Abstract 3106: Translational regulation of hypoxia-inducible factor-1α and 2α by mutant oncogenes in colon cancer

Abstract

Abstract Background: KRAS and BRAF mutations are frequently observed in human colon cancers. These mutations occur in a mutually exclusive manner, and each is associated with distinctive biological features. Many hypoxic responses are mediated by hypoxia-inducible factor (HIF)-1α and 2α, and mTOR pathways can regulate the translation of HIF proteins. We previously reported that HIF-1α promotes the growth of SW480 colon cancer cells but HIF-2α appears to restrain growth. We sought to determine the roles of KRAS, BRAF, and mTOR in the regulation of these two HIF isoforms. Methods: Colon cancer cell lines DLD-1 and HCT116 that carry mutant KRAS, HT29 and Colo205 that carry mutant BRAF and Caco2 that does not have mutation in either KRAS or BRAF gene were utilized. The cells were treated with siRNA directed against K-ras or BRAF or a non-specific control. Mutant K-ras and BRAF were lentivirally introduced in Caco2 cells. Following incubation in normoxia or hypoxia, HIF-1α and 2α expression were analyzed by western blotting and QRT-PCR. DKO-3 and HKe-3 cells are DLD-1 and HCT116 cells, respectively, in which the mutant KRAS gene is disrupted by homologous recombination. HIF-1α protein synthesis was measured by its accumulation during treatment with the proteasome inhibitor MG132. To analyze signaling pathways downstream of K-ras, the MEK inhibitor PD98059, PI3K inhibitor LY294002, and mTOR inhibitor Rapamycin were utilized. Results: Ectopic expression of mutant K-ras in Caco2 cells enhanced the hypoxic induction of only HIF-1α, whereas mutant BRAF enhanced both HIF-1α and 2α. Knockout of mutant KRAS in DLD-1 and HCT116 cells impaired the hypoxic induction of HIF-1α and the rate of HIF-1α protein synthesis. In contrast, knockdown of mutant BRAF in HT29 cells impaired the translation of HIF-2α. In DLD-1 and HCT116 cells, LY294002 suppressed the hypoxic induction of HIF-1α, but not HIF-2α. PD98059 strongly inhibited the hypoxic induction of HIF-1α as well as HIF-2α in HT29 and Colo205 cells. p70 S6 kinase, which regulates global translation and can be phosphorylated by mTOR, was inactivated in hypoxia. Inhibition of mTOR by rapamycin minimally suppressed HIF-1α induction but not HIF-2α. Similarly, knockdown of Raptor and Rictor, which are components of mTORC1 and mTORC2, respectively, only partially suppressed HIF induction. Conclusion: Mutant KRAS enhances the hypoxic induction of HIF-1α by regulating its translation through PI3K pathways. However, mutant BRAF can enhance HIF-2α translation by activating MAPK pathways. The mTOR pathway does not appear to be an important regulator of HIF-1α or 2α translation in colon cancer cells. Mutant oncogenes appear to interact with hypoxia to enhance HIF expression in an mTOR-independent manner. 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 3106.