ABSTRACT
Isocitrate dehydrogenase 1 (IDH1), a cytosolic enzyme that converts isocitrate to alpha-ketoglutarate, has been shown to be dysregulated during tumorigenesis. However, at what stage of cancer development IDH1 is dysregulated and how IDH1 may affect cell transformation and tumor promotion during early stages of cancer development are unclear. We used a skin cell transformation model and mouse skin epidermal tissues to study the role of IDH1 in early skin tumorigenesis. Our studies demonstrate that both the tumor promoter TPA and UVC irradiation decreased expression and activity levels of IDH1, not IDH2, in the tumor promotable JB6 P+ cell model. Skin epidermal tissues treated with dimethylbenz[α]anthracene/TPA also showed decreases in IDH1 expression and activity. In non-promotable JB6 P-cells, IDH1 was upregulated upon TPA treatment, whereas IDH2 was maintained at similar levels with TPA treatment. Interestingly, IDH1 knockdown enhanced, whereas IDH1 overexpression suppressed, TPA-induced cell transformation. Finally, manganese superoxide dismutase overexpression suppressed tumor promoter induced decreases in IDH1 expression and mitochondrial respiration, while intracellular alpha-ketoglutarate levels were unchanged. These results suggest that decreased IDH1 expression in early stage skin tumorigenesis is highly correlated with tumor promotion. In addition, oxidative stress might contribute to IDH1 inactivation, because manganese superoxide dismutase, a mitochondrial antioxidant enzyme, blocked decreases in IDH1 expression and activity.
Subject(s)
Antioxidants/metabolism , Cell Transformation, Neoplastic/metabolism , Isocitrate Dehydrogenase/metabolism , Keratinocytes/metabolism , Papilloma/metabolism , Skin Neoplasms/metabolism , Skin/metabolism , Superoxide Dismutase/metabolism , Animals , Blotting, Western , Down-Regulation , Fluorescent Antibody Technique , Mice , Mice, Inbred DBA , Oxidative Stress , Oxygen Consumption , RNA, Small Interfering , Skin/pathology , TransfectionABSTRACT
Differentiated cells primarily metabolize glucose for energy via the tricarboxylic acid cycle and oxidative phosphorylation, but cancer cells thrive on a different mechanism to produce energy, characterized as the Warburg effect, which describes the increased dependence on aerobic glycolysis. The M2 isoform of pyruvate kinase (PKM2), which is responsible for catalyzing the final step of aerobic glycolysis, is highly expressed in cancer cells and may contribute to the Warburg effect. However, whether PKM2 plays a contributing role during early cancer development is unclear. In our studies, we have made an attempt to elucidate the effects of varying mitochondrial respiration substrates on skin cell transformation and expression of PKM2. Tumorigenicity in murine skin epidermal JB6 P+ (promotable) cells was measured in a soft agar assay using 12-O-tetradecanoylphorbol-13-acetate (TPA) as a tumor promoter. We observed a significant reduction in cell transformation upon pretreatment with the mitochondrial respiration substrate succinate or malate/pyruvate. We observed that increased expression and activity of PKM2 in TPA-treated JB6 P+ cells and pretreatment with succinate or malate/pyruvate suppressed the effects. In addition, TPA treatment also induced PKM2 whereas PKM1 expression was suppressed in mouse skin epidermal tissues in vivo. In comparison with JB6 P+ cells, the nonpromotable JB6 P- cells showed no increase in PKM2 expression or activity upon TPA treatment. Knockdown of PKM2 using a siRNA approach significantly reduced skin cell transformation. Thus, our results suggest that PKM2 activation could be an early event and play a contributing role in skin tumorigenesis.
