:57–63.PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    DeClerck YA. Interactions between tumour cells and stromal cells and proteolytic modification of the extracellular matrix by metalloproteinases in cancer. Eur J Cancer. 2000;36:1258–68.PubMedCrossRefGoogle Scholar
  34. 34.
    Fregnani ER, Sobral LM, Alves FA, Soares FA, Kowalski LP, Coletta RD. Presence of myofibroblasts and expression of matrix metalloproteinase-2 (mmp-2) in ameloblastomas correlate with rupture of the osseous cortical. Pathol Oncol Res. 2009;15:231–40.PubMedCrossRefGoogle Scholar
  35. 35.
    Vered M, Shohat I, Buchner A, Dayan D. Myofibroblasts in stroma of odontogenic cysts and tumors can contribute to variations in the biological behavior of lesions. Oral Oncol. 2005;41:1028–33.PubMedCrossRefGoogle Scholar
  36. 36.
    de Oliveira RG, Costa A, Meurer MI, Vieira DS, Rivero ER. Immunohistochemical analysis of matrix metalloproteinases (1, 2, and 9), ki-67, and myofibroblasts in keratocystic odontogenic tumors and pericoronal follicles. J Oral Pathol Med. 2014;43:282–8.CrossRefGoogle Scholar
  37. 37.
    Iezzi G, Piattelli A, Rubini C, Artese L, Goteri G, Perrotti V, et al. Expression of transforming growth factor beta1 in ameloblastomas. J Craniofac Surg. 2008;19:1618–21.PubMedCrossRefGoogle Scholar
  38. 38.
    Karathanasi V, Tosios KI, Nikitakis NG, Piperi E, Koutlas I, Trimis G, et al. Tgf-beta1, smad-2/-3, smad-1/-5/-8, and smad-4 signaling factors are expressed in ameloblastomas, adenomatoid odontogenic tumors, and calcifying cystic odontogenic tumors: an immunohistochemical study. J Oral Pathol Med. 2013;42:415–23.PubMedCrossRefGoogle Scholar
  39. 39.
    Uttamsingh S, Bao X, Nguyen KT, Bhanot M, Gong J, Chan JL, et al. Synergistic effect between EGF and TGF-beta1 in inducing oncogenic properties of intestinal epithelial cells. Oncogene. 2008;27:2626–34.PubMedCrossRefGoogle Scholar
  40. 40.
    Xu Z, Jiang Y, Steed H, Davidge S, Fu Y. Tgfbeta and egf synergistically induce a more invasive phenotype of epithelial ovarian cancer cells. Biochem Biophys Res Commun. 2010;401:376–81.PubMedCrossRefGoogle Scholar
  41. 41.
    Richter P, Umbreit C, Franz M, Berndt A, Grimm S, Uecker A, et al. Egf/tgfbeta1 co-stimulation of oral squamous cell carcinoma cells causes an epithelial-mesenchymal transition cell phenotype expressing laminin 332. J Oral Pathol Med. 2011;40:46–54.PubMedCrossRefGoogle Scholar
  42. 42.
    Lu Z, Jiang G, Blume-Jensen P, Hunter T. Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase. Mol Cell Biol. 2001;21:4016–31.PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Kawamoto M, Matsunami T, Ertl RF, Fukuda Y, Ogawa M, Spurzem JR, et al. Selective migration of alpha-smooth muscle actin-positive myofibroblasts toward fibronectin in the Boyden’s blindwell chamber. Clin Sci (Lond). 1997;93:355–62.Google Scholar
  44. 44.
    Christian MM, Moy RL, Wagner RF, Yen-Moore A. A correlation of alpha-smooth muscle actin and invasion in micronodular basal cell carcinoma. Dermatol Surg. 2001;27:441–5.PubMedGoogle Scholar
  45. 45.
    Dilly M, Hambruch N, Haeger JD, Pfarrer C. Epidermal growth factor (EGF) induces motility and upregulates mmp-9 and timp-1 in bovine trophoblast cells. Mol Reprod Dev. 2010;77:622–9.PubMedCrossRefGoogle Scholar
  46. 46.
    Kumamoto H, Yamauchi K, Yoshida M, Ooya K. Immunohistochemical detection of matrix metalloproteinases (mmps) and tissue inhibitors of metalloproteinases (timps) in ameloblastomas. J Oral Pathol Med. 2003;32:114–20.PubMedCrossRefGoogle Scholar
  47. 47.
    Naka T, Kuester D, Boltze C, Schulz TO, Samii A, Herold C, et al. Expression of matrix metalloproteinases-1, -2, and -9; tissue inhibitors of matrix metalloproteinases-1 and -2; cathepsin b; urokinase plasminogen activator; and plasminogen activator inhibitor, type i in skull base chordoma. Hum Pathol. 2008;39:217–23.PubMedCrossRefGoogle Scholar
  48. 48.
    Wells A. EGF receptor. Int J Biochem Cell Biol. 1999;31:637–43.PubMedCrossRefGoogle Scholar
  49. 49.
    Howe AK, Aplin AE, Juliano RL. Anchorage-dependent ERK signaling—mechanisms and consequences. Curr Opin Genet Dev. 2002;12:30–5.PubMedCrossRefGoogle Scholar
  50. 50.
    Rothhut B, Ghoneim C, Antonicelli F, Soula-Rothhut M. Epidermal growth factor stimulates matrix metalloproteinase-9 expression and invasion in human follicular thyroid carcinoma cells through focal adhesion kinase. Biochimie. 2007;89:613–24.PubMedCrossRefGoogle Scholar
  51. 51.
    Lal A, Glazer CA, Martinson HM, Friedman HS, Archer GE, Sampson JH, et al. Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion. Cancer Res. 2002;62:3335–9.PubMedGoogle Scholar
  52. 52.
    Choe G, Park JK, Jouben-Steele L, Kremen TJ, Liau LM, Vinters HV, et al. Active matrix metalloproteinase 9 expression is associated with primary glioblastoma subtype. Clin Cancer Res. 2002;8:2894–901.PubMedGoogle Scholar
  53. 53.
    Kang CS, Pu PY, Li YH, Zhang ZY, Qiu MZ, Huang Q, et al. An in vitro study on the suppressive effect of glioma cell growth induced by plasmid-based small interference RNA (siRNA) targeting human epidermal growth factor receptor. J Neurooncol. 2005;74:267–73.PubMedCrossRefGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Marina Rolo Pinheiro da Rosa
    • 1
  • Aline Semblano Carreira Falcão
    • 1
  • Hellen Thais Fuzii
    • 2
  • Maria Sueli da Silva Kataoka
    • 1
  • André L. R. Ribeiro
    • 1
    • 3
  • Enrique Boccardo
    • 4
  • Adriane Sousa de Siqueira
    • 5
  • Ruy G. Jaeger
    • 5
  • João de Jesus Viana Pinheiro
    • 1
  • Sérgio de Melo Alves Júnior
    • 1
  1. 1.Department of Oral and Maxillofacial Pathology, School of DentistryFederal University of Pará-UFPABelémBrazil
  2. 2.Tropical Medicine InstituteFederal University of Pará-UFPABelémBrazil
  3. 3.Department of Oral and Maxillofacial SurgerySchool of Dentistry, University Center of Pará-CESUPABelémBrazil
  4. 4.Department of MicrobiologyInstitute of Biomedical Sciences, University of São PauloSão PauloBrazil
  5. 5.Department of Cell and Developmental BiologyInstitute of Biomedical Sciences, University of São PauloSão PauloBrazil

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