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An NF-?B pathway-mediated positive feedback loop amplifies Ras activity to pathological levels in mice.

ABSTRACT: Genetic mutations that give rise to active mutant forms of Ras are oncogenic and found in several types of tumor. However, such mutations are not clear biomarkers for disease, since they are frequently detected in healthy individuals. Instead, it has become clear that elevated levels of Ras activity are critical for Ras-induced tumorigenesis. However, the mechanisms underlying the production of pathological levels of Ras activity are unclear. Here, we show that in the presence of oncogenic Ras, inflammatory stimuli initiate a positive feedback loop involving NF-?B that further amplifies Ras activity to pathological levels. Stimulation of Ras signaling by typical inflammatory stimuli was transient and had no long-term sequelae in wild-type mice. In contrast, these stimuli generated prolonged Ras signaling and led to chronic inflammation and precancerous pancreatic lesions (PanINs) in mice expressing physiological levels of oncogenic K-Ras. These effects of inflammatory stimuli were disrupted by deletion of inhibitor of NF-?B kinase 2 (IKK2) or inhibition of Cox-2. Likewise, expression of active IKK2 or Cox-2 or treatment with LPS generated chronic inflammation and PanINs only in mice expressing oncogenic K-Ras. The data support the hypothesis that in the presence of oncogenic Ras, inflammatory stimuli trigger an NF-?B-mediated positive feedback mechanism involving Cox-2 that amplifies Ras activity to pathological levels. Because a large proportion of the adult human population possesses Ras mutations in tissues including colon, pancreas, and lung, disruption of this positive feedback loop may be an important strategy for cancer prevention.

SUBMITTER: Daniluk J

PROVIDER: S-EPMC3314459 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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An NF-κB pathway-mediated positive feedback loop amplifies Ras activity to pathological levels in mice.

Daniluk Jaroslaw J Liu Yan Y Deng Defeng D Chu Jun J Huang Haojie H Gaiser Sebastian S Cruz-Monserrate Zobeida Z Wang Huamin H Ji Baoan B Logsdon Craig D CD

The Journal of clinical investigation 20120312 4

Genetic mutations that give rise to active mutant forms of Ras are oncogenic and found in several types of tumor. However, such mutations are not clear biomarkers for disease, since they are frequently detected in healthy individuals. Instead, it has become clear that elevated levels of Ras activity are critical for Ras-induced tumorigenesis. However, the mechanisms underlying the production of pathological levels of Ras activity are unclear. Here, we show that in the presence of oncogenic Ras, ...[more]

PMID: 22406536

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Project description:BackgroundCancer cells avidly consume glucose and convert it to lactate, resulting in a low pyruvate level. This phenomenon is known as the Warburg effect, and is important for cell proliferation. Although cMyc has often been described as an oncoprotein that preferentially contributes to the Warburg effect and tumor proliferation, mechanisms of action remain unclear. Histone deacetylase 3 (HDAC3) regulates gene expression by removing acetyl groups from lysine residues, as well as has an oncogenic role in apoptosis and contributes to the proliferation of many cancer cells including cholangiocarcinoma (CCA). HDAC inhibitors display antitumor activity in many cancer cell lines. Cancer cells maintain low levels of pyruvate to prevent inhibition of HDAC but the mechanisms remain elusive. The purpose of our study was to explore the role of cMyc in regulating pyruvate metabolism, as well as to investigate whether the inhibitory effect of pyruvate on HDAC3 could hold promise in the treatment of cancer cells.MethodsWe studied pyruvate levels in CCA cell lines using metabolite analysis, and analyzed the relationship of pyruvate levels and cell proliferation with cell viability analysis. We cultivated CCA cell lines with high or low levels of pyruvate, and then analyzed the protein levels of HDAC3 and apoptotic markers via Western Blotting. We then explored the reasons of low levels of pyruvate by using seahorse analysis and 13C6 metabolites tracing analysis, and then confirmed the results using patient tissue protein samples through Western Blotting. Bioinformatics analysis and transfection assay were used to confirm the upstream target of the low levels of pyruvate status in CCA. The regulation of cMyc by HDAC3 was studied through immunoprecipitation and Western Blotting.ResultsWe confirmed downregulated pyruvate levels in CCA, and defined that high pyruvate levels correlated with reduced cell proliferation levels. Downregulated pyruvate levels decreased the inhibition to HDAC3 and consequently protected CCA cells from apoptosis. Synergistically upregulated LDHA, PKM2 levels resulted in low levels of pyruvate, as well as poor patient survival. We also found that low levels of pyruvate contributed to proliferation of CCA cells and confirmed that the upstream target is cMyc. Conversely, high activity of HDAC3 stabilized cMyc protein by preferential deacetylating cMyc at K323 site, which further contributed to the low pyruvate levels. Finally, this creates a positive feedback loop that maintained the low levels of pyruvate and promoted CCA proliferation.ConclusionsCollectively, our findings identify a role for promoting the low pyruvate levels regulated by c-Myc, and its dynamic acetylation in cancer cell proliferation. These targets, as markers for predicting tumor proliferation in patients undergoing clinical treatments, could pave the way towards personalized therapies.

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An NF-?B pathway-mediated positive feedback loop amplifies Ras activity to pathological levels in mice.

Publications

An NF-κB pathway-mediated positive feedback loop amplifies Ras activity to pathological levels in mice.

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