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

Introduction: In our cellular model of esophageal carcinogenesis immortalization was induced independent 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 activation of telomerase. This study identifies the cellular pathways involved in the activation of telomerase and 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 shown by co-immunoprecipitation. Cells were treated with respective inhibitors and analyzed for growth characteristics and telomere biology. Moreover, analysis of the hTert promoter 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. However, AKT was phosphorylated at Ser 473 indicating the activation of the PI3K/AKT pathway. In contrast, ERK 1/2 levels and phosphorylation status remained unchanged. IP-western blot analysis showed direct phosphorylation of hTert and Hif-1α by AKT. Analysis of the hTert promoter indicated an additional transcriptional regulation of telomerase reactivation depending on the presence of a Hif-1α binding site at the hTert promoter. According to these findings, protein levels of Hif-1α were increased in EGFR overexpressing cells.

Conclusion: EGFR activates telomerase in immortalized squamous epithelial cells mediated through the PI3K/AKT pathway. The activation is effected by two different mechanisms, namely direct phosphorylation of hTert by AKT as well as transcriptional regulation by Hif-1α. Surprisingly, Hif-1α is also phosphorylated by AKT and thereby stabilized. Whereas immortalization is induced by ALT, in vitro transformation is dependent on telomerase activation, indicating an additional role of telomerase in tumor progression besides elongating telomeres.