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Home , Parkin (ligase)

, a target , mediates the role of p53 in glucose and the Warburg effect

Cen Zhanga, Meihua Lina, Rui Wua, Xiaowen Wanga, Bo Yangb, Arnold J. Levinec,d,1, Wenwei Huc, and Zhaohui Fenga,1

aDepartment of Radiation Oncology and cDepartment of Pediatrics, Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08903; bCollege of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China; and dInstitute for Advanced Study, Princeton, NJ 08540

Contributed by Arnold J. Levine, August 24, 2011 (sent for review June 8, 2011)

Regulation of energy metabolism is a novel function of p53 in 17). Recently, Parkin has been suggested to be a potential tumor tumor suppression. Parkin (PARK2), a Parkinson disease-associated suppressor, and diminished expression and of the Parkin gene, is a potential tumor suppressor whose expression is fre- gene have been frequently observed in various tumors (18–22). quently diminished in tumors. Here Parkin was identified as a However, the mechanisms by which Parkin contributes to tumor p53 target gene that is an important mediator of p53’s function suppression and the regulation of Parkin are not well-understood. fi in regulating energy metabolism. The human and mouse Parkin Here we identi ed Parkin as a p53 target gene. p53 increases contain functional p53 responsive elements, and p53 the of Parkin gene both in vitro and in vivo. Parkin ’ increases the transcription of Parkin in both humans and mice. contributes to p53 s role in regulating glucose metabolism and fi Parkin contributes to the function of p53 in glucose metabolism; the Warburg effect; Parkin de ciency results in the Warburg fi effect, whereas restoration of Parkin expression reverses the Parkin de ciency activates and reduces mitochondrial ’ respiration, leading to the Warburg effect. Restoration of Parkin Warburg effect in cells. Parkin also mediates p53 s role in anti- expression reverses the Warburg effect in cells. Thus, Parkin de- oxidant defense. These results suggest that the functions of Parkin in regulating energy metabolism and defense ficiency is a novel mechanism for the Warburg effect in tumors. should contribute greatly to Parkin’s role in tumor suppression Parkin also contributes to the function of p53 in antioxidant and, furthermore, contribute to p53’s role as a tumor suppressor. defense. Furthermore, Parkin deficiency sensitizes mice to γ-irra- diation-induced tumorigenesis, which provides further direct evi- Results dence to support a role of Parkin in tumor suppression. Our results Human Parkin Gene Is a p53 Target Gene. As a transcription factor, suggest that as a novel component in the p53 pathway, Parkin p53 binds to the p53 responsive elements (REs) in its target genes contributes to the functions of p53 in regulating energy metabo- to transcriptionally regulate their expression in response to stress lism, especially the Warburg effect, and antioxidant defense, and (23). The p53MH algorithm is a computational program developed thus the function of p53 in tumor suppression. for genome-wide scanning for potential p53 targets by identifying putative p53 REs in genes (24), which has been successfully applied etabolic alterations are a hallmark of tumor cells (1, 2). to identify new p53 targets (11, 25, 26). Using this algorithm, we MWhereas normal cells use mitochondrial respiration to identified human Parkin gene as a potential p53 target. provide energy, the majority of tumor cells preferentially use To investigate whether p53 transcriptionally regulates the aerobic glycolysis, a switch known as the Warburg effect (3). Parkin gene, Parkin expression was examined in various lines Because glycolysis produces ATP much less efficiently than exposed to different stress signals. A pair of isogenic p53 wild- mitochondrial respiration, tumor cells compensate by having a type and p53-deficient human lung cell lines, H460-con and much higher rate of glucose uptake and utilization than normal H460-p53siRNA, which stably express a control vector and a p53 cells (1, 2). Recent studies have strongly suggested that the shRNA vector in p53 wild-type H460 cells, respectively, were used. Warburg effect is a key contributor to malignant progression (1, H460-p53siRNA cells displayed greatly decreased p53 2), and reversing the Warburg effect inhibits the tumorigenicity levels and decreased induction of p53 target in response to of cancer cells (4, 5). However, the underlying mechanisms for Etoposide and H2O2 compared with H460-con cells (Fig. 1A). the Warburg effect are not well-understood (1, 2). Etoposide and H2O2 clearly induced Parkin expression in H460- p53 plays a central role in tumor prevention. As a transcription con cells at both protein and mRNA levels (Fig. 1 A and B). This factor, in response to stress, p53 transcribes its target genes to induction is p53-dependent because no clear induction of Parkin start various cellular responses, including cell-cycle arrest, apo- was observed in H460-p53siRNA cells. Furthermore, p53 increased ptosis, and/or senescence, to prevent tumor formation (6, 7). the basal levels of Parkin under nonstressed conditions. In addition Recent studies have revealed that regulating energy metabolism to these stress signals, Nutlin-3a, a nongenotoxic small molecule and the Warburg effect is a novel function of p53 in tumor that activates p53 through disruption of p53-MDM2 interaction suppression (2, 8). p53 induces TIGAR (TP53-induced glycolysis (27), clearly induced Parkin expression in a p53-dependent and regulator) to reduce glycolysis (9), and induces manner in H460 cells (Fig. 1 C and D). The regulation of Parkin by SCO2 (10) and GLS2 (11, 12) to promote mitochondrial respi- p53 activation was also confirmed by immunofluorescence (IF) ration. Loss of p53 results in decreased mitochondrial respiration staining (Fig. 1E). Results in Fig. 1E further show that Parkin is and enhanced glycolysis, leading to the Warburg effect. Fur- mainly localized in mitochondria and cytoplasm, and not in nuclei. thermore, regulating antioxidant defense has recently been Similar results were observed in p53 wild-type HCT116-con and revealed as another novel function for p53 (8, 13). p53 induces p53-deficient HCT116-p53siRNA (Fig. S1). These results to- several antioxidant genes, including Sestrins (14), TIGAR (9), ALDH4 (15), and GLS2 (11, 12), to reduce the levels of (ROS) and DNA damage in cells, which con- Author contributions: A.J.L., W.H., and Z.F. designed research; C.Z., M.L., R.W., and X.W. tributes greatly to the role of p53 as a tumor suppressor. performed research; C.Z., M.L., R.W., X.W., B.Y., A.J.L., W.H., and Z.F. analyzed data; and Parkin (PARK2) was first identified as a gene associated with A.J.L., W.H., and Z.F. wrote the paper. Parkinson disease (PD), a neurodegenerative disease. Mutations of The authors declare no conflict of interest. Parkin account for most autosomal recessive forms of juvenile 1To whom correspondence may be addressed. E-mail: [email protected] or fengzh@umdnj. Parkinson disease. Parkin deficiency leads to mitochondrial dys- edu. function and enhanced in neuronal cells in Dro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CELL BIOLOGY sophila and mice, which are believed to contribute greatly to PD (16, 1073/pnas.1113884108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1113884108 PNAS | September 27, 2011 | vol. 108 | no. 39 | 16259–16264 Downloaded by guest on September 29, 2021 H460 con p53siRNA H460 con p53siRNA PuPuPuC(A/T)(T/A)GPyPyPy (N)0-14 PuPuPuC(A/T)(T/A)GPyPyPy A H2O2 0h 18h 24h 0h 18h 24h A Etp 0h 18h 24h 0h 18h 24h - 3332 TAACTTGCTT CGTGAT AAGCTTGTTC +2629 GGGCTAGCTT G AAGCAAGCTT Parkin Parkin Exon1 Exon2 p53 p53 H460-Con H460-p53siRNA B C H1299 MDM2 MDM2 30 Mut p53 WT p53 25

Ab Anti-p53 Input Anti-p53 Anti-p53 IgG Actin Actin Anti-p53 IgG 20 Intron 1 15 10 Parkin B 4 3 5 Con p53siRNA Con p53siRNA 0 3 MDM2 luciferase Relative activity 2 p53RE Promoter Intron 1 2 Fig. 2. p53 binds to and transactivates the p53 responsive element in hu- 1 1 man Parkin gene. (A) The putative REs in human Parkin gene predicted by Relative Parkin levels mRNA 0 0 the p53MH algorithm. Numbers indicate the nucleotide position relative to Etp 0h 6h 18h 24h H2O2 0h 6h 18h 24h the ATG site. (Upper) Sequences of the p53 RE. N, any nucleotide; Pu, purine; Py, pyrimidine. (B) ChIP analysis of interactions between p53 and the p53 RE H460 con p53siRNA in human Parkin gene in cells. H460-con and H460-p53siRNA cells were C D μ Nutlin 0h 18h 24h 0h 18h 24h treated with Etoposide (10 M for 16 h) before ChIP assays. Input DNA, 1/20 Parkin 6 DNA of ChIP. (C) p53 activates the luciferase reporter vector containing the Con 5 putative p53 RE in intron 1 of human Parkin gene. Reporter vectors were p53siRNA transfected into p53 null H1299 cells along with a wild-type (WT p53) or p53 4 3 R273H mutant (Mut p53) p53 expression plasmid. The relative luciferase p21 2 activity in cells cotransfected with mutant p53 was designated as 1. Data are Relative Parkin levels mRNA 1 presented as mean ± SD (n =3). Actin 0 Nutlin Con 18h 24h ments containing one copy of these two putative p53 REs were E Parkin Mitochondria Nuclei Merge inserted into the promoter of PGL2 luciferase reporter vector. con p53 null human lung H1299 cells were cotransfected with the reporter vectors and a vector expressing either wild-type or R273H mutant p53. Compared with mutant p53, the expression of

H460-con wild-type p53 greatly enhanced luciferase activities of the reporter Etp vector containing the putative p53 RE in the Parkin intron 1 (by >20-fold), but not in the promoter region (by less than twofold) (Fig. 2C). Taken together, our data demonstrate that human Parkin gene is a p53 target gene; p53 binds to the p53 RE in con Parkin intron 1 and increases Parkin transcription in cells.

Parkin Etp Mouse Gene Is a p53 Target Gene. The p53MH algorithm H460-p53siRNA also showed that mouse Parkin gene contains three putative p53 REs in its promoter region, including RE A and two overlapped Fig. 1. p53 regulates the expression of human Parkin gene. H460-con and REs, B and C (Fig. 3A). This suggested that mouse Parkin gene H460-p53siRNA cells were treated with Etoposide (Etp, 10 μM) or H2O2 (200 μ was a potential p53 target. Two luciferase reporter vectors were M) for different amounts of time. The protein levels were determined by constructed that contained a copy of RE A and REs B+C. Lu- Western blot assays. mRNA levels of Parkin were measured by real-time PCR and normalized with actin. Data are presented as mean ± SD (n = 3). (A and ciferase reporter assays showed that the expression of wild-type p53 in p53 null H1299 cells and mouse embryonic fibroblasts B) p53 induces Parkin expression at both protein (A) and mRNA (B) levels in −/− H460-con cells under both nonstressed and stressed conditions. (C and D) p53 (p53 MEF) clearly enhanced the luciferase activities of both activation by Nutlin-3a (5 μM) induces Parkin expression at both protein (C) reporter vectors by approximately four- to sixfold compared with and mRNA (D) levels in cells. (E) p53 induces Parkin expression in H460-con mutant p53 (Fig. 3B). Furthermore, H2O2 clearly induced Parkin μ expression at both mRNA and protein levels in p53+/+ MEF but cells treated with Etoposide (10 M for 24 h) as detected by IF staining. − − not p53 / MEF cells (Fig. 3 C and D). p53+/+ MEF cells also − − displayed a higher level of basal Parkin expression than p53 / gether demonstrate that p53 increases Parkin expression under MEF cells. These results demonstrate that mouse Parkin is a p53 both stressed and nonstressed conditions. target gene; p53 increases Parkin transcription through the reg- ulation of p53 REs in Parkin promoter region. Human Parkin Gene Contains a Functional p53 Responsive Element. To investigate whether p53 activation induces Parkin expres- +/+ −/− To investigate whether p53 regulates Parkin expression through sion in vivo, p53 and p53 C57BL6/J mice were subjected to γ its direct binding to the two putative p53 REs in human Parkin whole-body -irradiation (IR) (4 Gy). Parkin was clearly induced promoter region and intron 1 predicated by the p53MH algo- at both mRNA and protein levels (by approximately four- to sixfold at 20 h after IR) in the spleen and thymus, two highly radio- rithm (Fig. 2A), H460-con and H460-p53siRNA cells were sensitive tissues that display the clearest p53 responses to IR, in − − treated with Etoposide to activate p53, and chromatin immu- p53+/+ but not p53 / mice (Fig. 3 E and F). The p53-dependent noprecipitation (ChIP) assays were performed. The anti-p53 induction of Parkin by IR appears to be tissue-specific because fi antibody speci cally pulled down the DNA fragment containing Parkin was not induced in the cortex of brain, liver, or kidney. the putative p53 RE in Parkin intron 1 in H460-con cells treated Together, these results demonstrate that the regulation of Parkin with Etoposide, but not in H460-p53siRNA cells (Fig. 2B). These by p53 is evolutionarily conserved from mice to humans. results demonstrate that p53 protein physically interacts with the putative p53 RE in human Parkin intron 1 in vivo. Parkin Contributes to the Function of p53 in Regulating Glucose To further investigate whether these two putative p53 REs Metabolism and the Warburg Effect. p53 has been reported to re- confer p53-dependent transcriptional activities, the DNA frag- duce glycolysis and promote mitochondrial respiration in cells.

16260 | www.pnas.org/cgi/doi/10.1073/pnas.1113884108 Zhang et al. Downloaded by guest on September 29, 2021 Fig. 3. p53 regulates the A B CD expression of mouse Par- 7 Mut p53 WT p53 MEF p53+/+ p53-/- MEF A: -2972 TTGCAAGTTT GAGCTAGTCA 6 4 kin gene both in vitro and H2O2 0h 18h 24h 0h 18h 24h 5 p53+/+ p53-/- in vivo. (A) The putative Parkin 3 4 p53 REs in mouse Parkin Exon 1 3 p53 2 gene predicted by the B: -794 GGGCTTGTCA TCCTCTACAA CCACATGTCC 2 p53MH algorithm. Num- 1 p21 1 C: -774 CCACATGTCC CCTGATTCG GAGCAAGTGC Luciferase activities 0 bers indicate the nucleo- p53 RE A B+C A B+C 0

Actin Relative Parkin mRNA levels tide position relative to H1299 p53-/- MEF H2O2 Con 6h 18h 24h the initiation codon ATG site. (B) p53 activates the EF p53+/+ spleen p53-/- spleen IR - - - + + + - - - + + + luciferase reporter vec- 18 3 Parkin 16 p53+/+ p53-/- tors containing putative 14 Parkin p21 Parkin p21 p53 12 2 p53 REs in mouse Parkin 10 gene. Reporter vectors 8 p21 6 1 were transfected into p53 4

−/− Actin mRNA induction fold 2 null H1299 and p53 MEF 0 0 cells along with a p53 Spleen Thymus Cortex liver kidney Spleen Thymus Cortex liver kidney wild-type (WT p53) or R273H mutant (Mut p53) expression plasmid. Data are presented as mean ± SD (n = 3). (C and D) p53 induces the expression of Parkin in +/+ −/− MEF cells at both protein (C) and mRNA (D) levels. p53 and p53 MEFs were treated with H2O2 (200 μM), and Parkin mRNA levels were measured by real- − − time PCR and normalized to actin. (E and F) p53 induces mouse Parkin expression in vivo in response to IR. p53+/+ and p53 / C57BCL/6 mice were irradiated (4 Gy). The levels of Parkin protein (E) and mRNA (F) in different tissues were measured by Western blot and real-time PCR assays, respectively, at 20 h after IR (n = 6). Represented is the induction of Parkin protein by IR in three mouse spleen tissues in a p53-dependent manner (E). The mRNA levels of Parkin and p21 were normalized to actin. The mRNA induction fold of Parkin and p21 was calculated as the mRNA levels in IR-treated mice compared with those in control mice without IR. Data are presented as mean ± SD (n =6).

p53 deficiency leads to the Warburg effect in tumors, which is levels of acetyl-CoA and glucose metabolism in cells. Results in characterized by higher glucose uptake, a higher rate of glycol- Fig. 5 A–D clearly show that Parkin knockdown in H460-con ysis, and higher lactate production in tumor cells than normal cells decreased PDHA1 protein levels, the activity of the PDH cells (9, 10). We found that Parkin, as a downstream target of complex, and levels of acetyl-CoA, whereas ectopic Parkin ex- p53, contributes to the role of p53 in regulating glucose metab- pression in H460-p53siRNA cells increased PDHA1 protein olism and the Warburg effect in cells. As shown in Fig. 4 A–C, levels, the activity of the PDH complex, and levels of acetyl-CoA. ectopic expression of Parkin in H460-p53siRNA cells signifi- Consistently, PDHA1 levels were much higher in Parkin+/+ − − cantly decreased glucose uptake, the rate of glycolysis, and lac- MEF than Parkin / MEF cells (Fig. 5A). Furthermore, PDHA1 tate production. Furthermore, knockdown of endogenous Parkin knockdown in H460-con cells significantly reduced mitochon- in H460-con cells significantly enhanced glucose uptake, the rate drial respiration (Fig. 5E), which in turn increased glucose up- of glycolysis, and lactate production. Similar effects of Parkin on −/− take, the rate of glycolysis, and lactate production, leading to the the Warburg effect were also observed in Parkin MEF com- Warburg effect (Fig. 5F). It is still unclear how Parkin regulates +/+ – pared with Parkin MEF cells (Fig. 4 A C, Right). These PDHA1. Parkin does not regulate PDHA1 expression at the fi results demonstrate that Parkin de ciency results in the Warburg mRNA level (Fig. S2A). Knockdown of Parkin or PDHA1 did effect, whereas restoration of Parkin expression reverses the not decrease intracellular ATP levels (Fig. S2B), which suggests Warburg effect in cells. Furthermore, Parkin mediates the role of that enhanced glycolysis compensates the decreased mitochon- p53 in glucose metabolism. As shown in Fig. 4D, p53 knockdown drial respiration for ATP generation in cells. significantly enhanced glucose uptake, the rate of glycolysis, and lactate production in H460 cells. Simultaneous knockdown of Parkin Contributes to the Role of p53 in Regulating Antioxidant p53 and Parkin results in higher glucose uptake, rate of glycol- Defense. The antioxidant function is a novel mechanism for p53 ysis, and lactate production compared with individual knock- in tumor suppression (8, 13). Parkin regulates antioxidant func- down of p53 or Parkin in cells, but the effects are less than tion in neuronal cells (16). To investigate whether the induction additive effects. These results suggest that Parkin is one of the of Parkin by p53 also contributes to the role of p53 in antioxidant important mediators for p53’s role in glucose metabolism. fi defense, Parkin was overexpressed or knocked down in cells. Parkin de ciency leads to mitochondrial dysfunction in neu- fi ronal cells, which contributes to the development of PD (16). Ectopic Parkin expression signi cantly reduced ROS levels in Therefore, the role of Parkin in preventing the Warburg effect H460-p53siRNA cells (Fig. 6A) treated with or without H2O2. Parkin knockdown in H460-con (Fig. 6B) and Parkin knockout in could be mainly due to its function in maintaining mitochondrial fi respiration. Consistent with the role of p53 in enhancing mito- MEF cells (Fig. 6C) signi cantly increased ROS levels. Fur- chondrial respiration (Fig. 4E, first panel), ectopic expression of thermore, Parkin mediates the role of p53 in ROS regulation. As Parkin in H460-p53siRNA cells enhanced oxygen consumption shown in Fig. 6D, simultaneous knockdown of p53 and Parkin (Fig. 4E, second panel). Furthermore, Parkin knockdown in results in higher intracellular ROS levels than individual − − H460-con and Parkin knockout in MEF cells (Parkin / MEF) knockdown of p53 or Parkin, but the effect is less than additive. decreased oxygen consumption (Fig. 4E, third and fourth pan- These results suggest that Parkin is one of the important medi- els), which indicates reduced mitochondrial respiration. Parkin ators for p53’s role in ROS regulation in cells. knockout in mice was reported to result in the decreased ex- Reduced (GSH) is an important antioxidant mol- pression of several mitochondrial , including pyruvate ecule and a scavenger for ROS. GSH:GSSG (oxidized glutathi- dehydrogenase E1α1 (PDHA1), in the mouse brain as detected one) balance reflects the redox state of cells. p53 has been reported by 2D gel electrophoresis and mass spectrometry analysis (16). to up-regulate GSH levels and the GSH:GSSG ratio in cells (11, PDHA1 is a critical component of the pyruvate dehydrogenase 12). As shown in Fig. 6 E and F, H460-con displayed significantly (PDH) complex, which catalyzes the conversion of pyruvate into higher GSH levels and GSH:GSSG ratio than H460-p53siRNA acetyl-CoA and serves as a critical link between glycolysis and cells. Ectopic Parkin expression significantly increased GSH levels mitochondrial respiration. It is unknown whether Parkin can and GSH:GSSG ratio in H460-p53siRNA cells. Furthermore, regulate the expression of PDHA1 in human cells, which may in Parkin knockdown in H460-con and Parkin knockout in MEF cells CELL BIOLOGY turn affect the activity of the PDH complex and therefore the significantly decreased GSH levels and the GSH:GSSG ratio (Fig.

Zhang et al. PNAS | September 27, 2011 | vol. 108 | no. 39 | 16261 Downloaded by guest on September 29, 2021 150 200 200 A H460-p53siRNA H460-con * MEF # 150 150 100 * 100 100 50 50 50

0 0

Relative glucose uptake 0 Vector Con Parkin siRNA Con Parkin Parkin +/+ Parkin -/-

150 200 200 B H460-p53siRNA H460-con MEF 150 100 150 100 100 50 50 50

Relative glycolysis rate 0 0 0 Vector Con Parkin siRNA Con Parkin Parkin +/+ Parkin -/-

150 300 250 C H460-p53siRNA H460-con MEF 250 200 100 200 150 150 100 50 100 50 50

Relative lactate levels 0 0 0 Con Parkin siRNA Con Parkin Parkin +/+ Parkin -/-

D 250 200 300 # 200 # 250 # 150 200 150 Fig. 4. Parkin regulates the Warburg effect in cells. (A) 100 150 Parkin reduces glucose uptake in H460 and MEF cells. (B) Parkin 100 100 reduces the rate of glycolysis in H460 and MEF cells. (C)Parkin 50 50 50 reduces the lactate production of H460 and MEF cells. (D) glucose uptake Relative lactate levels 0 Relative glycolysis rate 0 0 Parkin contributes to the role of p53 in glucose metabolism. p53 p53 siRNA Con p53 Parkin siRNA Con p53 Parkin p53 siRNA Con p53 Parkin Simultaneous knockdown of p53 and Parkin in H460 cells +Parkin +Parkin +Parkin results in higher glucose uptake, rate of glycolysis, and lactate production than individual knockdown of p53 or Parkin, but E H460 H460-p53siRNA H460-con MEF the effects are less than the additive effects. (E) Parkin 10 20 10 5 con con-vector con-siRNA Parkin +/+ increases oxygen consumption of H460 and MEF cells. First 8 p53siRNA 15 Parkin-vector 8 Parkin-siRNA 4 Parkin -/- panel: p53 deficiency reduces oxygen consumption in cells. For p p p – 6 <0.01 p<0.01 6 <0.01 3 <0.01 A C and E, H460-p53siRNA cells were transfected with Parkin 10 expression vector, and H460-con cells were transfected with 4 4 2

consumption siRNA oligos against Parkin 24 h before assays. For D, H460 cells 2 5 O 2 2 1 were transfected with siRNA oligos against p53 and/or Parkin 24 h before assays. Three different siRNA oligos were used, and 0 0 0 0 Time(h) 01 2 3 4 5 6 similar results were obtained as the represented one. Data are 01234560123456012345678 presented as mean ± SD (n =3).#P < 0.05; *P < 0.01.

6 E and F). These results strongly suggest that as a target of p53, Discussion Parkin contributes greatly to the role of p53 in antioxidant defense. Parkin has recently been suggested to be a potential tumor suppressor (18–22). Here we demonstrate that Parkin deficiency Parkin Deficiency Sensitizes Mice to IR-Induced Tumorigenesis. Re- sensitizes mice to IR-induced tumorigenesis, providing further cent studies have suggested that Parkin is a potential tumor direct evidence to support a role of Parkin in tumor suppression. suppressor (18–22). It has been well-established that IR induces Diminished expression of Parkin has frequently been observed in tumorigenesis in mice. To study the impact of Parkin deficiency various tumors, but the mechanisms are not well-understood. − − upon tumorigenesis, 2-mo-old Parkin+/+ and Parkin / C57BL6/J Parkin mutations do not account for all of the decreased Parkin male mice (28) were subjected to a single dose of 4-Gy IR. As shown expression in tumors (18–22). Our finding that p53 regulates in Fig. 7A,Parkinknockoutdidnotenhancetherateofspontaneous Parkin expression not only provides a mechanism for the regu- − − tumors but sensitized mice to IR-induced tumorigenesis; Parkin / lation of Parkin but also suggests that loss of p53, a common mice displayed a shorter tumor latency induced by IR compared event in tumors, is an important mechanism contributing to the with wild-type mice (P < 0.01). The IR-induced tumor spectrum is frequently decreased expression of Parkin in tumors. − − Recently, Parkin was reported to transcriptionally repress p53 similar between Parkin / and wild-type mice; IR mainly induced (29). Our results show that Parkin does not repress p53 expres- lymphomas in the spleen in both mice (Fig. 7B). Interestingly, our fi sion in H460 or HCT116 cells in which p53 induces Parkin ex- results have shown that IR speci cally induced Parkin expression in pression (Fig. S3 A–D). Interestingly, in human neuroblastoma a p53-dependent manner in mouse spleen (Fig. 3 E and F), which SH-SH5Y cells, whereas Parkin represses p53 expression and suggests that the induction of Parkin by p53 in response to IR may transcriptional activity (Fig. S3 A–D), which is consistent with the contribute to the role of Parkin in preventing IR-induced lympho- previous report (29), p53 does not regulate Parkin expression mas in the spleen. These results provide further direct evidence to (Fig. S4 A and B). So far, no negative feedback loop between p53 support the role of Parkin as a potential tumor suppressor. and Parkin was observed in these three cell lines and several

16262 | www.pnas.org/cgi/doi/10.1073/pnas.1113884108 Zhang et al. Downloaded by guest on September 29, 2021 H460-con MEF H460-p53siRNA H460-con Our results demonstrate that as a newly identified important A siRNA Parkin Vector B siRNA Con Parkin +/+ -/- Con Parkin Con PDHA1 component of the p53 signaling pathway, Parkin contributes to Parkin the functions of p53 in both energy metabolism and antioxidant PDHA1 defense. Parkin deficiency results in the Warburg effect, whereas PDHA1 Parkin ectopic expression of Parkin reverses the Warburg effect in cells. Actin Actin These results indicate that decreased expression of Parkin, which has been frequently observed in tumors, should be an important 120 200 mechanism for the Warburg effect in tumors. The reduced mi- C H460-con H460-siRNA * 100 150 tochondrial respiration resulting from Parkin deficiency could be 80 * 60 100 an important mechanism that contributes to enhanced glycolysis 40 * 50 and the Warburg effect in tumor cells. The decreased expression 20 fi

Relative PDH activity of mitochondrial proteins resulting from Parkin de ciency, such

0 Relative PDH activity 0 siRNA Con PDHA1 Parkin Vector Con parkin as PDHA1, contributes to reduced mitochondrial respiration, which in turn promotes the Warburg effect. Recently, several D 200 E H460-con 12 additional mechanisms by which Parkin regulates mitochondrial * Con-siRNA 150 10 PDHA1-siRNA function have been proposed, including regulating to 8 p fi 100 <0.01 clear damaged mitochondria (30), promoting mitochondrial s- * 6

consumption sion (31), and maintaining mitochondrial genome integrity (32), 50 * 2 4 O 2 all of which may contribute to the role of Parkin in regulation of 0 Con- Parkin- Con- Parkin- Con- PDHA1- 0

Relative acetyl-coA levels the Warburg effect. p53 has also been reported to play similar vec t or vec t or siRNA siRNA siRNA siRNA Time (h) 0123456 H460-p53siRNA H460-con H460-con roles in some of these processes, such as autophagy and main- taining mitochondrial genome integrity (6). It is possible that 200 200 400 F H460-con H460-con H460-con Parkin also contributes to the role of p53 in the regulation of * * * 150 150 300 these processes. Thus, maintaining the of energy 100 100 200 metabolism and preventing the Warburg effect could be an im-

50 50 100 portant mechanism contributing to the tumor-suppressive func- Relative lactate levels 0 Relative glycolysis rate 0 0 tion of Parkin. Furthermore, Parkin enhances GSH levels and Relative glucose uptake siRNA Con PDHA1 siRNA Con PDHA1 siRNA Con PDHA1 decreases ROS levels. Considering the important role of ROS in Fig. 5. The regulation of PDHA1 by Parkin contributes to the role of Parkin in tumorigenesis, the antioxidant function of Parkin should also regulating the Warburg effect in cells. (A) Parkin regulates the expression of contribute greatly to its role in tumor suppression. Thus, as a PDHA1. (B) PDHA1 knockdown by siRNA in H460-con cells. (C) The levels of Parkin direct p53 target, Parkin contributes to the functions of p53 in and PDHA1 affect PDH complex activity in H460 cells. (D) Parkin and PDHA1 tumor suppression through the regulation of energy metabolism, regulate the intracellular levels of acetyl-coA. (E) PDHA1 knockdown reduces especially the Warburg effect, and antioxidant defense. oxygen consumption in H460-con cells. (F) PDHA1 knockdown results in enhanced glucose uptake, rate of glycolysis, and lactate production in H460-con cells. Three Materials and Methods different siRNA oligos against Parkin or PDHA1 were used for all assays, and similar Cell Culture. p53-deficient H460-p53siRNA cells were established by stable results were observed. Data are presented as mean ± SD (n =3).*P < 0.01. transduction of a p53 shRNA retroviral vector (pSuper-puro-si-p53) in p53 wild- type H460 cells. H460-con cells are H460 cells with stable transduction of acontrolretroviralvector.p53 wild-type HCT116-conandp53-deficientHCT116- other cell lines we tested. These results strongly suggest that the p53siRNA cells were generous gifts from M. Oren (Weizmann Institute of Sci- − − regulation of Parkin by p53, and the regulation of p53 by Parkin, ence, Rehovot, Israel) (33). MEF Parkin+/+ and Parkin / cells were established could be cell type- or tissue-specific. from wild-type and Parkin−/− C57BL6/J mice (The Jackson Laboratory) (28).

A B C H460-p53siRNA H460-con 80 H460-p53siRNA H460-con MEF 200 80 250 250 Parkin-vector con-vector con-siRNA Parkin-siRNA * # 150 200 200 - * - * # 2

O 150 150 2 2 100 * H O 0 1 2 3 2 0 1 2 3 10 10 10 10 100 100 H 80 10 10 10 10 50 80 Parkin-vector con-vector con-siRNA Parkin-siRNA 50 50 Relative ROS levels Relative Relative ROS levels Relative

+ ROS levels Relative + 0 0 0 Vector Con Parkin Con Parkin siRNA Con Parkin Con Parkin parkin+/+ parkin -/- parkin +/+ parkin -/- H2O2 - - + + 0 1 2 3 H2O2 H2O2 - - + + 10 0 10 1 10 2 10 3 10 10 10 10 - - + + DFE 300 3.5 # 3.5 * 3 * 250 # * 3 2.5 2.5 200 * 2 2 150 1.5 1.5 100 1 * * 1 * * * 50 0.5 GSH/GSSG ratio 0.5 *

Relative ROS levels Relative 0 0 0 Relative GSH levels Con p53 parkin p53 Con p53 parkin p53 Con- Parkin- Con- Parkin- Parkin Parkin Con p53- Con- Parkin- Con- Parkin- Parkin Parkin Con p53- +Parkin +Parkin vector vector siRNA siRNA +/+ -/- siRNA vector vec t or siRNA siRNA +/+ -/- siRNA H2O2 - - - - + + + + H460-p53siRNA H460-con MEF H460 H460-p53siRNA H460-con MEF H460

Fig. 6. Parkin reduces ROS levels and increases GSH levels in cells. (A and B) Parkin reduces ROS levels in cells. H460-p53siRNA cells were transfected with Parkin

expression vectors (A), and H460-con cells were transfected with siRNA oligos against Parkin (B) for 24 h. The cells were then treated with H2O2 (200 μM) for 6 h before assays. (C) Parkin knockout increases ROS levels in MEF cells. (D) Parkin contributes to the role of p53 in ROS regulation. Simultaneous knockdown of p53 and Parkin in H460 cells results in higher ROS levels than individual knockdown of p53 or Parkin, but the effect is less than additive. (E and F) Parkin regulates GSH levels (E) and the GSH:GSSG ratio (F) in H460 and MEF cells. The levels of GSH and GSSG were measured in cells at 24 h after . Three different siRNA CELL BIOLOGY oligos against Parkin were used for all assays, and similar results were observed. Data are presented as mean ± SD (n =3).#P < 0.05; *P < 0.01.

Zhang et al. PNAS | September 27, 2011 | vol. 108 | no. 39 | 16263 Downloaded by guest on September 29, 2021 Measurement of Glucose Uptake, Glycolysis Rate, Lactate Production, and A Oxygen Consumption. Glucose uptake was measured by determining the uptake of 2-[3H]deoxyglucose (American Radiolabeled Chemicals) by cells as previously described (34). Glycolysis rate was measured by monitoring the 3 3 p>0.05 conversion of 5-[ H]glucose to H2O as described (5, 9). Lactate levels in the culture media of cells were determined by using a Lactate Assay Kit (Bio- p<0.01 vision). Oxygen consumption in cells was measured by using the BD Oxygen Biosensor System (BD Biosciences) as described (11).

Measurements of Activity of PDH and Levels of Acetyl-CoA, ATP, ROS, and GSH.

Wild type IR Parkin-/- IR The activity of PDH was measured by using a PDH Activity Assay Kit B Tumor % (n) % (n) (MitoSciences). The intracellular levels of acetyl-CoA were measured by using Lymphoma 66.7 (4) 78.5 (11) an Acetyl-CoA Assay Kit (Biovision). The intracellular levels of ATP were Sarcoma 33.3 (2) 14.2 (2) measured by using an ATP Bioluminescence Assay Kit (Roche) as described Other 0 (0) 7.3 (1) (11). ROS levels were measured by flow cytometry as described (9, 11). The Total 100 (6) 100 (14) levels of GSH and GSSG were measured by using a glutathione detection kit (Biovision) as described (9, 11). Fig. 7. Loss of Parkin sensitizes mice to IR-induced tumorigenesis. Two- − − month-old wild-type and Parkin / C57BL6/J male mice were subjected to γ-Irradiation and IR-Induced Tumorigenesis of Mice. Two-month-old p53+/+ − − a single dose of 4-Gy IR and monitored for survival. (A) Kaplan–Meier curve and p53 / male C57BL6/J mice were treated with IR (4 Gy). Mice were killed −/− demonstrates that Parkin mice had a significantly shorter tumor latency at different times after IR (n = 6 for each time point), and different tissues < induced by IR compared with wild-type mice (P 0.01). (B) The similar IR- were collected to determine Parkin expression. For tumorigenesis assays, 2- −/− induced tumor spectrum between Parkin and wild-type mice. mo-old wild-type and Parkin−/− C57BL6/J mice (28) (The Jackson Laboratory) were subjected to 4-Gy IR. Mice were examined three times/wk until mori- bund. The statistical differences in tumor latency were analyzed by Kaplan– ChIP and Luciferase Activity Assays. ChIP assays were performed in H460-con Meier analysis. and H460-p53siRNA cells treated with Etoposide (10 μM for 16 h) to activate See SI Materials and Methods for details. p53 as described (11, 25). The pGL2 firefly luciferase reporter containing putative p53 REs in human and mouse Parkin genes was constructed, and luciferase activity assays were performed as described (11, 25). ACKNOWLEDGMENTS. This work was supported by grants from the National Institutes of Health (1R01CA143204-01), New Jersey Commission on Cancer Research (NJCCR), and Foundation of the University of Medicine and Real-Time PCR, Western Blot Analysis, and IF Staining. TaqMan real-time PCR Dentistry of New Jersey (to Z.F.), and by a grant from the National Institutes was performed as described (11, 25). Western blot analysis and IF staining of Health (1P30CA147892-01) (to A.J.L. and W.H.). C.Z. is supported by a were performed as previously described (11). postdoctoral fellowship from the NJCCR.

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