EGFR-overexpression induces telomerase reactivation and cell cycle progression as distinct steps in a cellular model of oral esophageal carcinogenesis

Introduction: In our cellular model of oral-esophageal carcinogenesis EGFR-overexpression plays a crucial role in in vitro transformation, telomerase-reactivation and enhanced proliferation of immortalized cyclinD1-overexpressing/p53-inactivated keratinocytes (OKF6-D1/d.n.p53). We previously demonstrated that this is mediated by the activation of the PI3K/AKT-pathway. Our aim was to characterize the cellular mechanisms involved in the telomerase-reactivation and acceleration of the proliferative capacity induced by EGFR-overexpression leading to in vitro transformation.

Methods: We overexpressed EGFR in immortalized OKF6D1/d.n.p53 cells by retroviral mediated gene transfer. Expression levels and phosphorylation status of potential downstream targets of the PI3K-AKT pathway and cell cycle regulating proteins were analyzed by western blotting and RT-PCR. Protein-protein-interactions were demonstrated by co-immunoprecipitation. Cellular localization of cell cycle regulators was demonstrated by immunofluorescence. Analysis of the hTERT- and cyclinD1-promoter including site directed mutagenesis was performed.

Results: Co-immunoprecipitation showed a direct phosphorylation of hTERT and Hif-1alpha by AKT. Promoter-analysis indicated that hTERT-expression is additionally regulated transcriptionally involving a Hif-1alpha binding-site within the hTERT core promoter. Moreover, p21 is phosphorylated by AKT and phospho-p21 is translocated to the cytosol. Western blot analysis showed elevated levels of endogenous cyclinD1 in EGFR-overexpressing cells. Preliminary data suggested that cyclinD1 is regulated transcriptionally.

Conclusion: During malignant transformation EGFR-overexpression leads to telomerase-reactivation and increased proliferation mediated through the PI3K/AKT-pathway. hTERT is activated by AKT-mediated phosphorylation and transcriptional regulation via Hif-1alpha. Increased proliferation and activation of the cell-cycle are mediated by phosphorylation and consecutive translocation of p21 from the nucleus. Furthermore, endogenous cyclin D1 was upregulated. In this study we defined the cellular mechanisms leading to telomerase activation, increased proliferation and in vitro transformation as distinct steps of carcinogenesis, induced by EGFR-overexpression.