Malignant transformation of primary human oral squamous epithelial cells using a defined set of genetic alterations

Normal keratinocytes display a restricted replicative life span. Therefore, senescence has been thought to form a barrier against carcinogenesis and the acquisition of cellular immortalization is considered an essential step in the malignant transformation of normal cells. Several groups have demonstrated that the serial introduction of the SV40 early region, H-rasV12 and h-TERT sufficed to transform several types of human cells in vitro, although this combination is rarely seen in human cancer. In contrast, cyclin D1 overexpression, inactivation of p53, EGFR overexpression as well as c-myc overexpression are common genetic alteration in human oral-esophageal squamous cell carcinomas. Previously, we demonstrated that cyclin D1 overexpression and functional p53 inactivation induces immortalization of oral keratinocytes independent of telomerase activation but involving the alternative pathway of telomere maintenance (ALT). We now aimed to induce malignant transformation of previously normal oral keratinocytes using the genetically defined alterations frequently found in the corresponding cancer. We ectopically overexpressed cyclin D1, dominant negative p53, EGFR and c-myc in oral keratinocytes (OKF6) in order to evaluate cell cycle kinetics, signaling, growth characteristics, telomere biology, anchorage independent growth as well as tumor formation in mice. OKF6 D1/del.p53/EGFR/c-myc cells show an accelerated population doubling time, which can be further stimulated by addition of exogenous EGF when compared to immortal OKF6 D1/del.p53 cells. In addition, EGFR is functional in these cells and leads to an induction of PI3K and phosphorylation of AKT. EGFR overexpression induces a robust reactivation of telomerase in these cells. Furthermore, OKF6 D1/del.p53/EGFR/c-myc cells induce malignant growth in mice and form colonies in soft agar, a hallmark of malignant transformation at almost the same rate as human oral squamous cancer cells (SCC). We therefore conclude that the genetic alterations induced in normal human oral keratinocytes, which recapitulate the genetic events frequently found in the human disease, namely cyclin D1 overexpression, p53 inactivation EGFR overexpression and c-myc overexpression lead malignant transformation of this particular cell type.

Keywords: cellular model, esophageal cancer