Rai P, Young JJ, Burton DG, Giribaldi MG, Onder TT, Weinberg RA.. Enhanced elimination of oxidized guanine nucleotides inhibits oncogenic RAS-induced DNA damage and premature senescence. Oncogene 30: 1489-1496

Division of Gerontology and Geriatric Medicine, Department of Medicine, University of Miami, Miller School of Medicine, Miami, FL 33136, USA.
Oncogene (Impact Factor: 8.46). 11/2010; 30(12):1489-96. DOI: 10.1038/onc.2010.520
Source: PubMed


Approximately 20% of tumors contain activating mutations in the RAS family of oncogenes. As tumors progress to higher grades of malignancy, the expression of oncogenic RAS has been reported to increase, leading to an oncogene-induced senescence (OIS) response. Evasion of this senescence barrier is a hallmark of advanced tumors indicating that OIS serves a critical tumor-suppressive function. Induction of OIS has been attributed to either RAS-mediated production of reactive oxygen species (ROS) or to induction of a DNA damage response (DDR). However, functional links between these two processes in triggering the senescent phenotype have not been explicitly described. Our previous work has shown that, in cultured untransformed cells, preventing elimination of oxidized guanine deoxyribonucleotides, which was achieved by suppressing expression of the cellular 8-oxo-dGTPase, human MutT homolog 1 (MTH1), sufficed to induce a DDR as well as premature senescence. Here, we demonstrate that overexpression of MTH1 can prevent the oncogenic H-RAS-induced DDR and attendant premature senescence, although it does not affect the observed elevation in ROS levels produced by RAS oncoprotein expression. Conversely, we find that loss of MTH1 preferentially induces an in vitro proliferation defect in tumorigenic cells overexpressing oncogenic RAS. These results indicate that the guanine nucleotide pool is a critical target for intracellular ROS produced by oncogenic RAS and that RAS-transformed cells require robust MTH1 expression to proliferate.

  • Source
    • "Suppression of the mutator phenotype in mismatch repairdefective colorectal cancer cells has been achieved by overexpressing MTH1 [26]. Reversal of RAS-induced senescence by reducing the level of DNA damage has also been demonstrated [27]. MTH1 overexpression promoted longevity, reduced anxiety [28], and protected from neurodegeneration [29]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: MTH1 protein sanitizes the nucleotide pool so that oxidized 2'-deoxynucleoside triphosphates (dNTPs) cannot be used in DNA replication. Cancer cells require MTH1 to avoid incorporation of oxidized dNTPs into DNA that results in mutations and cell death. Inhibition of MTH1 eradicates cancer, validating MTH1 as an anticancer target. By overexpressing MTH1, cancer cells may mediate cancer growth and resist therapy. To date, there is unreliable evidence suggesting that MTH1 is increased in cancer cells, and available methods to measure MTH1 levels are indirect and semi-quantitative. Accurate measurement of MTH1 in disease-free tissues and malignant tumors of patients may be essential for determining if the protein is truly upregulated in cancers, and for the development and use of MTH1 inhibitors in cancer therapy. Here, we present a novel approach involving liquid chromatography-isotope-dilution tandem mass spectrometry to positively identify and accurately quantify MTH1 in human tissues. We produced full length (15)N-labeled MTH1 and used it as an internal standard for the measurements. Following trypsin digestion, seven tryptic peptides of both MTH1 and (15)N-MTH1 were identified by their full scan and product ion spectra. These peptides provided a statistically significant protein score that would unequivocally identify MTH1. Next, we identified and quantified MTH1 in human disease-free breast tissues and malignant breast tumors, and in four human cultured cell lines, three of which were cancer cells. Extreme expression of MTH1 in malignant breast tumors was observed, suggesting that cancer cells are addicted to MTH1 for their survival. The approach described is expected to be applicable to the measurement of MTH1 levels in malignant tumors vs. surrounding disease-free tissues in cancer patients. This attribute may help develop novel treatment strategies and MTH1 inhibitors as potential drugs, and guide therapies. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jun 2015 · DNA repair
  • Source
    • "The fraction of cells in the S phase started to decline 6e8 days post-induction (Figure 1B), presumably reflecting initial phases of the Ras oncogeneinduced senescence (OIS) program (Bartkova et al., 2006). It is known that cellular senescence induced by Ras is accompanied by increased DNA damage response (Bartkova et al., 2006; Di Micco et al., 2006; Rai et al., 2011) and that such DNA damaging impact is preserved even in secondary/bystander senescence caused by Ras (Hubackova et al., 2012). Using a high-throughput microscopy analysis, we found gradual accumulation of the DNA damage signaling marker gH2AX from the day 6 of Ras overexpression (Figure 1C). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Both Myc and Ras oncogenes impact cellular metabolism, deregulate redox homeostasis and trigger DNA replication stress (RS) that compromises genomic integrity. However, how are such oncogene-induced effects evoked and temporally related, to what extent are these kinetic parameters shared by Myc and Ras, and how are these cellular changes linked with oncogene-induced cellular senescence in different cell context(s) remain poorly understood. Here, we addressed the above-mentioned open questions by multifaceted comparative analyses of human cellular models with inducible expression of c-Myc and H-RasV12 (Ras), two commonly deregulated oncoproteins operating in a functionally connected signaling network. Our study of DNA replication parameters using the DNA fiber approach and time-course assessment of perturbations in glycolytic flux, oxygen consumption and production of reactive oxygen species (ROS) revealed the following results. First, overabundance of nuclear Myc triggered RS promptly, already after one day of Myc induction, causing slow replication fork progression and fork asymmetry, even before any metabolic changes occurred. In contrast, Ras overexpression initially induced a burst of cell proliferation and increased the speed of replication fork progression. However, after several days of induction Ras caused bioenergetic metabolic changes that correlated with slower DNA replication fork progression and the ensuing cell cycle arrest, gradually leading to senescence. Second, the observed oncogene-induced RS and metabolic alterations were cell-type/context dependent, as shown by comparative analyses of normal human BJ fibroblasts versus U2-OS sarcoma cells. Third, the energy metabolic reprogramming triggered by Ras was more robust compared to impact of Myc. Fourth, the detected oncogene-induced oxidative stress was due to ROS (superoxide) of non-mitochondrial origin and mitochondrial OXPHOS was reduced (Crabtree effect). Overall, our study provides novel insights into oncogene-evoked metabolic reprogramming, replication and oxidative stress, with implications for mechanisms of tumorigenesis and potential targeting of oncogene addiction.
    Full-text · Article · Nov 2014 · Molecular Oncology
  • Source
    • "Despite the efficient reduction in ATM levels and effective suppression of IL-6 (Supplemental Figure S2C), lamin B1 still declined markedly, similar to its behavior in normal senescent cells (Figure 2F). Finally, reactive oxygen species (ROS) are elevated in senescent cells, and ROS signaling is believed to mediate the senescence growth arrest (Lu and Finkel, 2008; Jun and Lau, 2010; Passos et al., 2010; Rai et al., 2011). We inhibited ROS by continuously treating cells with 10 mM N-acetyl cysteine (NAC), starting 2 d before inducing senescence by XRA or MKK6EE expression. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cellular senescence is a potent tumor-suppressive mechanism that arrests cell proliferation and has been linked to aging. However, studies of senescence have been impeded by the lack of simple, exclusive biomarkers of the senescent state. Senescent cells develop characteristic morphological changes, which include enlarged and often irregular nuclei and chromatin reorganization. Because alterations to the nuclear lamina can affect both nuclear morphology and gene expression, we examined the nuclear lamina of senescent cells. We show here than lamin B1 is lost from primary human and murine cell strains when they are induced to senesce by DNA damage, replicative exhaustion, or oncogene expression. Lamin B1 loss did not depend on the p38 mitogen-activated protein kinase, nuclear factor-κB, ataxia telangiectasia-mutated kinase, or reactive oxygen species signaling pathways, which are positive regulators of senescent phenotypes. However, activation of either the p53 or pRB tumor suppressor pathway was sufficient to induce lamin B1 loss. Lamin B1 declined at the mRNA level via a decrease in mRNA stability rather than by the caspase-mediated degradation seen during apoptosis. Last, lamin B1 protein and mRNA declined in mouse tissue after senescence was induced by irradiation. Our findings suggest that lamin B1 loss can serve as biomarker of senescence both in culture and in vivo.
    Full-text · Article · Apr 2012 · Molecular biology of the cell
Show more