| In1920s, Otto Warburg observed that most cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol even in the presence of sufficient oxygen, whereas most of pyruvate is oxidized in the mitochondria in normal cells, this phenomenon was known as Warburg effect (Aerobic glycolysis). Tumor cells rely on this altered metabolic program to meet the metabolites and energy needs of fast proliferation. Pyruvate kinase M2(PKM2) is necessary for the re-programming and maintenance of cancer metabolism. Meanwhile, PKM2could regulate activities of multiple cancer-related transcriptional factors. Knockdown of PKM2holds the promise of disrupting metabolic pathways and signaling pathways that tumor rely on, suppressing tumor growth and survival. During the past several years, several research groups had adapted RNA interference (RNAi) technology to knockdown the expression of PKM2for the aim of killing tumors, while the results are controversy. We think that a fully understanding of the signaling transduction events upon PKM2knockdown is necessary for the application of PKM2knockdown in treating cancers. In present work, we carefully analyzed the effect of PKM2knockdown on cell signaling pathways.First, using a lentiviral vector based RNAi system, we successfully depleted PKM2in H1299and A549cells. By protein dilution and Western Blot, we showed that the knockdown efficiency is higher than95%. We didn’t observe significant difference in the proliferation rate between wild type cells and PKM2knockdown cells; however, cell morphology was quite different between whild type cells and PKM2knockdown cellsWe next analyzed whether depletion of PKM2affects the expression of other glycolysis related enzymes. We found that in PKM2knockdown cells Hexokinase I and Hexokinase II expression levels were decreased, while Pyruvate dehydrogenase and CoxIV expression levels were increased. At the same time, global protein post-translational modifications, such as tyrosine phosphorylation, serine/threonine phosphorylation, acetylation and O-linked GlcNAcylation, all underwent significant changes in PKM2knockdown cells.To further investigate possibly changed signaling pathways in PKM knockdown cells, we tested the activation status of PI3K-AKT signaling pathway. Phosphorylation of AKT was significantly increased in PKM2knockdown cells, while the levels of total AKT was not changed. PKM2deprivation induced AKT phosphorylation can lead to the phosphorylation of AKT downstream targets GSK3(3and TSC2. We also found that this observation is not limited to H1299cells, as knockdown of PKM2in multiple cancer cells all leads to AKT activation.We sought to determine the mechanism by which AKT phosphorylation was up-regulated in PKM2knockdown cells. It has been known for a long while that inhibition of glycolysis will lead to AKT activation. In our experimental system, wild type cells response to glucose starvation by up-regulating AKT phosphorylation, but it doesn’t happen to PKM2knockdown cells. When PKM2expression was restored in PKM2knockdown cells, it facilitates AKT phosphorylation in PKM2knockdown cells to response to glucose starvation. We also test the effect of2-deoxy-D-glucose (2-DG), a glycolysis inhibitor, on Akt phosphorylation. Similar to glucose deprivation,2-DG treatment also led to increased AKT phosphorylation in control cells but not in PKM2knockdon cells. We think that PKM2knockdown suppresses glycolysis in the cells and leads to AKT activation as a stress response.We then evaluated sensitivity of AKT phosphorylation to different metabolic and signaling pathway inhibitors in PKM2knockdown cells and found that AKT phosphorylation is sensitive to both PI3K and Akt inhibitors. Inhibiton of PI3K-AKT signaling pathway in PKM2knockdown cells leads to significant growth inhibition and induction of apoptosis. Thus, PKM2knockdown cells reley on activation of PI3K-AKT signaling pathway for their survival. |
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