The Uric Acid Transporter SLC2A9 Is a Direct Target Gene of the Tumor Suppressor P53 Contributing to Antioxidant Defense

ORIGINAL ARTICLE The uric acid transporter SLC2A9 is a direct target gene of the tumor suppressor p53 contributing to antioxidant defense

Y Itahana1, R Han1, S Barbier2,ZLei1,3, S Rozen1,3 and K Itahana1

Only humans and higher primates have high uric acid blood levels. Although high uric acid causes gout, it has been linked with human longevity because of its hypothetical antioxidant function. Recent studies reveal that p53 has significant roles in cellular metabolism. One example of this is an antioxidant function that potentially contributes to tumor suppression. Here, we reported a first beneficial link between p53 and uric acid. We identified the uric acid transporter SLC2A9 (also known as GLUT9) as a direct p53 target gene and a key downstream effector in the reduction of reactive oxygen species (ROS) through transporting uric acid as a source of antioxidant. Oxidative stress induced SLC2A9 expression in a p53-dependent manner, and inhibition of SLC2A9 by small interfering RNA (siRNA) or anti-gout drugs such as probenecid significantly increased ROS levels in an uric acid-dependent manner and greatly sensitized cancer cells to chemotherapeutic drugs. Conversely, expression of SLC2A9 reduced ROS and protected against DNA damage and cell death, suggesting its antioxidant function. The increased production of ROS because of p53 loss was rescued by SLC2A9 expression. Furthermore, decreased SLC2A9 expression was observed in several cancer types and was associated with a poorer prognosis. Our findings suggest that the p53-SLC2A9 pathway is a novel antioxidant mechanism that uses uric acid to maintain ROS homeostasis and prevent accumulation of ROS-associated damage that potentially contributes to cancer development.

Oncogene (2015) 34, 1799–1810; doi:10.1038/onc.2014.119; published online 26 May 2014

INTRODUCTION glycolysis and apoptosis), which reduces ROS by increasing p53 is an important tumor suppressor that is mutated in activity of the pentose phosphate pathway through its fructose- 9 10 approximately half of all cancer types. The roles of p53 in tumor 2,6-bisphosphatase activity. p53-dependent activity of sestrins, 11 suppression rely primarily on its ability to activate transcription of glutaminase-2 and aldehyde dehydrogenase 4 family member 12 diverse target genes.1 Recent evidence reveals that p53 performs A1 also contribute to the reduction of ROS. Evidence suggests important functions in cellular metabolism, including the regula- that, although p53 acts as a pro-oxidant when cells receive critical damage, it functions to reduce ROS when the stress is tion of oxidative phosphorylation; glycolysis; glutamine, serine and 6 fatty acid metabolism; and reactive oxygen species (ROS).2,3 Mice manageable. In fact, it has been shown that the antioxidant functions of p53 are important for preventing DNA damage, bearing mutations at three p53 acetylation sites, which lead to 13 abrogated p53 function in cell cycle arrest, apoptosis and mutations and tumor development. fi senescence, do not show the same early onset of tumors as Here, we demonstrate that a recently identi ed uric acid p53-null mice that rapidly develop lymphomas.4 Interestingly, transporter, SLC2A9 (also known as GLUT9), is a direct p53 target these mice retain the metabolic functions of p53, such as gene. In response to oxidative stress p53 induces SLC2A9, which performs an antioxidant function by transporting uric acid to inhibition of glycolysis and reduction of ROS, suggesting that fi the metabolic-related functions of p53 are potentially associated protect cells from oxidative stress-induced damage. Our ndings with tumor suppression. demonstrate a novel role of p53 in the regulation of ROS Although ROS acts as a second messenger in cell signaling and homeostasis through induction of SLC2A9. protein modification, excessive generation of ROS is toxic to cells through damage to macromolecules such as DNA, RNA, proteins and lipids. In fact, ROS are the major sources of DNA damage5 and, RESULTS therefore, antioxidant mechanisms must be tightly regulated to p53 induces SLC2A9 mRNA and protein prevent oxidative damage-causing DNA mutations and genomic The SLC2A (or GLUT) family of membrane glycoproteins includes instability that potentially leads to cancer development. Several 14 transporters that have critical roles in cellular metabolism by studies have shown that p53 has both pro-oxidant and transporting different sugars such as glucose and fructose. The antioxidant functions.6 p53 induces diverse target genes involved affinity for each type of sugar and the expression pattern in tissues in the generation of ROS, thus contributing to the sensitization of differ among individual transporters. Some of the SLC2A family cells to apoptosis when stress reaches critical levels.7,8 However, transporters have very low affinity for glucose and fructose, and several antioxidant genes have recently been identified as p53 their exact functions are still unclear. As recent evidence suggests target genes. p53 induces TIGAR (a p53-inducible regulator of that the metabolic role of p53 is important for tumor suppression,

1Cancer & Stem Cell Biology Program, Duke-NUS Graduate Medical School Singapore, Singapore, Singapore; 2Ofﬁce of Clinical Sciences, Center for Quantitative Medicine, Duke-NUS Graduate Medical School Singapore, Singapore, Singapore and 3Centre for Computational Biology, Duke-NUS Graduate Medical School Singapore, Singapore, Singapore. Correspondence: Professor K Itahana, Cancer & Stem Cell Biology Program, Duke-NUS Graduate Medical School Singapore, Room 07-18, Level 7, 8 College Road, Singapore 169857, Singapore. E-mail: [email protected] Received 8 January 2014; revised 4 March 2014; accepted 13 March 2014; published online 26 May 2014 The p53-SLC2A9 antioxidant pathway via uric acid Y Itahana et al 1800 a b Ad-GFP 5 Ad-GFP 30 Ad-p53 Ad-p53 4

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2 10 1 Relative mRNA levels Relative mRNA Relative mRNA levels Relative mRNA 0 0 SLC2A9 mRNA MDM2 mRNA SLC2A9 mRNA MDM2 mRNA SAOS2 IMR-90 normal fibroblasts

c d Plasma membrane fraction digestion (PNGaseF) Ad-CMV Ad-SLC2A9 Ad-CMV Ad-SLC2A9

Mr(K) Ad-CMV Ad-p53 150 digested 100 endo-SLC2A9 75 undigested exo-SLC2A9 digested endo- ATPase α1 50 Total digested 37 exo-SLC2A9 p53

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0 0 Mdm2: +/+ –/–+/+ –/– SLC2A9 mRNA MDM2 mRNA p53 mRNA Slc2a9 mRNA p21 mRNA p53 ER/– MEFs

Figure 1. p53 Induces SLC2A9 mRNA and protein. (a, b) SLC2A9 mRNA is induced by p53. Green-fluorescent protein (GFP) or p53 adenovirus was infected into p53-null H1299 cells (a) and IMR-90 normal human fibroblasts (p53 wild type) (b). mRNA levels of indicated genes were determined with quantitative RT–PCR (n = 3, mean ± s.d.). mRNA levels of the Mdm2 gene, a well-known p53 target, were served as a positive control. Control (GFP) is set at 1, and relative expression is shown. (c) U2OS cells were infected with control CMV or non-tagged SLC2A9 adenoviruses, and undigested or digested SLC2A9 with PNGase F was assessed using western blotting. (d) p53 induces SLC2A9 protein levels. WI-38 fibroblasts were infected with control CMV or p53 adenoviruses, and cell extracts were analyzed with western blotting. ATPase α1 was used as a loading control for the plasma membrane fraction. (e) Slc2a9 is induced by endogenous p53 activation. +/+ ER/À − / − ER/À Mdm2 ; p53 and Mdm2 ; p53 MEFs were treated with ethanol (−) or 100 nM 4-OHT for 24 h to activate p53. mRNA levels of indicated genes were analyzed with quantitative RT–PCR (n = 3, mean ± s.d.). mRNA levels of the p21 gene, a well-known p53 target, were served as a positive control. (f) Endogenous p53 in the absence of stress contributes to SLC2A9 expression. WI-38 fibroblasts were transfected with indicated siRNAs, and mRNA levels of SLC2A9 and MDM2 genes were analyzed as in (e)(n = 3, mean ± s.d.).

we examined whether p53 expression affects the expression of as described14 (Figure 1c). Thus, protein extracts were further SLC2A transporters. Among the family members, we found that digested with the deglycosylation enzyme peptide-N-glycosidase SLC2A9 mRNA was induced by p53 in p53-null SAOS2 osteosar- F, and SLC2A9 signals shifted to around 40 kD as reported coma cells (Figure 1a). SLC2A9 induction by p53 was also observed previously14 (Figure 1c). SLC2A9 knockdown (Supplementary in IMR-90 normal human lung fibroblasts (Figure 1b). Similar Figure S2a) reduced both undigested and digested SLC2A9 results were obtained in H1299 lung cancer cells and normal signals (Supplementary Figure S2b), suggesting that our antibody human forskin fibroblasts (Supplementary Figure S1). We were specifically recognizes endogenous SLC2A9. We then found that unable to detect endogenous SLC2A9 protein using commercial endogenous SLC2A9 protein was markedly induced by ectopic antibodies; therefore, we raised a rabbit polyclonal antibody p53 expression, similar to p21, another transcriptional target of against SLC2A9. SLC2A9 is highly glycosylated,14 and ectopic p53 (Figure 1d). To test whether SLC2A9 is induced physiologically expression of non-tagged SLC2A9 appears as smear above 55 kD by endogenous p53, we took advantage of a knock-in mouse

Oncogene (2015) 1799 – 1810 © 2015 Macmillan Publishers Limited The p53-SLC2A9 antioxidant pathway via uric acid Y Itahana et al 1801 model in which the endogenous p53 gene is replaced by a identified as a member of the facilitated glucose transporter gene-encoding p53ERTAM, a fusion protein containing a full-length family.21 However, biochemical analyses performed by five p53 fused at its C terminus with the hormone-binding domain of a different groups demonstrated that SLC2A9 is in fact a high- modified estrogen receptor.15,16 This modification allows p53 capacity uric acid transporter.22–26 Recent genome-wide associa- activity in mouse embryonic fibroblasts (MEFs) to be rapidly tion studies based on 15 study populations reported that plasma switched between wild-type and knockout states by administration uric acid level variations are associated with genetic variants and withdrawal of 4-hydroxytamoxifen (4-OHT), respectively. Slc2a9 within/near the SLC2A9 gene,24,27–29 suggesting the physiological mRNA was induced approximately fivefold by activating endogen- role of SLC2A9 in transporting uric acid. Uric acid is a weak acid ous p53 with 4-OHT in p53ER/À MEFs (Figure 1e). Induction of Slc2a9 (pKa1 = 5.75) and, because of its charge, it is difficult to pass mRNA by p53 was further confirmed using Mdm2− / − p53ER/À MEFs through plasma membranes in the absence of transporters.22 in which p53 is highly activated by 4-OHT because of a lack of Several in vitro biochemical analyses have suggested that uric Mdm2, which degrades p5316,17 (Figure 1e). SLC2A9 was acid is a powerful scavenger of singlet oxygen, peroxyl radicals significantly decreased by depleting endogenous p53 in WI-38 (RO•2), peroxynitrite (ONOO-) and hydroxyl radicals (•OH), and its normal lung fibroblasts, suggesting that baseline p53 levels also scavenging activity is greater than that of vitamin C.30,31 However, contribute to SLC2A9 expression (Figure 1f). little is known about if and how somatic cells in fact incorporate uric acid as a source of antioxidant. Therefore, we first tested the The SLC2A9 gene is a direct transcriptional target of p53 effect of uric acid on intracellular ROS. We removed serum from cultured IMR-90 normal human fibroblasts, as serum contains uric We next asked whether the SLC2A9 gene is a direct transcriptional acid. Serum starvation is known to induce oxidative stress,32,33 target of p53. We isolated the SLC2A9 promoter from − 1519 to although the underlying mechanisms are poorly understood. Flow +411 base pairs. As many potential p53-binding sites were cytometry analysis of dichlorodihydrofluorescein (DCF) labeling identified in this region using computer software (p53MH 18 showed that serum removal induced ROS (Figure 4a). Adding uric algorithm), we generated deletion constructs linked to the acid significantly attenuated ROS induction caused by serum luciferase reporter gene (Figure 2a). Co-transfection of the SLC2A9 fi − removal (Figure 4a). Uric acid did not signi cantly change the promoter in the region from 970 to +154 (ABCD) together with a levels of p53 and its target genes, suggesting that reduction of wild-type p53 expression vector into p53-null H1299 cells ROS by uric acid is not because of the increased p53 or SLC2A9 significantly increased luciferase activity (Figure 2b). Further − − levels. (Supplementary Figure S6). Similar results as Figure 4a were analysis revealed that the 386 to 117 region (C) in the SLC2A9 obtained in WI-38 normal human fibroblasts and HCT116 cancer promoter (Figure 2a) was responsible for p53-mediated transacti- cell lines (Supplementary Figure S7a). We next monitored ROS vation of the SLC2A9 gene (Figure 2b). As there is only one levels after augmenting uric acid levels in U2OS cancer cells. potential p53-binding site in this region, we created two mutant Interestingly, normal blood levels of uric acid (40–60 mg/l) elements (mt1 and mt2), as well as one that combined these optimally reduced ROS levels (Figure 4b), suggesting that uric fi two (mt3; Figure 2a). Both mt1 and mt2 signi cantly reduced acid in serum significantly contributes to the reduction of ROS. p53-mediated transactivation capacity, and mt3 showed no A similar result was observed in IMR-90 fibroblasts (Supplementary p53-mediated transactivation capacity (Figure 2c), demonstrating Figure S7b). We next added diamide and spermine NONOate in − − that the region from 147 to 128 is a putative p53-responsive the presence or absence of uric acid in serum-free media and element (Figure 2a). Similar conclusion was obtained using monitored ROS levels. NONOate is a donor of NO. Adding uric acid p53-null SOAS2 cells (Supplementary Figures S3a and b). significantly reduced diamide- and NONOate-induced ROS, Chromatin immunoprecipitation (ChIP) revealed that the SLC2A9 suggesting a protective role of uric acid against ROS induced by genomic fragment containing this candidate p53-binding site was oxidative stress (Figure 4c). specifically immunoprecipitated as DNA complex with ectopic p53 (Figure 2d) as well as endogenous p53 (Figure 2e) with a p53 Knockdown of uric acid transporter, SLC2A9, induces ROS antibody, suggesting that p53 directly binds to this element. Whereas most uric acid transporters such as URAT1 and OAT4 are specifically expressed in the kidney, SLC2A9 is the only known SLC2A9 is induced by oxidative stress in a p53-dependent manner ubiquitously expressed uric acid transporter.34 As we confirmed 19 As oxidative stress activates p53, we next studied whether that uric acid has an antioxidant function, SLC2A9 may have a SLC2A9 is also induced by oxidative stress in a p53-dependent novel function to reduce ROS levels by facilitating uric acid traffic manner. We analyzed the time course of SLC2A9 induction in in variety of cell types. Antioxidant function of SLC2A9 has not p53-positive U2OS cells treated with the nitric oxide (NO) donor, been reported yet. To test this, we knocked down SLC2A9 with S-nitrosoglutathione (GSNO). p53 protein and SLC2A9 were two independent small interfering RNAs (siRNAs; Supplementary progressively induced by GSNO in a manner similar to that of Figure S8) in IMR-90 fibroblasts and monitored ROS levels. SLC2A9 MDM2, another transcriptional target of p53 (Supplementary Figure knockdown resulted in a 50–60% ROS increase in IMR-90 S4; Figure 3a). SLC2A9 induction by GSNO was completely fibroblasts and U2OS cells, suggesting that endogenous SLC2A9 attenuated by p53 knockdown (Supplementary Figure S5), again contributes to ROS reduction (Figure 4d). We next added similar to MDM2 (Figure 3b). We next employed colon carcinoma NONOate to SLC2A9-siRNA-transfected IMR-90 fibroblasts and − − HCT116 (p53+/+) wild-type cells and HCT116 (p53 / ) cells.20 found that ROS were significantly increased in SLC2A9 knockdown SLC2A9 induction by GSNO was also attenuated in HCT116 cells, suggesting that SLC2A9 protects cells from oxidative stress − − (p53 / ) cells compared with HCT116 (p53+/+) cells (Figure 3c). (Figure 4e). Comparable results were obtained with a widely used thiol- oxidizing agent, diamide, that oxidizes glutathione, major anti- Ectopic SLC2A9 expression reduces ROS oxidant in cells, thereby inducing ROS intracellularly (Figure 3d). As knockdown of endogenous SLC2A9 led to increased ROS levels, These data suggest that oxidative stress induces SLC2A9 in a p53- we next studied whether ectopic SLC2A9 expression could dependent manner. reduce ROS. We performed immunofluorescence to confirm that SLC2A9 expressed by retroviruses mainly localizes at the plasma Physiological levels of uric acid reduce intracellular ROS membrane as reported14 (Supplementary Figure S9). Retroviral- As we established that SLC2A9 is a bona fide p53 target gene, we mediated SLC2A9 expression in H1299 cells successfully reduced next investigated the function of SLC2A9. SLC2A9 was originally ROS in the absence and presence of oxidative stress mediated by

-1519 -970 -695 -386 -117 +1 +154 +411 ACB D Luciferase construct ATG

SLC2A9 -147 -128 Exon1 gene p53 consensus Putative element mt1 mt2 mt3

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Western blotting Input ChIP with Antibody α-p53 Adeno- GFP p53GFP p53

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Figure 2. SLC2A9 is a direct transcriptional target gene of p53. (a) Luciferase assay reporter gene constructs containing the putative p53 element are shown. The location of the initiation codon (ATG) and exon 1, as well as the sequences of the p53 element and mutated residues, are depicted. (b, c) Reporter plasmids containing indicated deletion constructs (A,B,C and D regions) in (a) or ABC region with wild-type or mutant p53 element (mt1, mt2 and mt3) in (a) were transfected into H1299 cells with control or p53 plasmid, and luciferase activity was monitored (n = 3, mean ± s.d.). Note that the mutations caused low or no p53-mediated transactivation. (d) p53 binds to the SLC2A9 promoter. H1299 cells were infected with GFP or p53 adenoviruses, and ChIP assays were performed with p53 antibody or control IgG. The promoter regions of indicated genes were analyzed with PCR. Pull-down of p53 was conﬁrmed with western blotting. (e) ChIP assays were similarly performed against endogenous p53 in HepG2 cells as in (d).

antimycin A, an inhibitor of complex III of the electric transport sufficient to increase ROS. Similar results were obtained using chain (Figure 5a). Oxidative stressors such as diamide and benzbromarone, another gout drug that inhibits SLC2A9- NONOate produced similar effects (Figure 5b). These data suggest mediated uric acid transport22,26 (Supplementary Figure S10). that SLC2A9 expression protected cells from toxic levels of ROS These data suggest that SLC2A9-mediated uric acid transport produced by oxidative stressors. contributes to ROS reduction. We further confirmed this result by a different way. We first asked whether the ability of endogenous SLC2A9 to reduce ROS is Uric acid transport activity of SLC2A9 contributes to the reduction dependent on serum that contains uric acid. We knocked down of ROS SLC2A9 and measured ROS levels in IMR-90 fibroblasts in the To test whether SLC2A9 uric acid transport activity contributes to presence or absence of serum. Whereas ROS levels were different ROS reduction, we added the widely used gout drug probenecid, between control and SLC2A9 knockdown cells in the presence of which has been shown to inhibit SLC2A9-mediated uric acid serum (solid line in Figure 5d), there was no difference between transport.22,26 Increased probenecid concentrations significantly control and SLC2A9 knockdown cells on ROS levels in serum-free induced ROS in IMR-90 and WI-38 fibroblasts, as well as U2OS cells media (dashed line in Figure 5d), suggesting that serum contains (Figure 5c). A therapeutic plasma level of probenecid (~1 mM) was an antioxidant that is transported by SLC2A9. To test whether this

2 1 Relative mRNA levels

Relative mRNA levels 1

0 0 si-ctrl si-p53si-p53 si-ctrl si-p53 si-p53 Time (hrs) 0102 4 6 8 12 082 4 6 10 12 (1) (2) (1) (2) SLC2A9 mRNA MDM2 mRNA SLC2A9 mRNA MDM2 mRNA

- GSNO 2 + GSNO 6 - Diamide 5 + Diamide 4 1 mRNA levels 3

SLC2A9 2 Relative mRNA levels 1 0 GSNO Relative 0 2 3 0 2 3 0 2 3 0 2 3 0 (mM) HCT116 HCT116 HCT116 HCT116 si-ctrlsi-p53 si-p53 p53 +/+ p53 -/- p53 +/+ p53 -/- (1) (2) SLC2A9 mRNA MDM2 mRNA Figure 3. SLC2A9 is induced by oxidative stress in a p53-dependent manner. (a) SLC2A9 is induced by oxidative stress. U2OS cells were treated with 2 mM GSNO for the indicated time periods, and SLC2A9 and MDM2 expression levels were analyzed using quantitative RT–PCR (n = 3, mean ± s.d.). (b and d) SLC2A9 is induced by oxidative stress in a p53-dependent manner. U2OS cells transfected with indicated siRNAs were +/+ − / − treated with 2 mM GSNO for 12 h (b) or 250 μM diamide for 1 day (d). (c) HCT116 p53 or p53 cells were treated with indicated concentration of GSNO for 12 h. SLC2A9 and MDM2 mRNA levels were analyzed as in (a)(n = 3, mean ± s.d.). antioxidant is uric acid, we added uric acid to serum-free media transduced with SLC2A9-expressing retrovirus compared with and measured ROS levels. The difference on ROS levels in control control cells. and SLC2A9 knockdown cells became evident after adding uric It has been well established that accumulation of ROS enhances acid in serum-free media (solid line in Figure 5e), suggesting that cell death, and proteins that mediate antioxidant protect cells 6 uric acid is critical for endogenous SLC2A9 to reduce ROS. from cell death. For example, p53 target gene TIGAR reduces ROS, 9 In addition, uric acid reduces ROS more efficiently in control thereby protecting cells from cell death. To ask whether SLC2A9 siRNA-transfected cells (black lines) compared with si-SLC2A9- also functions as a protein that mediates antioxidant, we monitored transfected cells (red lines), indicating that the reduction of ROS cell death by propidium iodide (PI) exclusion assay after by uric acid is dependent on SLC2A9. adding chemotherapeutic reagent cisplatin to cells expressing ectopic SLC2A9 by retrovirus infection. SLC2A9 expression significantly attenuated cell death induced by cisplatin treatment Contribution of the p53-SLC2A9 pathway to reduction of ROS and (Supplementary Figure S11). Conversely, SLC2A9 knockdown protection from DNA damage and cell death greatly sensitized cells to cisplatin treatment (Figure 6c). Similar As p53 loss has been shown to increase ROS levels,13 we next results were obtained by probenecid that inhibits uric acid transport investigated whether SLC2A9 expression could prevent ROS activity of SLC2A9 (Supplementary Figure S12). These data suggest induction mediated by p53 loss in an uric acid-dependent that SLC2A9 protects from DNA damage and cell death, consistent manner. In serum-free media, uric acid reduced ROS in HCT116 with the role of SLC2A9 in an antioxidant pathway. (p53+/+) cells (first column Figure 6a) but not efficiently in HCT116 − − (p53 / ) cells (second column). These data suggest that uric acid Lower SLC2A9 expression is observed in several cancer types and reduces ROS in a p53-dependent manner. SLC2A9 expression can is associated with poor prognosis − / − rescue the inability of HCT116 (p53 ) cells to reduce ROS by These data prompted us to investigate the potential role of uric acid (third column), suggesting that the p53-SLC2A9 pathway SLC2A9 in tumor suppression. We measured SLC2A9 mRNA contributes to the reduction of ROS. expression in a variety of cancer and normal tissues (Origene To determine whether SLC2A9 protects against oxidant-induced Technologies) by real-time PCR. SLC2A9 mRNA was significantly DNA damage, we monitored histone H2AX phosphorylation on decreased in the kidney, adrenal gland, prostate and testis cancer serine 139 after adding the NO donor spermine NONOate to types compared with corresponding normal tissues (Figure 7a). p53-null H1299 cells. As shown in Figure 6b, H2AX phosphoryla- Interestingly, we observed that in gastric cancer patients, lower tion after adding NONOate was significantly reduced in cells SLC2A9 mRNA levels were associated with poor survival (P = 0.027,

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0 NONOate –++– Figure 4. Uric acid reduces intracellular ROS, and knockdown of uric acid transporter SLC2A9 increases ROS with or without oxidative stress. (a) IMR-90 fibroblasts were serum-starved for 1 day with or without 40 mg/l uric acid. After staining with CM-H2DCFDA, intracellular ROS levels were measured using flow cytometry. (b) U2OS cells were serum-starved for 5 h with or without uric acid at the indicated concentrations. ROS levels were indicated by the relative levels of the mean CM-H2DCFDA fluorescence in each sample compared with cells in serum-containing medium (n = 3, mean ± s.d.). (c) Uric acid protects cells from ROS induced by oxidative stress. U2OS cells were treated with 250 μM diamide or 0.5 mM spermine NONOate with or without 40 mg/l uric acid under serum-starved conditions for 1 day, and ROS levels were measured (n = 3, mean ± s.d.). (d) IMR-90 fibroblasts or U2OS cells were transfected with indicated siRNAs, and ROS levels were measured. Representative graph is shown in left panel, and histogram shows the mean fluorescence in each sample compared with control cells (n = 5, mean ± s.d.). (e) IMR-90 fibroblasts were transfected with indicated siRNAs and treated with 0.8 mM spermine NONOate for 4 h, and ROS levels were measured (n = 3, mean ± s.d.).

log-rank test, Figure 7b). A Weibull proportional-hazards model Despite uric acid’s negative image as a cause of gout and that accounted for the known prognostic factor tumor-node- kidney stones, interestingly, blood uric acid levels correlate with metastases (TNM) stage indicated a hazard ratio of 0.281 for primate longevity.30,38 Therefore, it has been proposed that SLC2A9 mRNA level (P = 0.021; Supplementary Table S1). These uricase mutations that cause high blood uric acid levels in data are consistent with the potential role of SLC2A9 in tumor humans might confer a selective advantage in primate evolution, suppression. and uric acid could contribute to the increased lifespan of primates compared with other vertebrates.30,38 However, the underlying mechanisms are poorly understood. One potential DISCUSSION mechanism is the antioxidant property of uric acid. Here we Uric acid is the final enzymatic product of purine nucleosides in showed that physiological levels of uric acid efficiently reduce both humans and higher primates. In mammals other than the intracellular levels of ROS in culture (Figures 4a–c). Our results primates, uric acid is converted to the more water-soluble form, identify the p53-regulated uric acid transporter SLC2A9, as a allantoin, by the enzyme uricase. The gene encoding uricase mediator of the antioxidant function of uric acid to protect cells acquired eight independent nonsense mutations and was from ROS elevation. High ROS levels damage mitochondria and inactivated during primate evolution.35,36 This inactivation results macromolecules, such as DNA, RNA, lipids and proteins, and are in fivefold higher blood uric acid levels in humans compared with associated with neurodegenerative diseases, aging and cancer. lower mammals,37 leading to near saturated blood uric acid levels It is logical that humans have developed strong antioxidative and an increased risk of gout, which is caused by needle-like uric mechanisms to support their remarkable longevity compared with acid crystals. other mammals. In fact, abundant evidence suggests that low

500 pBabe pBabe pBabe-SLC2A9 pBabe-SLC2A9 400 pBabe +Antimycin A pBabe-SLC2A9 +Antimycin A 300 Counts 200

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si-ctl si-ctl - serum - serum si-SLC2A9 si-SLC2A9 - serum - serum si-ctrl si-ctrl - serum + serum +Uric Acid Counts si-SLC2A9 Counts + serum si-SLC2A9 - serum +Uric Acid

FL-1 FL-1 Figure 5. SLC2A9 reduces intracellular ROS levels by transporting uric acid. (a, b) SLC2A9 expression reduces ROS. H1299 cells infected with control or FLAG-tagged SLC2A9 retroviruses were treated with 5 μg/ml antimycin A or 300 μM diamide for 1 day or 1.5 mM spermine NONOate (NONOate) for 4 h, and ROS levels were measured. Representative graph is shown in a, and histogram (b) shows the mean fluorescence (n = 3-8, mean ± s.d.). (c) SLC2A9 inhibitors induce ROS. Indicated cells were treated with probenecid at the indicated concentrations for 1 day, and ROS levels were measured (n = 3, mean ± s.d.). (d, e) Uric acid is critical for endogenous SLC2A9 to reduce ROS. IMR-90 fibroblasts transfected with indicated siRNAs were maintained in serum-containing, serum-free or serum-free media with 30 mg/l of uric acid for 5 h, and ROS levels were measured (n = 3, mean ± s.d.). blood uric acid levels (hypouricemia) are associated with the This idea is consistent with our results showing that the reduction development and progression of a variety of diseases, such as of ROS by uric acid is dependent on SLC2A9 (Figure 5e). Lack of multiple sclerosis,39 Alzheimer's disease,40 Parkinson's disease41 SLC2A9 may delay the transportation of unoxidized uric acid into and cancer.42 the cells and cause the intracellular accumulation of oxidized uric It is difficult for uric acid to cross plasma membranes without acid that lacks scavenging activity. Therefore, pharmacological transporters. SLC2A9 belongs to the solute carrier (SLC) group of inhibition of SLC2A9-transporting activity by gout drugs may have membrane transporters that facilitate bidirectional transport of a side effect of increasing ROS. Indeed, adding a therapeutic metabolites across the plasma membrane. Uric acid is an organic concentration of the widely used gout drug, probenecid, that anion at physiological pH and entry of uric acid into cells will be inhibits SLC2A9 increased ROS in a variety of cell types (Figure 5c). electrogenic and opposed by the plasma membrane potential. Similar results were also obtained with another gout drug, However, oxidation of uric acid to allantoin by intracellular ROS benzbromarone, which also inhibits SLC2A9 (Supplementary may neutralize its charge and promote additional uptake of Figure S10). Interestingly, it was recently shown that probenecid unoxidized uric acid. The antioxidant capacity of uric acid may also is able to chemosensitize cancer cells, although the underlying depend on the ability of SLC2A9 to facilitate bidirectional diffusion mechanisms were not described.43 Consistent with this, we show of oxidized and unoxidized uric acid across the plasma membrane. that inhibition of SLC2A9 by siRNA or probenecid induced ROS

© 2015 Macmillan Publishers Limited Oncogene (2015) 1799 – 1810 The p53-SLC2A9 antioxidant pathway via uric acid Y Itahana et al 1806 and sensitized cancer cells to cisplatin-induced cell death the potential contribution of SLC2A9 on tumor suppression. (Figure 6c and Supplementary Figure S12). This is consistent with SLC2A9 can protect cells from DNA damage and cell death SLC2A9 functioning in an antioxidant pathway to protect from cell (Figures 6b and c and Supplementary Figures S11 and S12), and death. These data also suggest the therapeutic potential of lower SLC2A9 expression was observed from several cancer tissues inhibiting SLC2A9 for chemosensitization of cancer cells by compared with corresponding normal tissues (Figure 7a). In increasing ROS levels. Conversely, agents that block uric acid addition, lower SLC2A9 expression is associated with poor patient production such as the widely used drug allopurinol may also prognosis (Figure 7b and Supplementary Table 1). increase ROS. Regardless, the impact of gout drugs on ROS Although we demonstrated that p53 induces SLC2A9 in MEFs metabolism in various tissues in vivo remains to be elucidated. (Figure 1e), a mouse model may be inappropriate to elucidate the Serum removal has been known to induce ROS levels;32,33 antioxidant function of SLC2A9. Whereas SLC2A9 knockout mice however, the underlying mechanisms are unclear. Our results develop gout and nephropathy,44 heterozygosity for a loss-of- provide a potential novel mechanism by which uric acid in serum function SLC2A9 mutation in humans results in the opposite contributes to ROS reduction in cell culture. We also demonstrated phenotype, hypouricemia.25 Mice handle uric acid very differently

+ NONOate pBabe pBabe-SLC2A9 hrs 0 0.5 1 2 3 4 0 0.5 1 2 3 4 γ 100 -H2AX HCT116 p53 +/+ Actin pBabe HCT116 4 pBabe 50 p53 -/- pBabe-SLC2A9 pBabe

ROS levels HCT116 3 p53 -/-

(% of untreated control) pBabe-SLC2A9 0 2 -H2AX levels

Uric Acid treatment γ (60 mg/L) in serum free media 1 Relative

0 hrs 0 0.5 1 2 3 4

si-ctrl si-SLC2A9 (1)

80 3.28% 2.82% si-ctrl si-SLC2A9 (1) si-SLC2A9 (2)

Count Count 60 no treatment

40 F2-L FL-2

si-ctrl si-SLC2A9 (1)

PI-positive dead cells (%) 20 21.8% 64.4%

Count

Count 0 Cisplatin no treatment Cisplatin

FL-2 FL-2 Figure 6. Contribution of the p53-SLC2A9 pathway to reduction of ROS and protection of DNA damage and cell death. (a) SLC2A9 expression can rescue the defect of HCT116 (p53− / −) cells in reducing ROS by uric acid. HCT116 p53+/+ or p53− / − cells infected with control or FLAG- tagged SLC2A9 retroviruses were serum-starved for 6 h with or without 60 mg/l uric acid, and ROS levels were measured. The histogram shows the mean ﬂuorescence that was normalized to cells without uric acid (n = 3, mean ± s.d.). (b) SLC2A9 expression reduces DNA damage. H1299 cells infected with control or FLAG-tagged SLC2A9 retroviruses were treated with 1 mM spermine NONOate and harvested at the indicated time point. γ-H2AX levels were analyzed with western blotting, and relative levels were shown in the lower panel. (c) SLC2A9 protects cells from cell death as a protein that mediates antioxidant. U2OS cells were transfected with control or two independent SLC2A9 siRNAs and treated with 10 μg/ml cisplatin for 16 h. The percentage of dead cells was measured by propidium iodide (PI) exclusion assay using ﬂow cytometry. Representative plots are shown in the left panels, and histogram of the percentage of PI-positive dead cells after indicated treatments are shown in the right panel (n = 6, mean ± s.d.).

Oncogene (2015) 1799 – 1810 © 2015 Macmillan Publishers Limited The p53-SLC2A9 antioxidant pathway via uric acid Y Itahana et al 1807 Kidney Testis 3.0 2.5 P<0.0001 P=0.0006 2.0 2.0 1.5 SLC2A9 SLC2A9 1.0 1.0 mRNA levels mRNA levels 0.5 Relative Relative

0 0 Normal (n=5) Tumor (n=18)Normal (n=6) Tumor (n=19)

Prostate Adrenal gland 2.0 2.5 P=0.001 P=0.0018 1.5 2.0 1.5 SLC2A9 SLC2A9 1.0 1.0 mRNA levels mRNA levels 0.5

Relative 0.5 Relative

0.0 0.0 Normal (n=5) Tumor (n=21)Normal (n=6) Tumor (n=10)

1.00

High SLC2A9

Low SLC2A9 0.80

0.60

0.40 Kaplan−Meier survival estimates 0.20

Log-rank analysis: P=0.027, N =186 0.00

0 50 100 150 Time (months)

Uric Acid

SLC2A9

Nucleus

p53 SLC2A9

ROS

Figure 7. Decreased SLC2A9 expression is observed in several cancer types and is associated with poor prognosis. (a) SLC2A9 mRNA expression is decreased in cancer tissues. The mean value of SLC2A9 expression in normal tissues was set at 1, and relative expression of SLC2A9 mRNA is shown for each sample. The results were analyzed using unpaired t-tests and P-values of normal versus tumor tissue are shown. (b) Gastric cancer patients (n=186) were divided by SLC2A9 mRNA levels: 93 above the median and 93 below. Log-rank analysis (P = 0.027) indicated a better survival for patients with high SLC2A9 mRNA levels compared with those with low levels. (c) A conclusion model is shown.

© 2015 Macmillan Publishers Limited Oncogene (2015) 1799 – 1810 The p53-SLC2A9 antioxidant pathway via uric acid Y Itahana et al 1808 than humans because they possess uricase to convert uric acid MACSQuant VYB (Miltenyi Biotec). The software FlowJo (Tree Star) was to the more water-soluble form, allantoin, whereas human lost used for data analysis. All flow cytometry data are representative of at least uricase during evolution. three independent experiments. Regardless, our collective findings reveal that SLC2A9 is a novel direct target of p53-mediated transcription (Figures 1 and 2), and Quantitative real-time PCR oxidative stress induced SLC2A9 in a p53-dependent manner Total RNA was extracted using the TRIzol (Life Technologies) and RNeasy (Figures 3b–d). These data are consistent with a model in which Mini Kit (Qiagen, Limburg, Netherlands). cDNA was synthesized using an p53 activates SLC2A9 transcription, which enhances uric acid iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Quantitative trafficking across the plasma membrane to reduce ROS levels and RT–PCR was performed with SYBR Green (KAPA Biosystems, Woburn, MA, protect cells from oxidative damage (Figure 7c). Several lines of USA) using the CFX96 System (Bio-Rad). Relative expression was calculated evidence suggest that p53 has antioxidant functions9,10,13 that using GAPDH or TBP as an internal control using Bio-Rad CFX manager contribute to tumor-suppressive functions.4 A lack of p53 results in software. The primers used are shown in Supplementary Material. Tissue increased ROS levels, leading to DNA oxidation, mutations and Scan Cancer and Normal Tissue cDNA array were purchased from Origene instability. p53 may also prevent premature aging by reducing (Rockvile, MD, USA) to examine the expression of SLC2A9. ROS levels.45 Interestingly, almost all samples from four different cancer tissues showed lower SLC2A9 expression compared with RNA interference corresponding normal tissues (Figure 7a). Given that p53 is To knockdown genes, cells were transfected with siRNA or Dicer-substrate mutated in about half of all cancer types, there may be additional siRNAs (DsiRNAs) using Lipofectamine RNAiMax (Life Technologies). The factors contributing to reduced SLC2A9 expression in addition sequences of siRNAs used are listed in Supplementary Material. Silencer to p53 mutations. The relationship between p53 mutations negative control no. 1 (NC1) siRNA (control siRNA) and NC1 negative and SLC2A9 expression in various cancer tissues remains to be DsiRNA (control DsiRNA) were purchased from Life Technologies and investigated. Integrated DNA Technologies (Corallville, IA, USA), respectively. Endogenous ROS can modify ~ 20 000 bases of DNA per day in a single cell.13,46 Therefore, intracellular ROS scavengers are critically Protein analysis important for ameliorating ROS-mediated damage to maintain Cells were lysed in 2% sodium dodecyl sulphate (SDS) lysis buffer (2% SDS, tissue homeostasis and support human longevity. In summary, 50 mM Tris-HCl (pH 6.8), 10% glycerol) for western blotting. In case of our findings reveal that the p53-SLC2A9 pathway is a novel plasma membrane-enriched fractionation, cells were collected into cold antioxidant mechanism to maintain ROS homeostasis and fractionation lysis buffer (10 mM HEPES (pH 7.5), 1 mM EDTA, 250 mM potentially prevent ROS-associated diseases, aging and cancer. sucrose, protease inhibitor cocktail, 1 mM PMSF and 1 mM Na3VO4) and homogenized by passing through a 27½-gauge needle 20 times. The homogenate was then centrifuged at 1000 × g for 5 min at 4 °C to remove MATERIALS AND METHODS nuclei and debris. The supernatant was further centrifuged at 100 000 × g Cell lines, cell culture and reagents for 1 h at 4 °C. The pellets were then resuspended in 2% SDS lysis buffer. To remove N-linked glycosylation, total cell lysates or plasma membrane- U2OS, SAOS2, H1299 and HepG2 were obtained from ATCC. WI-38 and enriched fractions were incubated with PNGase F (New England BioLabs, IMR-90 and foreskin fibroblasts NHF were obtained from the Coriell À − − À Ipswich, MA, USA) for 1 h at 37 °C. The reaction was stopped by adding Institute. Mdm2+/+; p53ER/ and Mdm2 / ; p53ER/ MEFs were kind gifts SDS–PAGE loading buffer. Proteins were analyzed with western blotting. In from Dr Gerard Evan (University of Cambridge). HCT116 (p53+/+) and − − case of horseradish peroxidase-labeled secondary antibodies, signals were HCT116 (p53 / ) cell lines were kind gifts from Dr Bert Vogelstein (Johns detected with chemiluminescence detection reagents (Pierce, Rockford, IL, Hopkins University). All cells were routinely maintained in a 37 °C incubator USA). In case of fluorescent-labeled secondary antibodies, signals were with 5% CO2 in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and penicillin/streptomycin. Uric acid, diamide, antimycin A, detected using Odyssey Infrared Imaging system (LI-COR Biosciences, probenecid, benzbromarone, cisplatin and 4-OHT were purchased from Lincoln, NE, USA). FLAG (M2, Sigma), p53 (DO1, Santa Cruz Biotechnology), α Sigma (St Louis, MO, USA). Spermine NONOate and GSNO were purchased p21(C-19, Santa Cruz Biotechnology), Na+/K+-ATPase 1 (C464.6, Santa from Cayman chemical (Ann Arbor, MI, USA) and Santa Cruz Biotechnology Cruz Biotechnology), phospho-Histone H2A.X (Ser139) (JBW301, Millipore) (Dallas, TX, USA), respectively. Percentage of cell death after cisplatin and actin (Millipore, Bilerica, MA, USA) antibodies were purchased treatment was measured using PI exclusion assay. In brief, attached and commercially. Rabbit polyclonal anti-SLC2A9 antibody was produced using dead floating cells were collected and washed twice in phosphate-buffered a synthetic peptide (CKRNKAYPPEEKIDSAVTDGKINGRP) corresponding to saline (PBS) before re-suspension in 2 μg/ml PI. The percentage of dead amino-acid residues 516–540 of human SLC2A9. cells up-taking PI was measured by flow cytometry using MACSQuant VYB (Miltenyi Biotec, Bergisch Gladbach, Germany) and analyzed with the ChIP analysis software FlowJo (Tree Star, Ashland, OR, USA). ChIP assay was performed as previously described with slight modifications.49 The detailed procedures are shown in Supplementary Plasmids, adenoviruses and retroviruses Material. To generate human SLC2A9 expression constructs, full-length SLC2A9 fi – complementary DNA (cDNA) was ampli ed with reverse transcriptase Luciferase reporter assays polymerase chain reaction (RT–PCR) using cDNA from HepG2 cells as a template. PCR products were cloned into pshuttle-CMV (Agilent pGL4.11-Luc reporter plasmids containing genomic fragments of the Technologies, Santa Clara, CA, USA) or pBabe-puro to generate adeno- SLC2A9 gene were transfected into cells along with CMV-p53 and pRL-CMV viruses or retroviruses, respectively. Recombinant adenoviruses and plasmid. The luciferase and renilla luciferase activities were measured 24 h retroviruses were produced as previously described.47,48 The promoter after transfection using a dual-luciferase reporter assay system (Promega). regions of the SLC2A9 gene were amplified with PCR from genomic DNA Renilla luciferase activity was used as an internal control to normalize isolated from WI-38 cells. PCR fragments were subcloned into a reporter transfection efficiency. plasmid, pGL4.11-Luc (Promega, Madison, MI, USA). Mutations (mt1, mt2 and mt3) were introduced by site-directed mutagenesis PCR. Primer Indirect immunofluorescence sequences for cloning are shown in Supplementary Material. Cultured cells were fixed in formalin (Sigma), permeabilized in 0.2% Triton X-100/PBS and incubated with blocking buffer (PBS containing 0.5% Measurement of intracellular ROS bovine serum albumin) before incubation with the primary antibody. To measure intracellular ROS, cells were trypsinized, washed and stained Cells were then incubated with Alexa Fluor 488-conjugated secondary with 10 μM CM-H2DCFDA (Life Technologies, Carlsbad, CA, USA) at 37 °C for antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA) 1h. After staining, fluorescence-based flow cytometry was performed using and counterstained with 4′, 6-diamidino-2-phenylindole.

Oncogene (2015) 1799 – 1810 © 2015 Macmillan Publishers Limited The p53-SLC2A9 antioxidant pathway via uric acid Y Itahana et al 1809 Patients and tumors, and cancer-specific survival analysis 19 Vousden KH, Ryan KM. p53 and metabolism. Nat Rev Cancer 2009; 9:691–700. The microarray data from gastric cancer patients were downloaded Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP et al. Requirement from the NCBI Gene Expression Omnibus repository (accession numbers: for p53 and p21 to sustain G2 arrest after DNA damage. Science 1998; 282: GSE15459 and GSE34942). Details of patient recruitment are described.50 1497–1501. We analyzed data for patients whose survival information was available 20 Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP et al. Requirement (n = 186). We divided the patients by SLC2A9 mRNA levels: 93 above the for p53 and p21 to sustain G2 arrest after DNA damage. Science 1998; 282: median and 93 below for log-rank analysis. Non-cancer deaths were 1497–1501. treated as censored. A Weibull proportional hazard model adjusted for 21 Phay JE, Hussain HB, Moley JF. Cloning and expression analysis of a novel TNM stages in Supplementary Table S1 was fit using Stata V11.2 (Stata member of the facilitative glucose transporter family, SLC2A9 (GLUT9). Genomics Corp, College Station, TX, USA). 2000; 66:217–220. 22 Anzai N, Ichida K, Jutabha P, Kimura T, Babu E, Jin CJ et al. Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) CONFLICT OF INTEREST in humans. J Biol Chem 2008; 283: 26834–26838. fi The authors declare no conflict of interest. 23 Caul eld MJ, Munroe PB, O'Neill D, Witkowska K, Charchar FJ, Doblado M et al. SLC2A9 is a high-capacity urate transporter in humans. PLoS Med 2008; 5: e197. 24 Vitart V, Rudan I, Hayward C, Gray NK, Floyd J, Palmer CN et al. SLC2A9 is a newly ACKNOWLEDGEMENTS identified urate transporter influencing serum urate concentration, urate excretion and gout. Nat Genet 2008; 40:437–442. We acknowledge funding from the Singapore Ministry of Education AcRF Tier 2 fund 25 Matsuo H, Chiba T, Nagamori S, Nakayama A, Domoto H, Phetdee K et al. (MOE2013-T2-1-123) and the Duke-NUS core grant. We thank Dr Gerald Evan for Mutations in glucose transporter 9 gene SLC2A9 cause renal hypouricemia. Am J providing Mdm2+/+; p53ER/À and Mdm2− / −;p53ER/À MEFs, Dr. Bert Vogelstein for Hum Genet 2008; 83: 744–751. HCT116 (p53+/+) and HCT116 (p53À / À) cell lines, Dr Yanping Zhang for p53 and 26 Bibert S, Hess SK, Firsov D, Thorens B, Geering K, Horisberger JD et al. Mouse green-fluorescent protein adenoviruses. We also thank Drs David Virshup, Patrick GLUT9: evidences for a urate uniporter. Am J Physiol Renal Physiol 2009; 297: Casey, Kanaga Sabapathy and Paul Yen for critical reading of the manuscript. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)