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

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Tumor Biology

Abstract

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.

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Acknowledgments

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

None.

Ethics

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

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Authors and Affiliations

  1. Department of Oral and Maxillofacial Pathology, School of Dentistry, Federal University of Pará-UFPA, Belém, Brazil

    Marina Rolo Pinheiro da Rosa, Aline Semblano Carreira Falcão, Maria Sueli da Silva Kataoka, André L. R. Ribeiro, João de Jesus Viana Pinheiro & Sérgio de Melo Alves Júnior

  2. Tropical Medicine Institute, Federal University of Pará-UFPA, Belém, Brazil

    Hellen Thais Fuzii

  3. Department of Oral and Maxillofacial Surgery, School of Dentistry, University Center of Pará-CESUPA, Belém, Brazil

    André L. R. Ribeiro

  4. Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

    Enrique Boccardo

  5. Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

    Adriane Sousa de Siqueira & Ruy G. Jaeger

Authors
  1. Marina Rolo Pinheiro da Rosa
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  2. Aline Semblano Carreira Falcão
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  3. Hellen Thais Fuzii
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  4. Maria Sueli da Silva Kataoka
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  5. André L. R. Ribeiro
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  6. Enrique Boccardo
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  7. Adriane Sousa de Siqueira
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  8. Ruy G. Jaeger
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  9. João de Jesus Viana Pinheiro
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  10. Sérgio de Melo Alves Júnior
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Correspondence to João de Jesus Viana Pinheiro.

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da Rosa, M.R.P., Falcão, A.S.C., Fuzii, H.T. et al. EGFR signaling downstream of EGF regulates migration, invasion, and MMP secretion of immortalized cells derived from human ameloblastoma. Tumor Biol. 35, 11107–11120 (2014). https://doi.org/10.1007/s13277-014-2401-3

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  • DOI: https://doi.org/10.1007/s13277-014-2401-3

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