Multiple mechanisms of ODC regulation by activated Ras: Studies in an epithelial cell model

A majority of human cancers carry activating mutations in the Ras oncogene. One of the key effectors of Ras that is essential for its tumorigenic properties is Ornithine Decarboxylase (ODC). Studies characterizing the effects of Ras on ODC synthesis have thus far been carried out in fibroblasts. However, since most Ras-activated cancers are epithelial in origin, we aimed to determine the regulation of ODC by activated Ras using an epithelial cell model, RIE-1 cells. Ras transformation greatly induced ODC activity, which was predominantly regulated at the level of RNA stability. Increased RNA stability correlated with increased levels of ODC RNA associated with the polysomes. Treatment with the mTOR inhibitor Rapamycin surprisingly inhibited this increase in RNA stability without affecting transcription or efficiency of ODC translation. Analysis of the ODC 5'UTR in control and Ras12V-transfected cells revealed the presence of four splice variants of variable length, with the presence or absence of two intronic sequences. The variants containing intronic sequences were more abundant in the Ras-transformed cells. All splice variants supported internal ribosome entry site (IRES)-mediated translation, and this activity was markedly elevated in cells transformed by Ras, which was further increased by dephosphorylation of eIF4E by inhibiting either MEK or its kinase Mnk even in RIE-1 cells. When both the Raf-kinase and PI3K/mTOR pathways were inhibited in normal cells, ODC IRES activity was very low, and most cells arrested in G1. However, when these pathways were inhibited in Ras-transformed cells, cell cycle arrest did not occur and ODC IRES activity increased, thus maintaining the high levels of ODC activity. In conclusion, our study for the first time shows that Ras predominantly regulates ODC synthesis both at the level of RNA degradation and also by a novel mechanism involving IRES-mediated translation. Understanding the regulation of ODC will help us to develop better therapeutic strategies to treat Ras-activated cancers.