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

Research Article

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

Pavel B. Kopnin,1,2 Larissa S. Agapova,3 Boris P. Kopnin,3 and Peter M. Chumakov1,4

1Engelhardt Institute of Molecular Biology; 2University of Oslo, Centre for Medical Studies in Russia; 3Institute of Carcinogenesis, Russian Blokhin Cancer Research Center, Moscow, Russia; and 4Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio

Abstract Introduction Oncogenic mutations within RAS genes and inactivation of The proteins of the Ras family (H-, K-, and N-Ras) function as key p53 are the most common events in cancer. Earlier, we regulators of signal transduction pathways that control cell reported that activated Ras contributes to chromosome proliferation, survival, migration, and differentiation (1–5). Various instability, especially in p53-deficient cells. Here we show extracellular signals reaching cell-surface receptors stimulate the that an increase in intracellular reactive oxygen species (ROS) conversion of Ras proteins from the inactive GDP-bound to the and oxidative DNA damage represents a major mechanism of active GTP-bound form. In the GTP-bound form, Ras stimulates Ras-induced mutagenesis. Introduction of oncogenic H- or downstream effectors, which, in turn, affect activities of numerous N-Ras caused elevated intracellular ROS, accumulation proteins, including large group of transcription factors. The of 8-oxo-2¶-deoxyguanosine, and increased number of biological effects of activated Ras proteins are mediated through chromosome breaks in mitotic cells, which were prevented several effectors that include Raf serine/threonine kinases, by antioxidant N-acetyl-L-cysteine. By using Ras mutants that phosphatidylinositol-3-kinases (PI3K), and RalGDS, guanine nucle- selectively activate either of the three major targets of Ras otide exchange factor (GEF) for small GTPases RalA and RalB (Raf, RalGDS, and phosphatidylinositol-3-kinase) as well as (2–6). Mutations at residues 12, 13, or 61 that constitutively activate dominant-negative Rac1 and RalA mutants and inhibitors of Ras proteins are found in 95% to 98% of pancreatic cancers and mitogen-activated protein kinase (MAPK)/extracellular 25% to 40% of many other tumor types (1, 7, 8). Substantial signal–regulated kinases kinase-1 and p38 MAPKs, we have experimental data indicate that aberrant Ras expression plays a shown that several Ras effectors independently mediate critical role in oncogenesis causing stimulation of cell proliferation, ROS up-regulation. Introduction of oncogenic RAS resulted angiogenesis, inhibition of apoptosis, and increased cell motility in repression of transcription from sestrin family genes (i.e., features that are responsible for tumor growth, invasion, and SESN1 and SESN3, which encode antioxidant modulators of metastasis; refs. 2–5). peroxiredoxins. Inhibition of mRNAs from these genes in In addition to these effects, the expression of Ras oncoproteins control cells by RNA interference substantially increased causes genetic instability (9–11), a feature that is considered as an ROS levels and mutagenesis. Ectopic expression of SESN1 engine for steadfast tumor progression (12–14). We have previously and SESN3 from lentiviral constructs interfered with Ras- found that the mutagenic effect of Ras is especially prominent in induced ROS increase, suggesting their important contribu- p53-deficient cells (11) and can be connected with both attenuation tion to the effect. The stability of Ras-induced increase in of DNA damage cell cycle checkpoints and up-regulation of ROS was dependent on a p53 function: in the p53-positive reactive oxygen species (ROS; refs. 15, 16). In fact, expression of Ras cells displaying activation of p53 in response to Ras, only oncoproteins increases ROS content (15, 17–20) and interferes with transient (4–7 days) elevation of ROS was observed, whereas DNA damage–induced arrest in G1 and G2 (11, 16), although the in the p53-deficient cells the up-regulation was permanent. mechanisms of these effects are poorly understood. The reversion to normal ROS levels in the Ras-expressing In this article, we present the data showing a critical role of ROS p53-positive cells correlated with up-regulation of p53- up-regulation in Ras-induced mutagenesis. We found that func- responsive genes, including reactivation of SESN1 gene. Thus, tional p53 is capable of counteracting the Ras-induced increase in changesinexpressionofsestrinscanrepresentanimportant ROS levels. We also propose a novel mechanism by which determinant of genetic instability in neoplastic cells showing oncogenic Ras up-regulates ROS and compromises genetic stability, simultaneous dysfunctions of Ras and p53. [Cancer Res which is connected to transcriptional repression of sestrin family 2007;67(10):4671–8] 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.

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. Materials and Methods 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 DNA constructs. The following previously developed and described M. Chumakov, Department of Molecular Genetics, Lerner Research Institute, The constructs were used: pBabe-puro/Ras retroviral vector expressing activated Cleveland Clinic Foundation, Cleveland, OH 44195. Phone: 216-444-9540; E-mail: human N-Ras D13 mutant (11, 22); the retroviral vector pLXSN-neo [email protected]. I2007 American Association for Cancer Research. containing human activated H-Ras V12 and its restricted effector specificity doi:10.1158/0008-5472.CAN-06-2466 mutants V12S35, V12G37, and V12C40 (ref. 23; provided by J. Downward, www.aacrjournals.org 4671 Cancer Res 2007; 67: (10). May 15, 2007

Cell cultures. Human p53-deficient MDAH041 immortalized fibroblasts derived from a Li-Fraumeni syndrome patient (25) and rat immortalized embryonic fibroblasts REF52 and Rat1 were used. Their derivatives with tetracycline-repressible expression of activated N-RasD13 mutant (MDAH041/ tet-Ras, REF52/tet-Ras, and Rat1/tet-Ras cell lines, respectively) were described by us earlier (11, 22). The cell sublines with constitutive expression of various H-Ras, N-Ras, RalA, and Rac1 mutants were previously described (11, 16). Briefly, the retroviral DNA constructs were transfected into Phoenix-ampho packaging cells using LipofectAMINE PLUS reagent (Invitrogen). Virus- containing supernatants collected 24 to 8 h after transfection were used to infect recipient cells in the presence of polybrene (8 Ag/mL). Infected cell cultures were selected in medium containing genticin (Invitrogen; 0.5 mg/mL for 14–20 days) or puromycin (Sigma; 1 Ag/mL for 5–6 days). High-titer stocks of recombinant lentiviruses pseudotyped with VSV-G protein were produced by cotransfection into 293T cells of the pLV or pLSLP constructs together with the pCMVy8.2R and pVSV-G packaging plasmids. Virus-containing supernatants were collected 24 to 48 h after transfection and used for infection of recipient cells. All the cells were cultured in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin. The MDAH041/tet-Ras, REF52/tet-Ras, and Rat1/tet-Ras cells were permanently maintained in medium supplemented with 1 Ag/mL of tetracycline (Sigma), and the expression of RAS oncogene was induced by removal of tetracycline from culture medium 2 to 15 days before the experiments. All experiments with the MDAH041 cell sublines sustaining constitutive expression of various Ras, Ral, or Rac1 mutants were done 7 to 20 days after retrovirus vector–mediated gene transfer. Measurement of intracellular ROS. Intracellular ROS was measured as described earlier (26). Briefly, the cells were incubated with 2¶-7¶ dichloro- dihydrofluorescein diacetate (DCF-DA; Molecular Probes) at 5 Amol/L for 30 min, washed with PBS, trypsinized, collected in 1 mL of PBS, transferred to polystyrene tubes with cell-strainer caps (Falcon), and subjected to fluorescence-activated cell sorting (FACS; Becton Dickinson FACScan) using CellQuest 3.2 (Becton Dickinson) software for acquisition and analysis. DNA oxidation. DNA oxidation was assessed by measuring 8-oxo-2¶- Figure 1. Effect of expression of activated N-RasD13 on ROS levels and deoxyguanosine content with Biotrin OxyDNA Kit (Biotrin). For the assay, oxidized DNA content in human and rat cells. A, influence of constitutive 6 expression of pBabe-puro/Ras construct (MDAH041/Ras cells) or incubation of 10 cells were processed for FACS analysis according to protocol provided MDAH041/tet-Ras, REF52/tet-Ras, and Rat1/tet-Ras cells in tetracycline-free by the manufacturer. medium for 5 d on expression of N-Ras protein (Western blot analysis was done Chromosome analysis. Chromosomal slides were prepared by routine 4 as described in Materials and Methods). B, distribution of 10 cells according to methods. To determine the frequency of chromosomal breaks, 50 to 100 the intensity of DCF-DA fluorescence in control and Ras-expressing cultures. Result of one of typical experiments. C, indices of total DCF-DA fluorescence of metaphases were karyotyped in each case. The unrepaired chromatid and 104 cells. Summarized data of two to three independent experiments. D, indices chromosome breaks and gaps were scored. of total fluorescence of 104 cells stained with FITC-conjugated antibodies to Detection of mRNA for sestrin genes by reverse transcription-PCR. ¶ 8-oxo-2 -deoxyguanosine (8-oxo-DG). Summarized result of two experiments. Total mRNA was isolated with SV Total RNA Isolation System (Promega) according to the manufacturer’s protocols. Reverse transcription-PCR (RT- PCR) analysis was done as described (24). To monitor expression of Cancer Research UK, London, United Kingdom); pLXSN-neo/RalA-V23 with corresponding genes by RT-PCR e, we used the following primers: for hSESN1 constitutively active V23 RalA mutant and lentiviral pLV-CMV/RalA-N28 (all isoforms), forward primer 5¶-CTTCTGGAGGCAGTTCAAGC-3¶ and reverse expressing dominant-negative N28 RalA mutant (16); and lentiviral pLSLP primer 5¶-TGAATGGCAGCCTGTCTTCAC-3¶;forSESN1 T1 isoform, forward vectors bearing short hairpin RNAs (shRNA) against p53 or human SESN1 5¶-CGGATGGGTTGAATAAGCTACT-3¶ and common (T1, T2, and T3 isoforms) and SESN2 genes (21, 24). Complementary 60-bp hairpin oligonucleotides reverse 5¶-CCAATGTAGTGACGATAATGTAGG-3¶ primer; for SESN1 T2 containing 19-nucleotide regions corresponding to the human SESN3 gene isoform, forward 5¶-CGACCAGGACGAGGAACTT-3¶;forSESN1 T3 isoform, (5¶-GACGAGGAGAAGAGCATTT-3¶ and 5¶-CCAGAGAGAGATCCAGAAA-3¶) forward 5¶-CCTGCAAGCTCTATGACGTTATG-3¶;forSESN2,forward were designed according to siRNA-scale algorithm5 and cloned into pLSLP 5¶-CAAGCTCGGAATTAATGTGCC-3¶ and reverse 5¶-CTCACACCATTAAG- vector for shRNA, as described earlier (24). The pMSCV-puro retroviral CATGGAG-3¶; and for SESN3, forward 5¶-GTTCACTGTATGTTTGGAAT- vectors expressing activated V23 RalB and dominant-negative N17 Rac1 CAGG-3¶ and reverse 5¶-GGGTGATACTTCAGGTCAAATG-3¶.Forratgenes, mutants were obtained from Dr. I. Zborovskaya (Russian Cancer Research we used the following primers: for Sesn1,forward5¶-GGACGAGGAACTTG- Center, Moscow, Russia). For expression of human SESN1 (T2 and T3 GAATCA-3¶ and reverse 5¶-CCCTCGATGTGTTCTTTGGT-3¶;forSesn2, isoforms), SESN2, and SESN3 genes, the appropriate cDNA was introduced forward 5¶-ACAGGTTTCCTCCGACACAC-3¶ and reverse 5¶- into cells using lentiviral expression vector pLV-CMV, as described CTCTGGAGTCTGCCTGTTCC-3¶; and for Sesn3, forward 5¶-CCTGGACAACAT- earlier (24). CACACAGG-3¶ and reverse 5¶-ACTAGCTCAGGCAAGGACCA-3¶.Todetectin human and rat cells a-tubulin mRNA, we used the same forward (5¶-GTTGGTCTGGAATTCTGTCAG-3¶)andreverse(5¶-AAGAAGTCCAAGCTG- GAGTTC-3¶)primers. Western blot analysis. Whole-cell extracts were lysed in ice-cold 5 http://gesteland.genetics.utah.edu/siRNA_scales/index.html radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.4),

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150 mmol/L NaCl, 1% deoxycholate-Na, 1% NP40, 0.1% SDS, 100 mmol/L suggesting that the increase in intracellular ROS can boost phenylmethylsulfonyl fluoride, 1 mmol/L pepstatin A, and 1 mmol/L E64]. chromosome instability in cells expressing oncogenic Ras. Protein concentration in the extracts was determined with a protein assay Several Ras effectors are responsible for ROS up-regulation system (Bio-Rad). Twenty micrograms of protein were separated on a and genetic instability. To assess the role of different Ras 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride effectors in ROS regulation, we introduced into MDAH041, REF52, membrane (Amersham GE Healthcare). The membranes were probed with antibodies specific to p21Ras (OP21, Calbiochem), p53 (PAb421, Calbio- and Rat1 cells the restricted effector specificity H-Ras mutants chem), p21Cip1/Waf1 (F-5, Santa Cruz Biotechnology), and p44/p42 V12S35, V12G37, and V12C40, which selectively activate Raf, extracellular signal–regulated kinases (ERK) phosphorylated at Thr202/ RalGDS, or PI3K, respectively (23). In all tested cell lines, expression Tyr204 (E10, Cell Signaling Technology). Membranes were treated with of each of these mutants caused notable increase in ROS levels, secondary antimouse-HRP or antirabbit-HRP antibodies (Amersham GE although its magnitude was lower than that observed after Healthcare). Filters were developed with enhanced chemiluminescence introduction of full-value activated Ras V12 (Fig. 2A, B, and E). reagents (Amersham GE Healthcare) according to the manufacturer’s Of note, in the cells expressing the H-Ras mutants, the frequency of protocol. chromosome breaks directly correlated with the ROS levels Ral activity assay. Cells were lysed in RAB buffer (Upstate Biotechnol- (Fig. 2A). This observation highlights a major role of elevated ogy) and the amount of GTP-bound Ral was determined using Ral ROS in Ras-induced genetic instability. Activation Assay Kit (Upstate Biotechnology) according to the manufacturer’s protocols. The effect of V12C40 mutant can be connected with activation of the PI3K-Rac1-NADPH-oxidase pathway, which is known to be responsible for ROS generation in cells with activated Ras (17, 18, Results 20). Accordingly, we have found that inactivation of Rac1 function Ras-induced chromosome breaks are associated with with dominant-negative Rac1 mutant caused a decrease of ROS elevated intracellular ROS and enhanced DNA oxidation. To levels in the cell lines expressing activated RasV12 (Fig. 2C). estimate contribution of increased intracellular ROS in Ras- Tentatively, up-regulation of ROS in response to the mutant Ras induced genomic instability, we used human MDAH041 and rat V12G37 can be mediated through activation of the Ras-RalGDS- REF52 and Rat1 cell lines. Constitutive or conditional tetracycline- RalA pathway. We found that in Rat1 cells, the constitutively regulated expression of activated Ras (H-RasV12 and N-RasD13) active RalA mutant V23 (but not the RalB mutant V23) caused coincided with increased intracellular ROS levels (DCF fluores- an increase in ROS level similar to that induced by the mutant cence), accumulation of the DNA oxidation product 8-oxo-2¶- H-Ras V12G37, whereas dominant-negative RalA mutant N28 led deoxyguanosine, and elevated frequency of chromosome breaks in to decreased ROS in the cells expressing activated Ras (Fig. 2E). mitoses (Fig. 1; Table 1). Treatment with 2 to 10 mmol/L of However, contribution of this pathway was less prominent in antioxidant N-acetyl-L-cysteine (NAC) for 12 to 24 h did not human MDAH041 cells showing a modest effect of mutant H-Ras influence the level of Ras protein (Supplementary Fig. S1) but V12G37 compared with that in the Rat1 cells. In the MDAH041 abolished the increase in intracellular ROS and led to almost cells, the constitutively active or dominant-negative RalA mutants complete attenuation of Ras-induced mutagenesis (Table 1), caused no notable changes in ROS levels, although both mutants

Table 1. Effects of Ras expression and NAC treatment on ROS levels and frequency of chromosome breaks in dividing cells

Cell line Exogenous Ras expression NAC DCF fluorescence Chromosome breaks

% Metaphases Breaks per metaphase

MDAH041 - - 57 F 933F 1.2 0.67 F 0.05 +52F 817F 1.0 0.36 F 0.04 MDAH041/Ras* + - 105 F 12 58 F 1.8 1.44 F 0.10 +65F 829F 3.0 0.72 F 0.05 c MDAH041/tet-Ras --65F 12 39 F 3.2 1.20 F 0.34 + - 125 F 18 63 F 4.0 2.30 F 0.52 c REF52/tet-Ras --24F 613F 1.4 0.16 F 0.02 +21F 510F 1.0 0.14 F 0.02 +-49F 822F 2.0 0.58 F 0.02 +28F 78F 0.1 0.08 F 0.01 c Rat1/tet-Ras --9F 413F 1.0 0.16 F 0.08 +7F 411F 1.0 0.13 F 0.02 +-66F 16 28 F 2.0 0.56 F 0.10 +25F 810F 1.0 0.16 F 0.04

*The MDAH041/Ras cells were analyzed 9 to 12 d after retroviral transfer of H-RasV12 (4–7 d after selection in the medium with puromycin). The data of two experiments are presented; in each experiment, 30 to 50 control and Ras-expressing metaphases were scored. cCell cultures were analyzed 5 to 7 d after removal of tetracycline from culture medium (REF52/tet-Ras cells were studied on the 5th day, i.e. before the development of Ras-induced cell cycle arrest; ref. 22). For each cell line, the data of two experiments are presented; in each experiment, 25 to 50 metaphases were scored.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. Cancer Research induced corresponding alterations in activity of RalA, as deter- for SESN3 gene; SESN2, on the contrary, showed minor repression mined by measurement of its GTP-bound forms (Fig. 2D). in human MDAH041 cells and no changes in rat REF52 and Rat1 Role of sestrins in Ras-induced ROS up-regulation and cells (Figs. 2A–B and 3). In the human MDAH041 cells, among mutagenesis. Recently, we identified a pathway that controls three alternative transcripts encoded by SESN1 gene (30), the T2 activity of peroxiredoxins, the enzymes responsible for decompo- and T3 SESN1 mRNAs showed clear down-regulation in Ras- sition of hydrogen peroxide produced in response to receptor expressing cells whereas the expression T1 was not affected signaling (27–29). Sestrins form a small family of redox enzymes, (Figs. 2A and 3). In Ras-expressing rat REF52 and Rat1 cells, the which, together with sulfiredoxin, participate in regeneration of only known SESN1 transcript was found to be strongly inhibited overoxidized peroxiredoxins, thus controlling their antioxidant (Figs. 2B and 3). function (21). To test for possible involvement of this pathway in The results obtained with restricted effector specificity H-Ras Ras-mediated up-regulation of ROS levels, we studied the effects of mutants and inhibitors of MAPK/ERK kinase (MEK)-1 (PD98059) various Ras mutants on the expression of sestrin family genes. We and p38 (SB203580) suggest that the repression of SESN3 gene is found that in all tested cell lines, constitutive expression of V12 H- mainly connected with activation of Ras-mitogen-activated protein Ras as well as conditional expression of D13 N-Ras causes a kinase (MAPK) pathways. In fact, the V12S35 Ras mutant retaining decrease in levels of mRNAs derived from the sestrin family genes the ability to interact with Raf (23) and activate ERK1/2 kinases (Figs. 2A–B and 3). In all cases, the inhibition was most prominent (Fig. 2A) was capable of repressing SESN3 gene transcription

Figure 2. Effect of changes in activity of various Ras effectors on expression of sestrin family genes, ROS levels, and mutagenesis. A, effect of constitutive expression of various H-Ras mutants on activity of ERK1/2 kinases; levels of SESN1, SESN2, SESN3 mRNAs; DCF-DA fluorescence; and chromosome breaks in human MDAH041 cells. The cell cultures were analyzed, as described in Materials and Methods, 7 d after retroviral transfer of corresponding pLXSN-neo constructs and selection in the medium with genticin. B, effect of various H-Ras mutants on expression of SESN1, SESN2, and SESN3 mRNAs and DCF-DA fluorescence in rat REF52 cells. The cell cultures were analyzed 7 d after introduction of corresponding retroviral pLXSN-neo vectors. C, effect of constitutive expression of dominant- negative Rac1N17 mutant on ROS levels in Ras-expressing MDAH041/tet-Ras, REF52/tet-Ras, and Rat1/tet-Ras cells. The cells were infected with pMSCV-puro/Rac1N17 vector and selected with puromycin in tetracycline-containing medium for 3 d. The DCF-DA fluorescence of Ras-expressing cells was analyzed 5 d after tetracycline withdrawal. D, effect of constitutively active V23 and dominant-negative N28 RalA mutants on amounts of functionally active GTP-bound forms of RalA and levels of ROS in human MDAH041 cells. The cell cultures expressing the RalA mutants were obtained and analyzed as described in Materials and Methods. E, effect of various H-Ras and RalA mutants on ROS levels in Rat1 cells. The cell cultures were obtained and analyzed as described in Materials and Methods. Typical results of one of two to three independent experiments (A–E).

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SESN1, SESN2, and SESN3 genes by lentiviral transduction of corresponding shRNA-expressing constructs (Supplementary Fig. S2). An inhibition of each of the sestrin genes in human MDAH041 cells caused ROS up-regulation and stimulation of mutagenesis similar to those observed in their Ras-expressing derivatives (Tables 1 and 2). Similar results were obtained with rat REF52 and Rat1 cells expressing an shRNA (21) to a conservative region of SESN1 and SESN3 mRNA, which simultaneously targets both transcripts (Supplementary Fig. S2; Table 2). p53-mediated reversion of the high ROS levels induced by oncogenic Ras. We monitored changes in intracellular ROS levels at different time intervals following onset of oncogenic Ras expression. We observed in the p53-deficient MDAH041/tet-Ras cells a stable increase in ROS. In the p53-positive REF52/tet-Ras cells, the increase was transient, and by day 15 the intracellular ROS content retreated to the initial level (Fig. 4B). The observed reversion in ROS level correlated with gradual accumulation of p53 and its target gene product p21Cip1/Waf1, as well as an increased expression of SESN1, a p53-responsive antioxidant gene (Fig. 4C). To test whether the reversion was dependent on a function of p53, we inhibited p53 in REF52/tet-Ras cells by expression of shRNA. There was no induction of p53 and p21Cip1/Waf1 and SESN1 in response to oncogenic H-Ras expression. Concomitantly, there was no significant reversion of elevated ROS as compared with that observed in the p53-positive REF52 cells (Fig. 4B).

Discussion Figure 3. Effect of conditional expression of N-RasD13 and inhibitors of MEK1 (PD98059) and p38 (SB203580) on amounts of SESN1, SESN2, and Previous studies indicate that expression of activated Ras SESN3 mRNAs and ROS levels. Ras-expressing REF52/tet-Ras and oncogene can cause an increase in intracellular ROS levels (15) Rat1/tet-Ras cells were analyzed on the 5th day after tetracycline withdrawal; and genetic instability (11), although underlying mechanisms as MDAH041/tet-Ras cells were incubated in tetracycline-free medium for 7 d; PD98059 (10 Amol/L; Promega) and SB203580 (0.5 Amol/L; Promega) were well as possible connection between these effects were not added for the last 48 h; and DCF-DA fluorescence and mRNAs were detected established. Here we present evidence for a substantial role of as described in Materials and Methods. ROS up-regulation in Ras-induced mutagenesis. Indeed, in all tested cell lines with constitutive and conditional expression of whereas the V12G37 and V12C40 H-Ras mutants, interacting with oncogenic Ras, we observed a direct correlation between elevated RalGDS and PI3K, respectively (23), caused substantially weaker levels of intracellular ROS, accumulation of 8-oxo-2¶-deoxyguano- ERK1/2 phosphorylation (Fig. 2A) and induced only minor sine (a mutagenic product of DNA oxidation; reviewed in ref. 31), repression of SESN3 gene transcription, both in human and in and frequent chromosome breaks in mitotic cells. Moreover, rat cells (Fig. 2A and B). Furthermore, inhibition of MEK1 by treatment with antioxidant NAC was capable of restoring normal PD98059 or p38a and p38h by SB203580 attenuated substantially levels of intracellular ROS, which also abrogated the Ras-induced Ras-induced SESN3 gene repression (Fig. 3). Probably both Ras- mutagenesis. Thus, the increased ROS level can be a major cause of Raf-MEK-ERK and Ras-p38 pathways contribute to SESN3 repres- genetic instability in cells expressing oncogenic Ras. sion. On the other hand, inhibition of MEK1 and p38 differentially Oncogenic Ras can induce an increase in intracellular ROS levels affected Ras-induced down-regulation of distinct SESN1 tran- by stimulation of membrane-bound NAD(P)H-dependent oxidases scripts: PD98059 attenuated repression of T2 but did not influence that generate superoxide anion from molecular oxygen (32, 33). T3 expression; the SB203580 counteracted the effect of Ras on NAD(P)H-dependent oxidases belong to a family of multi-subunit expression of T3 rather than of T2 (Fig. 3). MAPK-independent enzymes composed of a membrane-bound heme-containing SESN1 pathways seem to be also involved in regulation of gene: the cytochrome b558 complex (a catalytic gp91 tightly associated with RalGDS-interacting V12G37 Ras mutant repressed SESN1 gene in cytochrome b light chain p22), cytosolic p47 or Noxo1 (Nox- rat (Fig. 2B) and human cells, where the strongest down-regulation organizer), and p67 or Noxa1 (Nox-activator) proteins and a small was observed for the T3 transcript (Fig. 2A). GTPase of the Rac family (34, 35). Several lines of evidence To estimate possible contribution of repression of sestrins to suggested that expression of activated Ras can cause an increase in Ras-induced ROS up-regulation, we independently expressed ROS levels through activation of the PI3K-Rac1/NAD(P)H oxidase various isoforms of sestrins from introduced lentiviral constructs. pathway (17, 19, 20). Here, we found that in all tested cell lines, the The MDAH041/tet-Ras cells bearing SESN1, SESN2,orSESN3 transduction of dominant-negative Rac1N17, which inhibits the constructs were more resistant to ROS increase upon switched-on function of NAD(P)H oxidase (36), caused only partial (30–40%) expression of Ras (Fig. 4A). In Ras-expressing cells bearing reduction of Ras-induced increase in intracellular ROS levels. In exogenous SESN1 T2 or T3 cDNAs, the ROS level was increased addition, transduction of mutant RasV12C40 that selectively 1.3- to 1.4-fold, compared with a 3-fold increase in the Ras- activates PI3K resulted in only moderate increase in intracellular expressing control. We also studied the effects of inhibition of ROS. Therefore, we assumed that there might be additional www.aacrjournals.org 4675 Cancer Res 2007; 67: (10). May 15, 2007

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. Cancer Research mechanisms for Ras-induced ROS-up-regulation that are not SESN1 mRNA isoform T3. The data obtained with SB203580 related to activation of NAD(P)H oxidase through PI3K and Rac. inhibitor suggest that the Ras-p38 pathway might also participate We decided to test whether Ras can act downstream of NAD(P)H in the down-regulation of SESN1 mRNA isoform T3. At the same oxidase, possibly by interfering with the decomposition of time, the mutant RasV12S35 was more effective than RasV12G37 in produced hydrogen peroxide. Cytosolic peroxiredoxins play a repressing human SESN1 mRNA isoform T2. The result of major role in decomposition of endogenously produced H2O2 (37, experiment with MEK1 inhibitor PD98059 confirmed a major role 38) whereas their activity is regulated by combined action of of the Raf-MEK-ERK pathway in the down-regulation of human sulfiredoxin (21, 39, 40) and proteins of the sestrin family (21, 24). SESN1 mRNA isoform T2. The detailed mechanisms by which Ras- We found that expression of H-RAS and N-RAS oncogenes in induced activation of the MAPK and RalGDS pathways mediate human and rat fibroblast cells is accompanied by a decrease in repression of SESN3 and SESN1 genes remain to be established. expression of mRNAs derived from the sestrin family genes. SESN3 To assess possible biological significance of the Ras-induced gene showed the strongest repression in all tested cell lines. A decrease in expression of SESN3 and SESN1 mRNAs, we used two decrease in expression of SESN1 was also prominent whereas approaches. First, we studied how elimination of this effect can SESN2 gene showed only minor repression in human cells and no influence ROS up-regulation. For this purpose, we analyzed ROS visible changes in rat cells. The data obtained with restricted levels in Ras-expressing cells, which additionally express various effector specificity Ras mutants that selectively activate the Raf- exogenous sestrin cDNAs introduced by lentiviral transfer. This ERK, PI3K, and RalGDS pathways, as well as with selective experiment showed that restoration of expression of each of the inhibitors of MEK1 and p38 MAPKs, suggest that activation of the sestrin genes interfered with the ROS up-regulation, suggesting a Raf-MEK-ERK and p38 pathways may play a major role in substantial role of sestrin gene repression in Ras-induced ROS repression of the SESN3 gene. Repression of the SESN1 gene might increase. Then, using RNA interference, we studied the effects of be subject to a more complex regulation. Both the RasV12S35 repression of sestrin genes. In human MDAH041 cells, a shRNA- mutant that selectively activates the Raf-MEK-ERK pathway and induced decrease in expression of each of the SESN1, SESN2, and the RasV12G37 mutant that stimulates RalGDS were similarly SESN3 genes leads to a nearly 2-fold increase in ROS levels and a active in the down-regulation of rat SESN1 mRNA and human 2- to 3-fold increase in frequency of chromosome breaks in dividing

Figure 4. A, effects of sestrins on Ras-induced ROS up-regulation. MDAH041/tet-Ras cells infected with lentiviral vectors expressing SESN1, SESN2,orSESN3 cDNAs were introduced into MDAH041/tet-Ras cells using lentiviral vectors. On day 2 after infection, tetracycline was removed from culture medium and DCF fluorescence was assayed on day 7. Left, distribution of 104 cells according to the intensity of DCF-DA fluorescence in tested cell cultures; right, average indices of total DCF-DA fluorescence of 104 cells calculated from two independent experiments. B, kinetics of changes in ROS levels in Ras-expressing p53-positive REF52/tet-Ras cells, p53-deficient REF52/tet-Ras/si-p53 cells, and p53-negative MDAH041/tet-Ras cells. The cell cultures were analyzed 2 to 15 d after tetracycline withdrawal. Average result of two independent experiments. C, changes in expression of Ras, p53, and p21Cip1/Waf1 proteins and SESN1, SESN2, and SESN3 mRNAs in REF52/tet-Ras and REF52/tet-Ras/si-p53 cell cultures incubated without tetracycline for 2 to 15 d. Western blot analysis of Ras, p53, and p21Cip1/Waf1 proteins and RT-PCR detection of mRNAs.

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Table 2. Effect of inhibition of sestrin by shRNAs on ROS levels and frequency of chromosome breaks

Cell line DCF fluorescence Chromosome breaks

% Metaphases Breaks per metaphase

MDAH041 57 F 933F 1.2 0.67 F 0.05 MDAH041/shRNA SESN1 92 F 954 1.7 MDAH041/shRNA SESN2 101 F 11 58 1.8 MDAH041/shRNA SESN3 105 F 10 51 1.6 REF52 23 12 0.22 REF52/shRNA SESN1/3 35 26 0.40 REF52/shRNA SESN2 39 30 0.34 Rat1 10 12 0.12 Rat1/shRNA SESN1/3 39 24 0.36 Rat1/shRNA SESN2 45 28 0.52

NOTE: The cells were analyzed 7 to 10 d after lentiviral transfer of corresponding shRNAs. In each cell culture, 30 to 50 metaphases were analyzed.

cells, which is comparable to the effects produced by activated p53-deficient MDAH041 cells, the Ras-induced increase in ROS RAS. Similar effects were observed in rat cells with simultaneously level is permanent. Possibly, the observed reversion of elevated ROS inhibited expression of SESN1 and SESN3 mRNAs. These data to normal levels in Ras-expressing REF52/tet-Ras cells is connected suggest that down-regulation of sestrins could contribute to the with the up-regulation of a set of the transcriptional target genes of increased ROS level and accelerated mutagenesis in cells express- p53, which include those with antioxidant function, such as GPX1 ing oncogenic RAS. (42, 43), SOD2 (42), ALDH4A1 (44), SESN1, and SESN2 (21, 24). In The p53 function is known to be up-regulated in some types of fact, in the Ras-expressing p53-positive REF52 cells, we observed a Ras-expressing cells (41) including the REF52 cell line (11, 22). The gradual restoration of SESN1 gene expression, which was not seen effect of p53 on ROS level seems to depend on the degree of stress in the corresponding p53-deficient cultures. The p53-mediated and the degree of p53 activation, being pro-oxidant at high stresses feedback mechanisms counteracting Ras-induced ROS up-regula- and antioxidant at nearly physiologic conditions (24). Earlier, we tion can prevent the previously observed accelerated mutagenesis reported that the rate of Ras-induced mutagenesis is higher in in p53-deficient cells expressing oncogenic Ras (11). Collectively, p53-deficient cells as compared with isogenic cells expressing our results highlight the important role of Ras-induced inhibition functional p53 (11). In part, the synergy in the induction of of antioxidant defense in acceleration of genomic instability and chromosome instability by oncogenic RAS in the p53-null cancer progression. background is explained by the ability of Ras to attenuate the p53-independent components of the DNA damage–induced G1 and G2 phase checkpoints (11, 16). In the present study, we found a Acknowledgments connection between up-regulated ROS and inhibited expression of Received 7/5/2006; revised 1/18/2007; accepted 3/9/2007. sestrin family genes, which might represent an additional factor Grant support: Russian Foundation for Basic Research grants 05-04-48969 (L.S. Agapova) and 05-04-48269 (B.P. Kopnin), International Research Scholars Program enhancing Ras-induced mutagenesis in p53-deficient cells. In the of the Howard Hughes Medical Institute grants 55000321 (B.P. Kopnin) and cell lines with unimpaired p53 activation (particularly in the REF52 55005603 (P.M. Chumakov), and NIH R01 grants AG25278 and R01 CA10490 (P.M. cell line), the increase in ROS is temporal, returning to normal level Chumakov). The costs of publication of this article were defrayed in part by the payment of page after 10 to 15 days of oncogenic Ras expression. By contrast, in the charges. This article must therefore be hereby marked advertisement in accordance REF52 cells with inhibited p53, as well as in the human with 18 U.S.C. Section 1734 solely to indicate this fact.

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Pavel B. Kopnin, Larissa S. Agapova, Boris P. Kopnin, et al.

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