Tumor Protein 53–Induced Nuclear Protein 1 Is a Major Mediator of P53 Antioxidant Function Carla E

Research Article

Tumor Protein 53–Induced Nuclear Protein 1 Is a Major Mediator of p53 Antioxidant Function Carla E. Cano,1,2 Julien Gommeaux,1,2 Sylvia Pietri,3 Marcel Culcasi,3 Ste´phane Garcia,1,2 Myle`ne Seux,1,2 Sarah Barelier,1,2 Sophie Vasseur,1,2 Rose P. Spoto,1,2 Marie-Jose`phe Pe´busque,1,2 Nelson J. Dusetti,1,2 Juan L. Iovanna,1,2 and Alice Carrier1,2

1Institut National de la Sante´et de la Recherche Me´dicale,U624 ‘Stress cellulaire’; 2Aix-Marseille Universite´,Campus de Luminy; 3Sondes Mole´culaires en Biologie, Laboratoire Chimie Provence UMR 6264, Centre National de la Recherche Scientifique-Universite´sAix-Marseille I, II & III, Faculte´des Sciences de Saint-Je´roˆme, Marseille, France

Abstract response to cell injury involves key processes such as cell cycle arrest, DNA repair, replicative senescence, and apoptosis whenever p53 exerts its tumor suppressor function mainly through damage overwhelms repair. Furthermore, p53 continuously pro- transcriptional induction of target genes involved in several tects cells from endogenous, highly mutagenic reactive oxygen processes, including cell cycle checkpoints, apoptosis, and species (ROS), including free radicals, produced by the cell itself as regulation of cell redox status. p53 antioxidant function is respiration products and second messengers during cell signaling. dependent on its transcriptional activity and proceeds by Although cytoplasmic and mitochondrial functions have been sequential induction of antioxidant and proapoptotic targets. described for p53 (1, 2), its most relevant property seems to be However, none of the thus far renowned p53 targets have its transcriptional activity over target genes that mediate tumor- proved able to abolish on their own the intracellular reactive suppressive functions. A good example of this is the recently oxygen species (ROS) accumulation caused by p53 deficiency, reported physiologic antioxidant activity of p53, which is therefore pointing to the existence of other prominent and dependent on its DNA-binding domain (3). p53 response to yet unknown p53 antioxidant targets. Here, we show that elevated intracellular ROS concentration involves two waves of TP53INP1 represents such a target. Indeed, TP53INP1 tran- transcription induction. The first wave starts when cells endure script induction on oxidative stress is strictly dependent on low oxidant concentrations, in which case p53 induces transcrip- p53. Mouse embryonic fibroblasts (MEF) and splenocytes À À tion of genes with antioxidant and cell cycle regulatory properties, derived from TP53INP1-deficient (inp1 / ) mice accumulate such as sestrins and p21, to favor the restoration of physiologic intracellular ROS, whereas overexpression of TP53INP1 intracellular ROS levels (3, 4). When cells endure long-lasting in p53-deficient MEFs rescues ROS levels to those of and/or high concentrations of oxidant, p53 will induce a second p53-proficient cells, indicating that TP53INP1 antioxidant wave of transcription of proapoptotic genes, such as those function is p53 independent. Furthermore, accumulation of À À encoding Puma and Bax, to promote cell death (3, 5, 6). ROS in inp1 / cells on oxidant challenge is associated with Interestingly, none of the above-mentioned p53 targets induced decreased expression of p53 targets p21/Cdkn1a, Sesn2, TAp73, during the oxidative stress response is capable of fully restoring Puma, and Bax. Mutation of p53 Ser58 (equivalent to human physiologic ROS levels in the absence of p53, suggesting that a p53 Ser46) abrogates transcription of these genes, indicating yet unknown p53 target might be responsible for sensing and/or that TP53INP1-mediated p53 Ser58 phosphorylation is impli- eliminating ROS surplus. cated in this process. In addition, TP53INP1 deficiency results Tumor protein 53–induced nuclear protein 1 (TP53INP1; also in an antioxidant (N-acetylcysteine)-sensitive acceleration of known as TEAP, SIP, and p53DINP1) is a p53 target gene that cell proliferation. Finally, TP53INP1 deficiency increases encodes the TP53INP1 protein (7–9). Two isoforms of this protein oxidative stress–related lymphoma incidence and decreases À exist, TP53INP1a and TP53INP1h (18 and 27 kDa, respectively), survival of p53+/ mice. In conclusion, our data show that resulting from alternative splicing of the transcript and showing no TP53INP1 is a major actor of p53-driven oxidative stress known functional domain apart from a PEST motif characteristic of response that possesses both a p53-independent intracellular short half-life proteins (8, 9). TP53INP1 may act on a p53-positive ROS regulatory function and a p53-dependent transcription feedback through its interaction with protein kinases HIPK2 and regulatory function. [Cancer Res 2009;69(1):219–26] protein kinase Cy (PKCy; refs. 10, 11). These protein kinases enhance p53 protein stability and transcriptional activity through 46 Introduction phosphorylation of p53 Ser (10–12). TP53INP1 expression is induced by different cell stress agents (adriamycin, cisplatin, The TP53 gene, which encodes the p53 tumor suppressor, is the ethanol, and heat shock) and, most remarkably, by oxidants such most frequently mutated gene in human cancers. p53-dependent as hydrogen peroxide (H2O2) or conditions promoting their formation, such as exposure to UV light or g-rays (8, 9). In turn, Note: Supplementary data for this article are available at Cancer Research Online TP53INP1 induces the transcriptional activation of p53 target (http://cancerres.aacrjournals.org/). promoters, such as those of CDKN1A and Bax genes, when C.E. Cano and J. Gommeaux contributed equally to this work. expressed ectopically in cell lines (10). Furthermore, consistent Requests for reprints: Alice Carrier, Institut National de la Sante et de la Recherche Medicale U624, Case 915 Parc Scientifique de Luminy, 13288 Marseille with genotoxic stress-induced TP53INP1 transcriptional regulation Cedex 9, France. Phone: 33-4-91-82-88-29; Fax: 33-4-91-82-60-83; E-mail: alice.carrier@ by p53, p73, and E2F1 (which are transcription factors implicated inserm.fr. I2009 American Association for Cancer Research. in cell cycle control and tumor suppression), TP53INP1 over- doi:10.1158/0008-5472.CAN-08-2320 expression provokes a G1 cell cycle arrest and apoptosis in vitro, www.aacrjournals.org 219 Cancer Res 2009; 69: (1). January 1, 2009

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Cancer Research suggesting a tumor suppressor activity (10, 13, 14). We recently was analyzed using the Kaplan-Meier test. All values were expressed as showed that, compared with wild-type (wt), mice deficient for mean F SE, with significance set at P < 0.05. ESR data were analyzed using TP53INP1 present increased susceptibility to colon tumor devel- two-way ANOVA over the entire incubation period. If a difference was found opment in a chronic inflammation setting and a greater production (P < 0.05), a Bonferroni test was carried out to test for differences among mean values of all groups at each time point. of ROS in inflamed colon (15). Moreover, the low blood ascorbate concentration observed in deficient mice indicates a chronic and systemic oxidative stress, suggesting an antioxidant function for Results TP53INP1. In this work, we address the question of the implication TP53INP1 induction on oxidative stress is p53 dependent. of TP53INP1 in the p53-dependent oxidative stress response and To establish p53 dependence of TP53INP1 expression on oxidative intracellular ROS regulation. This is achieved by the analysis of stress, we derived fibroblasts (MEFs) from wt and p53-deficient À À primary cells derived from mice deficient for TP53INP1 and p53 (p53 / ) embryos, cultured them for 1 hour with or without and by the generation and analysis of mice and cells that are H2O2, and allowed cells to recover for 12 hours. Then, we assessed deficient for both of these stress proteins. TP53INP1 transcription levels by quantitative reverse transcription-PCR (qRT-PCR). Figure 1 shows the resulting expression data Materials and Methods that confirm our previous observations showing strong TP53INP1 À/À induction (>4-fold) in H2O2-treated wt cells (8). In contrast, Animals. TP53INP1-deficient (inp1 ) mice were described elsewhere À/À (15). TP53INP1- and p53-deficient [double knockout (DKO)] mice were TP53INP1 expression was severely compromised in p53 cells À À À À obtained by inbreeding inp1 / and p53 / mice (16). All mice were kept in the absence of stress (70% reduction) and on H2O2 challenge. within the animal facilities and according to the policies of the Laboratoire These data show that induction of TP53INP1 on oxidative stress d’Exploration Fonctionnelle de Luminy (Marseille, France). is totally dependent on p53, although a basal p53-independent Cells. Primary mouse embryonic fibroblasts (MEF) from 14.5-d post- TP53INP1 expression exists in unstressed and H2O2-treated MEFs. À À À À coitum wt, inp1 / , p53 / , and DKO embryos were prepared following TP53INP1 deficiency induces intracellular accumulation of a standard protocol (17). All experiments were performed using early- ROS. To determine whether TP53INP1 possesses an antioxidant À À À À passage MEFs of at least two independent preparations per genotype. function, we cultured wt, TP53INP1-deficient (inp1 / ), p53 / , TP53INP1a-inducible MiaPaCa2 cell line was described elsewhere (18). Cells and TP53INP1/p53 (DKO) MEFs with or without H O for 1 hour were cultured in DMEM and 10% (v/v) fetal bovine serum (FBS; Invitrogen) 2 2 and allowed cells to recover for 3 or 10 hours. Then, we assessed at 37jC, 5% CO2. Viral transduction of MEFs and transfection of MiaPaCa2 cells are described in Supplementary Materials and Methods. intracellular ROS content by flow cytometry using the DCF probe Oxidative stress and intracellular ROS detection. MEFs were platted and determined the percentage of ROS-containing cells (RCC) À/À in triplicate in 35-mm culture dishes (BD Biosciences) and treated with within each population. In mock conditions, only inp1 cells

50 Amol/L H2O2 in DMEM without FBS at 37jC. After 1 h, medium was showed a greater percentage of RCC than wt at any time point replaced by DMEM and 10% FBS with or without 5,000 units/mL catalase (Fig. 2A). H2O2 challenge increased the fraction of RCC in all (Sigma), and cells were allowed to recover for the times indicated. For genotypes tested, the highest increase being observed after 3 hours. À À intracellular ROS detection, cells were further cultured for 15 min at 37jC inp1 / cells showed more RCC than wt (2- and 1.7-fold at 3 and in DMEM, 10% FBS, and 5 Amol/L dichlorofluorescein (DCF) diacetate 10 hours, respectively) on H2O2 challenge. We found more RCC À À (Sigma), which is oxidized in the presence of ROS into green fluorescent among p53 / cells compared with wt after H O challenge, DCF, whose fluorescence was assessed by flow cytometry on a FACSCalibur 2 2 consistent with previous reports (3). Double deficiency for cytometer (BD Biosciences). ROS accumulation rate is expressed as the fold TP53INP1 and p53 resulted in RCC increase 3 and 10 hours after changes of DCF-positive cells between mock- and H2O2-treated MEFs. À/À Electron spin resonance measurements. Assessment of extracellular oxidative stress compared with wt and p53 cells, with only a À/À release of free radicals from H2O2-pretreated MEFs was performed using slight increase 3 hours after stress when compared with inp1 À/À À/À electron spin resonance (ESR) technology and the spin trap 5-(diethox- cells. Strikingly, there were more inp1 than p53 RCC with or yphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO). For more details, without H2O2 treatment (3- and 1.5-fold, respectively), suggesting see Supplementary Materials and Methods. Proliferation assays. One hundred thousand MEFs of each genotype were plated in triplicate in 35-mm dishes and cultured for up to 7 d in conventional medium with or without 10 mmol/L N-acetylcysteine (NAC; Sigma). Medium was renewed every 3 d. Cells were counted daily by trypan blue exclusion to establish growth curves. The number of population doublings (PD) was calculated using the formula PD = ln(Nf/Ni)/ln2, where Nf and Ni are final and initial cell numbers, respectively. RNA extraction and quantitative reverse transcription-PCR analysis. Total RNA was extracted using Trizol (Invitrogen) and cDNAs were prepared using ImProm-II kit (Promega) following the manufacturer’s instructions. Quantitative PCR was performed in a LightCycler (Roche) using the SYBR Premix Ex Taq (Takara Bio). For details of amplification, see Supplementary Materials and Methods. À À À Histologic analysis. p53 / , p53+/ , and TP53INP1/p53 double-deficient mice were sacrificed when showing illness and autopsied. Tissues/tumors were collected, formalin fixed, and paraffin embedded. Sections were stained with H&E (Vector Laboratories) and mounted in Eukitt solution (Vector Laboratories) before diagnosis. Figure 1. TP53INP1 transcription on oxidative stress is p53 dependent. Histogram shows relative transcript expression of TP53INP1 quantified by Statistics. All statistical analyses were performed using the StatView qRT-PCR in wt and p53À/À MEFs incubated in serum-free DMEM and without 5.0 software (SAS Institute, Inc.). Data on MEFs were analyzed using the (Mock)orwithH2O2 (50 Amol/L) for 1 h and then cultured in conventional nonparametric Mann-Whitney U test on triplicate measures. Mice survival medium for 12 h. *, P < 0.005, compared with wt.

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Figure 2. TP53INP1 deficiencycauses intracellular ROS accumulation. A, wt, inp1À/À, p53À/À, and DKO MEFs were incubated in DMEM without FBS and À/À without (Mock) or with H2O2 (50 Amol/L) for 1 h and then cultured in conventional medium for 3 and 10 h. Histogram shows percentage of DCF-positive wt, inp1 , p53À/À, and DKO RCCs determined byflow cytometry. B, E1A/rasV12-transformed p53À/À MEFs were transduced with emptyMSCV-neo, MSCV-TP53INP1 a, MSCV-TP53INP1h, emptypLPC, and pLPC-p53 retroviral vectors. Then, cells were submitted to H 2O2 treatment as indicated above and allowed to recover for 3 h before DCF staining. Histogram shows ROS accumulation rate (fold changes of mock: H2O2-treated DCF-positive cells) in these cells. *, P < 0.05, compared with p53-restored p53À/À MEFs (white column). C, histograms show DCF mean fluorescence intensity( MFI) of splenocytes, thymocytes, and RBCs from wt and inp1À/À mice stained ex vivo immediatelyafter sacrifice. D, left, dot plot showing side scatter (SSC), indicative of cell granularity, and forward scatter (FSC), indicative of cell size, which allows discrimination of myeloid and lymphoid compartments of the spleen for DCF fluorescence measurement; right, histogram shows DCF levels of spleen myeloid and lymphoid cells from wt and inp1À/À mice. Columns, mean of triplicates and are representative of three independent experiments; bars, SE. P < 0.05, compared with wt (*), inp1À/À (&), and p53À/À (#) MEFs.

that TP53INP1 action over ROS could be, at least in part, TP53INP1 deficiency affects the regulation of H2O2 content independent of p53. To test this hypothesis, we performed in MEFs. DCF is a general oxidant indicator rather than a specific V12 À/À retroviral transduction on E1A/ras -transformed p53 MEFs marker for H2O2 (19). We determined TP53INP1 implication on to introduce TP53INP1a or TP53INP1h alone or along with p53 intracellular H2O2 regulation by incubating H2O2-pretreated wt À/À and determined the ROS accumulation rate in these cells 3 hours and inp1 MEFs with catalase (an H2O2 scavenger) before RCC after H2O2 treatment (Fig. 2B). As expected, restoration of p53 assessment using DCF. Catalase treatment after H2O2 stress À À À À expression in p53 / cells led to a drop of ROS accumulation on abolished RCC differences between inp1 / and wt MEFs À/À oxidant challenge compared with p53 cells transduced with (Fig. 3A), indicating that TP53INP1 deficiency provokes H2O2 empty vectors. Transduction of either TP53INP1a or TP53INP1h accumulation. Evidence that TP53INP1 deficiency triggers an À/À was able to drop ROS accumulation in p53 cells even below abnormal extracellular release of H2O2-derived free radicals after levels reached by p53 restoration alone, and cotransduction of p53 H2O2 challenge was provided with spin trapping experiments and TP53INP1 isoforms did not significantly improve TP53INP1 in which the spin trap DEPMPO was added to H2O2-pretreated antioxidant effect. Therefore, once TP53INP1 is induced on MEFs during recovery. Regardless of the genotype, no ESR signal oxidative stress, it seems to play its antioxidant function was detected in medium samples collected up to 30 minutes after independently of p53. H2O2 removal (Fig. 3B, trace a). From 30 minutes, addition of We determined the physiologic relevance of TP53INP1 antiox- DEPMPO led to spin adducts formation, typical spectra of which À À idant function by measuring ROS content in splenocytes, are shown in Fig. 3B (traces b and d for inp1 / and wt cells, À À thymocytes, and RBCs from wt and inp1 / mice (Fig. 2C). We respectively). Computer simulation of the signals afforded hyper- observed a significant increase of intracellular ROS content in fine coupling constants consistent with mixtures of DEPMPO/ÁOH À/À N H splenocytes but not in thymocytes or RBC from inp1 mice as a superimposition of f64% trans (a = 14.06 G, a h = 12.73 G, P N H P compared with wt. Splenocytes of both the myeloid and lymphoid a = 47.23 G) and 36% cis (a = 14.06 G, a h = 14.06 G, a = 47.29 G) À À compartments were affected to the same extent in inp1 / diastereoisomers (20), and two DEPMPO/alkyl adducts [aN = 14.55 H P mice (Fig. 2D). Altogether, these data show that TP53INP1 is a (14.34) G, a h = 21.47 (19.34) G, a = 46.76 (47.14) G] accounting for negative regulator of intracellular ROS content both under 25% to 35% of the total signal (see Fig. 3B, traces c and e). ESR physiologic conditions in splenocytes and under oxidative stress signal was completely abolished when either catalase (Fig. 3B, in MEFs. trace f ) or the metal ion chelator deferoxamine (Fig. 3B, trace g) www.aacrjournals.org 221 Cancer Res 2009; 69: (1). January 1, 2009

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Cancer Research was added to the medium from the beginning of the experiment. significantly higher number of doublings than wt cells after NAC Figure 3C displays the time course of spin adduct formation treatment, suggesting that TP53INP1 may have a redox-indepen- in both genotypes, with a burst at 75 minutes following H2O2 dent role in cell cycle regulation. Moreover, TP53INP1 restoration À À À À removal and still detectable levels after 150 minutes. All along this reinstated wt-like proliferation rates in inp1 / but not in p53 / 150-minute period, DEPMPO adducts detected in samples from E1A/rasV12-transformed MEFs (Supplementary Fig. S1), whereas À À wt cells were significantly lower than those from inp1 / cells DKO proliferation was only partially reestablished. In conclusion, (P < 0.001, by two-way ANOVA). these data show that TP53INP1 possesses a physiologic growth- Increased proliferation is related to oxidative stress in inhibitory function related to its antioxidant properties. TP53INP1-deficient cells. We determined whether TP53INP1 TP53INP1 deficiency affects p53-dependent transcription on plays a physiologic role in cell proliferation by comparing growth oxidative stress. Because expression of p53 target genes could be À À À À rates of wt, inp1 / , p53 / , and DKO MEFs. Early-passage MEFs affected by TP53INP1 deficiency, we searched for modifications of À À were seeded at equal number and counted daily for 7 days. inp1 / the expression of well-known oxidative stress–induced p53 targets À À À À MEFs proliferate more rapidly than wt, although slower than p21 (Cdkn1a), sestrin 2 (Sesn2), Puma, and Bax in inp1 / , p53 / , À/À p53 , which proliferate slower compared with DKO (Fig. 4A). and DKO MEFs compared with wt, in mock- and H2O2-treated Consistently, the number of PDs of each genotype after 7 days cells, using qRT-PCR. Although none of the expressions of these confirmed the differences in growth rates shown by daily counts genes was modified in unstressed cells, we observed that their À À À À (Fig. 4B, left). Importantly, addition of the antioxidant NAC during transcripts were drastically reduced in inp1 / , p53 / , and DKO À/À À/À proliferation assays resulted in a restoration of inp1 , p53 , cells compared with wt after H2O2 challenge (Fig. 5A). Similarly, and DKO number of doublings back to that of wt (Fig. 4B, right), oxidative stress–induced expression of p53 homologue TAp73 was indicating that accelerated growth in deficient cells is related to abolished in the absence of TP53INP1 (Supplementary Fig. S2). oxidative stress. Nevertheless, DKO cells conserved a mild but Positive action of TP53INP1 on transcription on H2O2 challenge is

Figure 3. TP53INP1 deficiencyaffects antioxidant response to H2O2. A, histogram shows percentages of DCF-positive wt and À/À inp1 MEFs after 1 h mock or H2O2 (50 Amol/L) treatment and 3 h recovery in culture medium with or without 5,000 units/mL catalase. B, DEPMPO (10 mmol/L) spin trapping of free radicals released from H2O2-pretreated wt or inp1À/À cells. Representative ESR spectra were recorded from inp1À/À cells 15 min (a) or 75 min (b) after removal of H2O2; c, computer simulation of b showing the participation of DEPMPO/ÁOH (DEPMPO-OH; 71%) and DEPMPO/alkyls (DEPMPO-R; 29%); d, same conditions as b for wt cells; e, computer simulation of d corresponding to a mixture of DEPMPO/ ÁOH (68%) and DEPMPO/alkyls (32%); f and g, spectra from inp1À/À cells 75 min after H2O2 in the presence of catalase (5,000 units/mL) or deferoxamine (0.1 mmol/L), respectively. Instrumental settings: microwave power, 10 mW; modulation amplitude, 0.497 G; receiver gain, 3.2 Â 105; time constant, 20.48 ms; and sweep rate, 2.86 G/s. C, comparative time course of DEPMPO/ÁOH (top), DEPMPO-alkyls (middle), and total (bottom) spin adduct formation in inp1À/À and wt cells. Two-wayANOVA: P < 0.001 versus wt followed byBonferroni test (*, P < 0.05 versus wt). Columns, mean (n = 4–6); bars, SE.

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significantly altered by TP53INP1 deficiency (data not shown). Nevertheless, TP53INP1 deficiency affects p53 heterozygous mice À fate (Fig. 6B). Indeed, whereas >80% of inp1+/+ and inp1+/ mice À À À with a p53+/ background survived at 15 months, 50% of inp1 / À p53+/ animals had died by the same time (P < 0.001). Moreover, À À À lymphoma penetrance was increased in inp1 / p53+/ mice À À À compared with inp1+/ p53+/ and inp1+/+ p53+/ animals (75%, 29%, and 0%, respectively; Fig. 5C). As TP53INP1 deficiency causes increased oxidative load in vivo, these observations are in accordance with previous data associating lymphoma incidence in p53- deficient animals with increased endogenous oxidative stress (3).

Discussion In this work, we show that TP53INP1 is necessary for controlling Figure 4. TP53INP1 and p53 repress cell proliferation through their antioxidant intracellular ROS levels both in the absence of exogenous stress and À/À À/À function. Proliferation of early-passage wt, inp1 , p53 , and DKO MEFs after oxidant challenge, that its induction on oxidative stress is was assessed during 7 d. A, growth curves obtained from dailycell counts for each genotype. B, the histogram shows the number of MEF PDs after 7 d of strictly dependent on p53, and that it enhances p53-dependent culture in the presence or absence of 10 mmol/L NAC. Data in A and B are transcription on oxidative stress. This is the first report of means of triplicates F SE and are representative of three independent experiments. P < 0.05, compared with wt (*), inp1À/À (&), and p53À/À (#) MEFs. TP53INP1 cell-intrinsic antioxidant function, and it sheds light on our previous observations of chronic oxidative stress in TP53INP1-deficient mice (15). We previously reported that À À not generalized to all p53 targets because transcription of cyclin G1 inp1 / mice are more sensitive than wt to experimentally induced À À and Mdm2 was not affected in inp1 / cells (Supplementary colitis and colon carcinogenesis, associated with increased Fig. S3). Furthermore, we assessed the expression of the CD44 granulocyte infiltration and colon ROS release. Although in the À À gene whose expression is repressed by p53 (21) and noticed that case of colon inflammation increased ROS in inp1 / colons could À À À À CD44 repression was released in inp1 / , p53 / , and DKO cells in be attributed to a more aggressive response of ROS secreting the absence of stress (Fig. 5B). Interestingly, these cells failed to granulocytes and thus to an indirect effect of TP53INP1 on ROS up-regulate CD44 expression on oxidative stress. Finally, unstressed in vivo, data from primary fibroblasts clearly show that TP53INP1 À À inp1 / splenocytes exhibit higher levels of CD44 expression than plays an antioxidant function at the cell level. À À their wt counterparts (Supplementary Fig. S4). In conclusion, Intracellular ROS accumulation observed in inp1 / MEFs can our data show that TP53INP1 deficiency significantly affects p53- result from either an increased production or a defective dependent transcription on oxidant challenge. degradation of ROS. Although our experiments do not discriminate p53 Ser58 is essential for TP53INP1-mediated transcription between these processes, we can take out some clues about of oxidative stress–induced p53 targets. Because TP53INP1 TP53INP1 mechanism of action on regulation of cell ROS content. favors p53 Ser46 phosphorylation in humans (the homologue of Abrogation by catalase of differences in RCCs between wt and 58 58 À/À p53 Ser in mice; refs. 10, 22), we determined whether Ser inp1 cells indicates that H2O2 aberrantly accumulates in À À phosphorylation is necessary for TP53INP1 action on oxidative inp1 / cells after oxidant challenge. Because TP53INP1 protein stress–related p53 targets. For this purpose, we introduced sequence does not suggest a potential catalytic activity, it is expression vectors encoding either a wt or a Ser58 to Ala mutated unlikely that it possesses an intrinsic capacity to produce or p53 (S58A) into a TP53INP1-inducible MiaPaCa2 cell line that detoxify H2O2. Thus, it is possible that TP53INP1 enhances the lacks functional p53 and in which TP53INP1 expression can be activity of ROS regulatory enzymes. Indeed, such cofactor activity triggered by ponasterone A (18). The absence of TP53INP1 has already been described for TP53INP1 in the case of p53 expression in the absence of ponasterone A treatment was regulation, in which TP53INP1 binds HIPK2 and PKCy kinases confirmed by qRT-PCR (Supplementary Fig. S5). We quantified and p53 itself to favor p53 phosphorylation (10, 11). p21, Sesn2, Bax, and Puma transcripts in these cells (Fig. 5C) Two ways of H2O2 degradation can be foreseen in our and observed that, consistent with results in MEFs, TP53INP1 experimental conditions that can be discriminated by the outcome induction enhanced the transcription of these genes in MiaPaCa2 of ESR. The first way is provided by the endogenous antioxidant cells transfected with wt p53. In contrast, cells transfected with p53 defenses (i.e., catalase, glutathione peroxidases, and peroxiredo- S58A failed to induce transcription of the same genes indepen- xins) that decompose H2O2 into water and molecular oxygen. dently of TP53INP1 presence. In conclusion, our data show that Whenever this pathway is defective, excess H2O2 may engage into p53 Ser58 is crucial for the triggering of p21, Sesn2, Puma, and Bax the Fenton reaction in the presence of metals, yielding HOÁ that transcription and for TP53INP1-dependent enhancement of this will be trapped by DEPMPO to form ESR-detectable DEPMPO/ÁOH À À triggering. adducts. Here, we show that inp1 / cells release more DEPMPO TP53INP1 deficiency increases lymphoma incidence and trappable HOÁ than wt after oxidant challenge (Fig. 3B and C) and mortality in p53 heterozygous mice. Finally, we determined the that spin adduct formation is inhibited by the chelator deferox- À À effect of TP53INP1 deficiency on survival and tumor incidence in amine (Fig. 3B, trace g). Therefore, our data suggest that inp1 / mice deficient for p53. As shown in Fig. 6A, TP53INP1 deficiency MEFs can either produce more de novo H2O2 or release greater did not affect survival of homozygous p53-deficient mice (i.e., amounts of H2O2 and metal catalysts than wt, or both, which f50% of animals died by 5 months regardless of their TP53INP1 ultimately lead to the observed extracellular DEPMPO/ÁOH À À status). Tumor incidence and tumor type in p53 / mice were not buildup. Two mechanisms unrelated to direct trapping of HOÁ www.aacrjournals.org 223 Cancer Res 2009; 69: (1). January 1, 2009

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Cancer Research can yield DEPMPO/ÁOH: (a) metal-catalyzed nucleophilic addition we found a reduction of the expression of several p53 target genes À/À of water occurring at the double bond of pyrroline N-oxides (20, in H2O2-pretreated inp1 MEFs compared with wt, of which À 23–25) and (b) reduction of the protonated form of DEPMPO/O2Á some are cell cycle regulators (i.e., p21/Cdkn1a), some antioxidants by glutathione peroxidase (20, 23, 24). Because none of these two (Sesn2), and some proapoptotic (i.e., TAp53, Puma, and Bax). unspecific mechanisms can account for inhibitions by catalase Moreover, we report for the first time to our knowledge the and deferoxamine (Fig. 3B, traces f and g), a dominant pathway dependence of the expression of these genes on TP53INP1- for DEPMPO/ÁOH formation in our study is likely to involve mediated p53 Ser58 phosphorylation. These results reinforce our trapping of HOÁ formed by Fenton reactions with H2O2. This previous report of TP53INP1 ability to enhance p53 transcriptional pathway can also explain the concomitant inhibition of secondary activity (10) and indicate that TP53INP1 is the missing piece in DEPMPO/alkyls, which may result from hydrogen abstraction by Sablina’s picture of p53 antioxidant function (3). In this new HOÁ from a variety of cell components. In further support, cis:trans picture, oxidative stress induces p53 expression, which in turn ratios for DEPMPO/ÁOH seen here are rather in the range of those triggers expression of TP53INP1 that will then enhance, by a reported in vitro with Fenton reagents than of those observed when mechanism yet to be elucidated, the activity of the endogenous enzymatic reductive systems or nucleophilic synthesis is involved antioxidant machinery (Supplementary Fig. S6). In parallel, (20, 24). Therefore, the outcome of ESR assays strongly suggests TP53INP1 interaction with HIPK2 and PKCy will lead to p53 that TP53INP1 deficiency leads to an improper function of the Ser58 phosphorylation, thus allowing transcription of p21 and endogenous antioxidant machinery, directing H2O2 processing Sesn2 and subsequent cell cycle arrest and ROS detoxification. toward the Fenton reaction. As redox status is restored to physiologic one, TP53INP1 Is TP53INP1 action on ROS dependent on p53 function? degradation is driven by its PEST domain, and p53 is subsequently Although induction of TP53INP1 by oxidative stress is p53 degraded, shutting down the oxidative stress response. In dependent (Fig. 1), overexpression of TP53INP1 is sufficient to conditions where oxidative stress persists, TP53INP1 enhances decrease ROS content in p53-deficient cells to levels comparable transcription of Puma and Bax to induce cell death. with p53-proficient cells (Fig. 2B). Therefore, TP53INP1 proceeds Another interesting finding is the implication of TP53INP1 in to its antioxidant function without direct participation of p53. repression of CD44 expression in unstressed MEFs and splenocytes, Nevertheless, TP53INP1 seems to collaborate with p53 in as it settles the notion of a transcription regulatory function of transcription induction in response to oxidative stress. Indeed, TP53INP1 in basal conditions without exogenous stresses as it has

Figure 5. TP53INP1 deficiencyalters expression of oxidative stress–induced p53 targets. Histograms show relative transcript expression of p21/Cdkn1a, Se sn2, À/À À/À Bax, and Puma in A, and of CD44 in B, as quantified byqRT-PCR in wt, inp1 , p53 , and DKO MEFs without stress and after H2O2 (50 Amol/L) treatment for the times indicated (* and & are P < 0.05 compared with wt and inp1À/À MEFs, respectively; note that Puma data are Â10À2). Histograms in C show relative transcript expression of p21/Cdkn1a, Sesn2, Bax, and Puma in TP53INP1a-inducible MiaPaCa2 cells transfected with pCAG 3.1 vectors encoding wt p53 or p53 S58A, which were previouslytreated for 24 h with ponasterone A (10 Amol/L) for TP53INP1a induction, or with a vehicle (DMSO). Bar colors in C indicate homolog to genotypes in A. *, P < 0.05. Target transcript expression was normalized bythe corresponding cyclophilinor TBP values. Data are means of triplicates F SE.

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antioxidant properties. This is particularly remarkable because the action of these proteins at the G1 checkpoint has thus far been attributed exclusively to their transcriptional activity on cell cycle regulatory genes. In fact, antioxidant and transcriptional activities of these two proteins may act synergistically on cell cycle because low concentrations of oxidant are necessary for S-phase progression (26). In this way, TP53INP1 and p53 could control cell cycle progression by keeping ROS in check and, when necessary, by inducing G1 checkpoint. Finally, TP53INP1 deficiency aggravates the outcome of p53 heterozygous mice as it enhances lymphomagenesis (Fig. 6), presumably promoted by chronic oxidative stress. These results are consistent with our previous report of TP53INP1 role in colon tumor suppression through ROS regulation (15). Although TP53INP1 deficiency alone is not sufficient to induce spontaneous tumors in mice, it seems to be a suitable ‘‘second hit’’ that allows tumor promotion after a first transforming event, such as chemical mutagenesis or loss of a p53 allele. Based on the data presented in this study, we hypothesize that loss of TP53INP1 favors malignant transformation by increasing genetic instability through ROS accumulation and by allowing uncontrolled proliferation of a damaged cell. Accordingly, we observed the loss of TP53INP1 during the progression of several cancers, particularly during pancreatic carcinogenesis, and we showed that loss of the TP53INP1 protein is caused by the action of protumoral miRNA- 155 (18). Therefore, reestablishment of TP53INP1 expression seems as a promising therapeutic strategy for the treatment of cancers in which oxidative stress is recognized as a promoting factor, such as those related to chronic inflammation.

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Figure 6. TP53INP1 deficiencydecreases survival of heterozygous p53-deficient mice. Cumulative survival of inp1+/+, inp1+/À, and inp1À/À mice on a p53-deficient homozygous (A) or heterozygous background (B). C, histogram shows number of cases of different tumor types within cohorts of inp1+/+, inp1+/À, Acknowledgments and inp1À/À mice on a p53+/À background. Received 6/18/2008; revised 9/26/2008; accepted 10/21/2008. Grant support: Institut National de la Sante´et de la Recherche Me´dicale,Centre National de la Recherche Scientifique, Institut National du Cancer, and La Ligue been described for p53 (21). Moreover, CD44 expression has been Nationale Contre le Cancer. C.E. Cano and J. Gommeaux were supported by La Ligue related to enhanced proliferation, which could provide a piece of Nationale Contre le Cancer and the Association pour la Recherche sur le Cancer. The costs of publication of this article were defrayed in part by the payment of page an explanation for the enhanced proliferation without p21 charges. This article must therefore be hereby marked advertisement in accordance À À expression alteration in inp1 / MEFs. with 18 U.S.C. Section 1734 solely to indicate this fact. We thank S. Soddu for wt p53 and S58A expression vectors, G. Warcollier and About cell proliferation, we show for the first time here that p53 F. Gianardi for animal care, and M.N. Lavaud for technical assistance in histologic and TP53INP1 negatively regulate cell growth through their analysis.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Tumor Protein 53−Induced Nuclear Protein 1 Is a Major Mediator of p53 Antioxidant Function

Carla E. Cano, Julien Gommeaux, Sylvia Pietri, et al.

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