Abstract 4116: Relationship between K-ras 4A and superoxide in lung adenocarcinoma cell lines

Abstract

Abstract The K-ras gene is mutated in 30-50% of human lung adenocarcinomas, and even more frequently in cancers of colon and pancreas. It exists in two protein isoforms, K-ras 4A and 4B. Their amino acid sequence is very similar, except for marked differences in the hypervariable domain near the carboxyl terminus. K-ras 4B, with a polylysine tail, is unique among the ras gene products and is the dominant K-ras isoform in most tissues. Wild-type K-ras 4B has tumor suppressor activity (Zhang et al. Nature Gen. 29:25, 2001), whereas K-ras 4A was recently found to be necessary for lung tumor development in mice (To et al. Nature Gen. 40:1240, 2008). In search of an oncogenic function for K-ras 4A in lung cancer, we previously demonstrated a highly significant (P<0.01) positive correlation between K-ras 4A expression, at both mRNA and protein levels, and superoxide amounts in a panel of 11 human lung adenocarcinoma cell lines (Proc. Am. Assoc. for Cancer Research 50: (#437)). Superoxide has been widely implicated in cancer causation and progression. In the present study, we test whether there is a cause-effect relationship between expression of K-ras 4A and superoxide production. Cell line H441 expresses high levels of both superoxide and K-ras 4A. Superoxide levels were determined using a nitroblue tetrazolium (NBT) assay and K-ras 4A protein levels quantified by immunoblot with a specific antibody. Results are means ± SEMs. The effect of reduced levels of K-ras 4A on superoxide production was determined using a K-ras 4A silencing siRNA, which reduced K-ras 4A protein by 60.2 ± 3.0% after 72 hours of siRNA treatment. This reduction had no effect on superoxide (control 1.13 ± 0.08, siRNA 1.17 ± 0.08 nmol NBT reduced/ug protein). The effect of reduced levels of superoxide on K-ras 4A expression was determined using 5 mM Tiron, an electron scavenger. A 36% decrease in superoxide did not affect K-ras 4A protein levels (control 26.90 ± 0.89 vs. Tiron-treated 26.89 ± 1.91, arbitrary units). If these preliminary results are confirmed with additional cell lines and reagents, they may suggest that both expression of K-ras 4A and production of superoxide are controlled by another, pro-oncogenic factor or set of conditions. In view of the importance of both K-ras and superoxide in cancer, discovery of this putative central pro-oncogenic regulator might offer an important preventive and therapeutic target in lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4116.