A global comparison of mRNA from PDTC versus IL-6 identified a highly significant difference of dysregulated gene expression in HepG2 cells. The human HepG2 hepatoma cell line was used because it secretes a variety of major acute phase proteins (e.g., fibrinogen, plasminogen, α2-macroglobulin) and will respond to stimulation with IL-6. HepG2 cells are also used as a suitable in vitro model system for studies of liver metabolism, hepatocarcinogenesis, and for drug targeting studies. Here, an unbiased pathway analysis using the Parameterized Analysis of Gene Set Enrichment method detected the mammalian target of rapamycin (mTOR) pathway, amino acid metabolism and mitochondrial oxidative function as the most differentially regulated pathways between PDTC and IL-6 treatments. Recent studies have indicated the role of mTOR signaling in limiting mammalian lifespan through the control of mitochondrial oxidative function. Moreover, many human disorders occur as a result of dysregulated cellular signal transduction through mTOR, the key player that coordinates proper cell growth and proliferation by regulating ribosomal biogenesis and protein translation. Interestingly, we identified DDIT4 as an early-immediate gene whose transcript, termed REDD1, was drastically up- and down-regulated by PDTC and IL-6 in HepG2 cells, respectively. The REDD1 tumor suppressor protein tightly controls mTOR. To assess the relevance of these observations, we performed immunoblot analysis and found that PDTC inhibits whereas IL-6 augments p70 S6 kinase phosphorylation at Ser 371, a known target of mTOR kinase activity. These and other results identify dysregulated mTOR activity as an important component of PDTC-mediated inhibition of IL-6 signaling through the control of DDIT4 (REDD1) expression. Little is known about the signaling pathways and post-translational modifications that regulate REDD1 expression and function. We are currently investigating the role of phosphorylation and ubiquitination in proteasome-mediated degradation of REDD1 in cells exposed to PDTC and/or IL-6. Genetic, biochemical and pharmacological approaches are also being used to study the effects of the JAK/STAT pathway in controlling DDIT4 (REDD1) expression at transcriptional and post-transcriptional levels. Taken together, our findings thus suggest that the degradation of REDD1 we detect with IL-6 may trigger proliferative and survival signals in cancer cells through up-regulation of the mTOR pathway. REDD1 and its signaling cascade may represent novel potential therapeutic targets for human diseases.
