EGFR overexpression induces activation of telomerase via PI3K/AKT-mediated phosphorylation and transcriptional regulation through Hif1-alpha in a cellular model of oral-esophageal carcinogenesis.

Telomerase plays an important role during immortalization and malignant transformation as crucial steps in the development of human cancer. In a cellular model of oral-esophageal carcinogenesis, recapitulating the human disease, immortalization occurred independent of the activation of telomerase but through the recombination-based alternative lengthening of telomeres (ALT). In this stepwise model, additional overexpression of EGFR led to in vitro transformation and activation of telomerase with homogeneous telomere elongation in already immortalized oral squamous epithelial cells (OKF6-D1_dnp53). More interestingly, EGFR overexpression activated the PI3K/AKT pathway. This strongly suggested a role for telomerase in tumor progression in addition to just elongating telomeres and inferring an immortalized state. Therefore, we sought to identify the regulatory mechanisms involved in this activation of telomerase and in vitro transformation induced by EGFR. In the present study we demonstrate that telomerase expression and activity are induced through both direct phosphorylation of hTERT by phospho-AKT as well as PI3K-dependent transcriptional regulation involving Hif1-alpha as a key transcription factor. Furthermore, EGFR overexpression enhanced cell cycle progression and proliferation via phosphorylation and translocation of p21. Whereas immortalization was induced by ALT, in vitro transformation was associated with telomerase activation, supporting an additional role for telomerase in tumor progression besides elongating telomeres.

Differential transcriptional regulation of human telomerase in a cellular model representing important genetic alterations in esophageal squamous carcinogenesis.

Telomerase activity is observed in approximately 90% of human cancer including esophageal squamous cell cancer. Normal somatic cells do not display telomerase activity on a regular basis. The major mechanism to regulate telomerase activity in human cells is the transcriptional control of the catalytic subunit, the human reverse transcriptase gene hTERT. However, the manner in which telomerase activity is regulated during malignant transformation and whether this regulation is influenced by single genetic alterations important in this process are not well understood. In this study we investigated the transcriptional regulation and activity of human telomerase in a cellular model representing important known genetic alterations observed in esophageal cancer. We characterized the respective cells with regard to their telomere biology and telomerase expression, transcriptional regulation using promoter--as well as electrophoretic mobility shift assay--analyses and their promoter methylation status. We could demonstrate that telomerase expression and subsequent activity are differentially regulated in the progression from normal esophageal epithelial cells to genetically defined esophageal cells harboring a specific genetic alteration frequently found in esophageal cancer and compared those changes with esophageal cancer cells. Whereas primary esophageal cells are mainly regulated by Sp1, in cells harboring a genetic alteration as cyclin D1 overexpression other transcription factors like E2F and c-myc as well as promoter methylation influence hTERT transcription. This model demonstrates that the transcriptional regulation of telomerase is influenced by a given genetic alteration important in esophageal cancer, and therefore provides new insight in telomerase regulation during carcinogenesis.

Creating oral squamous cancer cells: a cellular model of oral-esophageal carcinogenesis.

Immortalization and malignant transformation are important steps in tumor development. The ability to induce these processes from normal human epithelial cells with genetic alterations frequently found in the corresponding human cancer would significantly enhance our understanding of tumor development. Alterations in several key intracellular regulatory pathways (the pRB, p53, and mitogenic signaling pathways and the telomere maintenance system) appear to be sufficient for the neoplastic transformation of normal human cells. Nevertheless, in vitro transformation models to date depend on viral oncogenes, most prominently the simian virus 40 early region, to induce immortalization and malignant transformation of normal human epithelial cells. Here, we demonstrate a transformation model creating oral-esophageal cancer cells by using a limited set of genetic alterations frequently observed in the corresponding human cancer. In a stepwise model, cyclin D1 overexpression and p53 inactivation led to immortalization of oral keratinocytes. Additional ectopic epithelial growth factor receptor overexpression followed by c-myc overexpression as well as consecutive reactivation of telomerase induced by epithelial growth factor receptor sufficed to transform oral epithelial cells, truly recapitulating the development of the corresponding human disease.