Purpose: To evaluate the oncogenic impact of p53 knockdown, mutant K-RASV12, mutant EGFR alone and their combination on tumorigenicity of a newly developed immortalized human bronchial epithelial cell line. Background: Molecular analysis of lung cancer has revealed several genetic and epigenetic alterations in the multistep pathogenesis of lung cancer. However, little is known about the relative importance of each individual alteration on the tumorigenic process. One approach is to use a model system in which the contribution of each genetic alteration to lung tumorigenesis can be assessed individually and combinatorially. We have developed an in vitro model system using normal human bronchial epithelial cells that overexpress Cyclin-dependent kinase 4 and human telomerase. Ectopic expression of these two genes enables the cells to bypass the growth inhibitory signals of the p16/Rb pathway and also replicative senescence induced by shortened telomeres. We manipulated this cell line (HBEC3), by stably knocking down p53 using RNA interference, by introducing a mutant form of K-RASV12, and by introducing a mutant epidermal growth factor receptor (EGFR), alone and in combination. Methods: pSuper.retro vector was used for p53 knockdown and a mutant K-RASV12 and mutant EGFRs (EGFRdel746-750 and EGFRL858R) were introduced using viral expression vectors. To test the genetically manipulated lines for changes in their malignant potential, growth in liquid culture, colony formation ability, growth factor dependence, and growth as xenografts in immuno-compromised mice were performed. To determine the effect of tyrosine kinase inhibitors (TKIs) (gefitinib and erlotinib) on manipulated HBEC3 cells and to assess their potential as chemopreventive agents, the cells were evaluated both in the presence or absence of these drugs for colony forming ability. Results: Stable knockdown of p53 and overexpression of mutant EGFR were confirmed by immunoblotting in manipulated cells. K-RASV12 expression was confirmed by RFLP analysis. p53 knockdown and K-RASV12 alone caused cells to be less sensitive to contact inhibition and to be capable of partial anchorage-independent growth. The combination of p53 knockdown and K-RASV12 further enhanced the tumorigenic phenotype by growth in soft agar but failed to cause cells to form tumors in nude mice. In addition, the ability of both anchorage-dependent and-independent cell growth of HBEC3 cells was highly dependent on epidermal growth factor (EGF) and this ability was completely inhibited with TKIs. Both types of mutant EGFRs enhanced the tumorigenic phenotype by growth in soft agar. Importantly, introduction of mutant EGFRL858R into the HBEC3-p53 knockdown cells abrogated EGF dependence as measured by anchorage-independent colony formation ability. These results demonstrate that this isogenic in vitro model system is a powerful approach for studying lung cancer development.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]