K-ras is frequently mutated in lung adenocarcinomas but the nature of its contribution to the development and maintenance of the malignant phenotype is poorly understood. Wild-type K-ras is tumor suppressive in lung.How is mutant K-ras aggressively oncogenic? We hypothesized that mutant K-ras protein is oncogenic in part by induction of reactive oxygen species (ROS) and DNA damage, contributing to malignant transformation. To investigate mechanisms leading to K-ras oncogenicity in lung we transfected mouse nontransformed peripheral lung epithelial cells (E10 line) with a human K-rasV12 cDNA in a tetracycline regulated retroviral expression vector. Induction of K-rasV12 protein expression/activity resulted in significant increase in reactive oxygen species (ROS) generation as analyzed by intracellular oxidation of H2DCFDA. ROS generation resulted in DNA strand break damage measured by comet assay. The increases in ROS/DNA damage had a peak at 24 hours after K-rasV12 induction was initiated. Furthermore, we observed significant increase in the expression of cyclooxygenase-2 (COX-2), a potential source of ROS. Prostaglandin E2 (PGE2) level was also elevated. Levels of COX-2 protein, PGE2, ROS and DNA strand break damage were attenuated by treatment with COX-2 specific inhibitor SC58125, confirming the enzyme as a source of reactive oxygen species. There is evidence that induction of cyclooxygenase-2 (COX-2)and high PGE2 levels contribute to the pathogenesisof non-small cell lung cancer. To investigate cellular pathways leading to upregulation of COX-2 by mutant K-rasV12 we compared gene expression profiles of K-rasV12 expressing cells and uninduced control cells using Affymetrix high density oligonucleotide microarray (Mouse Genome 430 2.0). The array revealed five-fold higher expression of Hmga1 (high mobility group AT-hook 1) and 3.6-fold higher expression of Ereg (epiregulin) transcripts in the cells expressing K-rasV12 in comparison with uninduced control. Both proteins have been reported to induce COX-2 expression. Experiments to confirm this involvement are in progress. The results show that mutant K-ras through activation of Hmga1 and epiregulin expressions might lead to COX-2 upregulation, which in turn generates reactive oxygen species. ROS may in part contribute to active oncogenicity of mutant K-ras in lung. Funded in part by NO1CO-2008-00001

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4354.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO