Up-regulation and Genetic Instability Oncogenic Ras-Induced Reactive Oxygen Species Repression of Sestrin Family Genes Contributes to

Oncogenic mutations within RAS genes and inactivation of p53 are the most common events in cancer. Earlier, we reported that activated Ras contributes to chromosome instability, especially in p53-deficient cells. Here we show that an increase in intracellular reactive oxygen species (ROS) and oxidative DNA damage represents a major mechanism of Ras-induced mutagenesis. Introduction of oncogenic Hor N-Ras caused elevated intracellular ROS, accumulation of 8-oxo-2¶-deoxyguanosine, and increased number of chromosome breaks in mitotic cells, which were prevented by antioxidant N-acetyl-L-cysteine. By using Ras mutants that selectively activate either of the three major targets of Ras (Raf, RalGDS, and phosphatidylinositol-3-kinase) as well as dominant-negative Rac1 and RalA mutants and inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinases kinase-1 and p38 MAPKs, we have shown that several Ras effectors independently mediate ROS up-regulation. Introduction of oncogenic RAS resulted in repression of transcription from sestrin family genes SESN1 and SESN3 , which encode antioxidant modulators of peroxiredoxins. Inhibition of mRNAs from these genes in control cells by RNA interference substantially increased ROS levels and mutagenesis. Ectopic expression of SESN1 and SESN3 from lentiviral constructs interfered with Rasinduced ROS increase, suggesting their important contribution to the effect. The stability of Ras-induced increase in ROS was dependent on a p53 function: in the p53-positive cells displaying activation of p53 in response to Ras, only transient (4–7 days) elevation of ROS was observed, whereas in the p53-deficient cells the up-regulation was permanent. The reversion to normal ROS levels in the Ras-expressing p53-positive cells correlated with up-regulation of p53responsive genes, including reactivation of SESN1 gene. Thus, changes in expression of sestrins can represent an important determinant of genetic instability in neoplastic cells showing simultaneous dysfunctions of Ras and p53. [Cancer Res 2007;67(10):4671–8] Introduction The proteins of the Ras family (H-, K-, and N-Ras) function as key regulators of signal transduction pathways that control cell proliferation, survival, migration, and differentiation (1–5). Various extracellular signals reaching cell-surface receptors stimulate the conversion of Ras proteins from the inactive GDP-bound to the active GTP-bound form. In the GTP-bound form, Ras stimulates downstream effectors, which, in turn, affect activities of numerous proteins, including large group of transcription factors. The biological effects of activated Ras proteins are mediated through several effectors that include Raf serine/threonine kinases, phosphatidylinositol-3-kinases (PI3K), and RalGDS, guanine nucleotide exchange factor (GEF) for small GTPases RalA and RalB (2–6). Mutations at residues 12, 13, or 61 that constitutively activate Ras proteins are found in 95% to 98% of pancreatic cancers and 25% to 40% of many other tumor types (1, 7, 8). Substantial experimental data indicate that aberrant Ras expression plays a critical role in oncogenesis causing stimulation of cell proliferation, angiogenesis, inhibition of apoptosis, and increased cell motility (i.e., features that are responsible for tumor growth, invasion, and metastasis; refs. 2–5). In addition to these effects, the expression of Ras oncoproteins causes genetic instability (9–11), a feature that is considered as an engine for steadfast tumor progression (12–14). We have previously found that the mutagenic effect of Ras is especially prominent in p53-deficient cells (11) and can be connected with both attenuation of DNA damage cell cycle checkpoints and up-regulation of reactive oxygen species (ROS; refs. 15, 16). In fact, expression of Ras oncoproteins increases ROS content (15, 17–20) and interferes with DNA damage–induced arrest in G1 and G2 (11, 16), although the mechanisms of these effects are poorly understood. In this article, we present the data showing a critical role of ROS up-regulation in Ras-induced mutagenesis. We found that functional p53 is capable of counteracting the Ras-induced increase in ROS levels. We also propose a novel mechanism by which oncogenic Ras up-regulates ROS and compromises genetic stability, which is connected to transcriptional repression of sestrin family genes. The products of these genes participate in antioxidant defense by modulating regeneration of peroxiredoxins (21). The presented results highlight the importance of antioxidant mechanisms in controlling genetic stability. Materials and Methods DNA constructs. The following previously developed and described constructs were used: pBabe-puro/Ras retroviral vector expressing activated human N-Ras D13 mutant (11, 22); the retroviral vector pLXSN-neo containing human activated H-Ras V12 and its restricted effector specificity mutants V12S35, V12G37, and V12C40 (ref. 23; provided by J. Downward, Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). B.P. Kopnin and P.M. Chumakov contributed equally to this work. Requests for reprints: Boris P. Kopnin, Engelhardt Institute of Molecular Biology, 119991 Moscow, Russia. Phone: 7-095-324-1739; E-mail: [email protected] or Peter M. Chumakov, Department of Molecular Genetics, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195. Phone: 216-444-9540; E-mail: [email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-2466 www.aacrjournals.org 4671 Cancer Res 2007; 67: (10). May 15, 2007 Research Article American Association for Cancer Research Copyright © 2007 on February 23, 2013 cancerres.aacrjournals.org Downloaded from DOI:10.1158/0008-5472.CAN-06-2466