gms | German Medical Science

27. Deutscher Krebskongress

Deutsche Krebsgesellschaft e. V.

22. - 26.03.2006, Berlin

EGFR reactivates telomerase in immortalized squamous epithelial cells through PI3K/AKT-mediated phosphorylation and transcriptional regulation via Hif-1alpha

Meeting Abstract

  • corresponding author presenting/speaker Steffen Heeg - Department of Medicine, University of Freiburg, Freiburg, Germany
  • Alexander von Werder - Department of Medicine, University of Freiburg, Freiburg Germany
  • Michaela Doebele - Department of Medicine, University of Freiburg, Freiburg Germany
  • Michael Quante - Department of Medicine, University of Freiburg, Freiburg Germany
  • Erdmann Scheffer - Department of Medicine, University of Freiburg, Freiburg Germany
  • Gitta Goessel - Department of Medicine, University of Freiburg, Freiburg Germany
  • Christine Fulda - Department of Medicine, University of Freiburg, Freiburg Germany
  • Heike Kunert - Department of Medicine, University of Freiburg, Freiburg Germany
  • Hiroshi Nakagawa - Gastroenterology Division, University of Pennsylvania, Philadelphia, PA, USA
  • Roderick Beijersbergen - Netherlands Cancer Center, Amsterdam, NL
  • Hubert E. Blum - Department of Medicine, University of Freiburg, Freiburg Germany
  • Oliver G. Opitz - Department of Medicine, University of Freiburg, Freiburg Germany

27. Deutscher Krebskongress. Berlin, 22.-26.03.2006. Düsseldorf, Köln: German Medical Science; 2006. DocOP168

Die elektronische Version dieses Artikels ist vollständig und ist verfügbar unter: http://www.egms.de/de/meetings/dkk2006/06dkk278.shtml

Veröffentlicht: 20. März 2006

© 2006 Heeg et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielf&aauml;ltigt, verbreitet und &oauml;ffentlich zug&aauml;nglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

Introduction: In our cellular model of oral-esophageal carcinogenesis immortalization was induced independent of the activation of telomerase but through alternative lengthening of telomeres (ALT). In this stepwise model additional overexpression of EGFR in immortalized cyclin D1 overexpressing/p53 inactivated cells (OKF6 D1/del.p53) led to in vitro transformation and reactivation of telomerase. Therefore, we sought to identify the cellular pathways involved in the reactivation of telomerase and the in vitro transformation induced by EGFR.

Methods: We overexpressed EGFR in immortalized OKF6 D1/del.p53 cells by retroviral mediated gene transfer. Expression levels and phosphorylation status of the PI3K/AKT and MAPK/ERK pathways as well as respective downstream targets were analyzed by western blot and RT-PCR. Protein-protein-interactions were demonstrated by co-immunoprecipitation. Cells were additionally treated with respective inhibitors and analyzed for growth characteristics and telomere biology. Furthermore, analysis of the hTert promoter including site-directed-mutagenesis was performed.

Results: EGFR overexpression induced a robust telomerase activity in immortalized squamous epithelial cells. The level of AKT expression was unchanged compared to OKF6 D1/del.p53 cells. Nevertheless, AKT was phosphorylated at Ser 473 and Thr 308 indicating the activation of the PI3K/AKT pathway. In contrast, ERK 1/2 levels and phosphorylation status remained unchanged. IP-western blot analysis showed a direct phosphorylation of hTert as well as Hif-1alpha by AKT. Analysis of the hTert promoter indicated an additional transcriptional regulation of telomerase reactivation depending on the presence of a Hif-1alpha binding site at the hTert core promoter. Corresponding to these findings, protein levels of Hif-1alpha were increased in EGFR overexpressing cells.

Conclusion: We were able to show that EGFR reactivates telomerase in immortalized squamous epithelial cells mediated through the PI3K/AKT pathway. This reactivation is effected by two different mechanisms, namely direct phosphorylation of hTert by AKT as well as transcriptional regulation by Hif-1alpha as key transcription factor. Surprisingly, Hif-1alpha is also phosphorylated by AKT and thereby stabilized. Whereas immortalization is induced by ALT, in vitro transformation is dependent on telomerase reactivation, indicating an additional role of telomerase in tumor progression besides elongating telomeres.