Wild-Type p53 Promotes Cancer Metabolic Switch by Inducing PUMA-Dependent Suppression of Oxidative Phosphorylation

•WTp53-PUMA pathway drives cancer metabolic switch•PUMA suppresses mitochondrial pyruvate uptake by inactivating MPC•IKKβ-mediated phosphorylation of PUMA is important for PUMA-MPC interaction•High levels of PUMA in HCC are correlated with poor prognosis of HCC patients The tumor suppressor p53 is somatically mutated in half of all human cancers. Paradoxically, the wild-type p53 (WTp53) is often retained in certain human cancers, such as hepatocarcinoma (HCC). We discovered a physiological and oncogenic role of WTp53 in suppressing pyruvate-driven oxidative phosphorylation by inducing PUMA. PUMA inhibits mitochondrial pyruvate uptake by disrupting the oligomerization and function of mitochondrial pyruvate carrier (MPC) through PUMA-MPC interaction, which depends on IκB kinase-mediated phosphorylation of PUMA at Ser96/106. High expression levels of PUMA are correlated with decreased mitochondrial pyruvate uptake and increased glycolysis in HCCs and poor prognosis of HCC patients. These findings are instrumental for cancer drug discovery aiming at activating WTp53 or restoring WTp53 activity to p53 mutants. The tumor suppressor p53 is somatically mutated in half of all human cancers. Paradoxically, the wild-type p53 (WTp53) is often retained in certain human cancers, such as hepatocarcinoma (HCC). We discovered a physiological and oncogenic role of WTp53 in suppressing pyruvate-driven oxidative phosphorylation by inducing PUMA. PUMA inhibits mitochondrial pyruvate uptake by disrupting the oligomerization and function of mitochondrial pyruvate carrier (MPC) through PUMA-MPC interaction, which depends on IκB kinase-mediated phosphorylation of PUMA at Ser96/106. High expression levels of PUMA are correlated with decreased mitochondrial pyruvate uptake and increased glycolysis in HCCs and poor prognosis of HCC patients. These findings are instrumental for cancer drug discovery aiming at activating WTp53 or restoring WTp53 activity to p53 mutants. Glycolysis is a hallmark of cancer metabolism and is required for tumorigenesis. In contrast to the current paradigm in which WTp53 plays multiple roles in promoting oxidative phosphorylation and inhibiting glycolysis by regulating the expression or activity of metabolic enzymes, we discovered a dominant metabolic role of WTp53 in promoting cancer metabolic switch from oxidative phosphorylation to glycolysis by inducing PUMA-mediated disruption of mitochondrial pyruvate uptake. This role of WTp53 can resolve several long-lasting paradoxes in p53 biology and will be instrumental in the development of cancer therapy, especially in the context of the highly pursued strategies to eliminate human cancer by either activating WTp53 or restoring WTp53 function to p53 mutants in cancers. It has been well established that p53 is critical to suppress cancer development in humans. As the “guardian of the genome,” p53 plays complex roles in cell-cycle arrest, apoptosis, and senescence, all of which likely contribute to the protection of the genome from accumulating mutations and passing these mutations to the daughter cells (Kastenhuber and Lowe, 2017Kastenhuber E.R. Lowe S.W. Putting p53 in context.Cell. 2017; 170: 1062-1078Abstract Full Text Full Text PDF PubMed Scopus (960) Google Scholar, Vousden and Prives, 2009Vousden K.H. Prives C. Blinded by the light: the growing complexity of p53.Cell. 2009; 137: 413-431Abstract Full Text Full Text PDF PubMed Scopus (2325) Google Scholar). p53 also plays important roles in maintaining genomic stability of pluripotent stem cells by coordinating the DNA damage responses with pluripotency (Lin et al., 2005Lin T. Chao C. Saito S. Mazur S.J. Murphy M.E. Appella E. Xu Y. p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression.Nat. 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In contrast, PUMA is overexpressed in many human cancers (Cai et al., 2013Cai W. Li Q. Yang Z. Miao X. Wen Y. Huang S. Ouyang J. Expression of p53 upregulated modulator of apoptosis (PUMA) and C-myb in gallbladder adenocarcinoma and their pathological significance.Clin. Transl. Oncol. 2013; 15: 818-824Crossref PubMed Scopus (20) Google Scholar, Du et al., 2012Du Q.H. Zhang K.J. Jiao X.L. Zhao J. Zhang M. Yan B.M. Xu Y.B. Prognostic significance of PUMA in pancreatic ductal adenocarcinoma.J. Int. Med. Res. 2012; 40: 2066-2072Crossref PubMed Scopus (4) Google Scholar, Kim et al., 2007Kim M.R. Jeong E.G. Chae B. Lee J.W. Soung Y.H. Nam S.W. Lee J.Y. Yoo N.J. Lee S.H. Pro-apoptotic PUMA and anti-apoptotic phospho-BAD are highly expressed in colorectal carcinomas.Dig. Dis. Sci. 2007; 52: 2751-2756Crossref PubMed Scopus (18) Google Scholar) and the loss of PUMA ablates tumorigenesis in certain mouse models (Michalak et al., 2010Michalak E.M. Vandenberg C.J. Delbridge A.R. Wu L. Scott C.L. 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Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (43102) Google Scholar), accumulating data support the notion that p53 might confer tumor suppression by inhibiting glycolysis and promoting oxidative phosphorylation (Shen et al., 2012Shen L. Sun X. Fu Z. Yang G. Li J. Yao L. The fundamental role of the p53 pathway in tumor metabolism and its implication in tumor therapy.Clin. Cancer Res. 2012; 18: 1561-1567Crossref PubMed Scopus (61) Google Scholar). In this context, numerous reports have shown that p53 exerts this metabolic regulation by regulating the expression of metabolic genes, such as p53-induced glycolysis and apoptosis regulator (TIGAR) and glucose transporters; synthesis of cytochrome c oxidase 2, glutaminase 2, and malic enzyme; or protein-protein interactions with metabolic enzymes, such as glucose-6-phosphate dehydrogenase, peroxisome-proliferator-activated receptor γ coactivator-1, and SREBP (Humpton and Vousden, 2016Humpton T.J. Vousden K.H. Regulation of cellular metabolism and hypoxia by p53.Cold Spring Harb. Perspect. Med. 2016; 6https://doi.org/10.1101/cshperspect.a026146Crossref PubMed Scopus (87) Google Scholar, Kruiswijk et al., 2015Kruiswijk F. Labuschagne C.F. Vousden K.H. p53 in survival, death and metabolic health: a lifeguard with a licence to kill.Nat. Rev. Mol. Cell Biol. 2015; 16: 393-405Crossref PubMed Scopus (706) Google Scholar). These data support the current paradigm in which wild-type p53 (WTp53) suppresses tumorigenesis by inhibiting cancer metabolic switch from oxidative phosphorylation to glycolysis (Kruiswijk et al., 2015Kruiswijk F. Labuschagne C.F. Vousden K.H. p53 in survival, death and metabolic health: a lifeguard with a licence to kill.Nat. Rev. Mol. Cell Biol. 2015; 16: 393-405Crossref PubMed Scopus (706) Google Scholar). Consistent with the notion that the inactivation of WTp53 is required for cancer initiation and development, genomic DNA sequencing of human cancers indicates that the p53 gene (TP53) is somatically mutated in over 50% of all human cancers (Bykov et al., 2017Bykov V.J.N. Eriksson S.E. Bianchi J. Wiman K.G. Targeting mutant p53 for efficient cancer therapy.Nat. Rev. Cancer. 2017; 18: 89-102Crossref PubMed Scopus (497) Google Scholar). In addition to the loss of WTp53 activity, the expressed p53 mutants gain oncogenic activities to promote drug resistance, glycolysis, and other aspects of tumorigenesis (Bykov et al., 2017Bykov V.J.N. Eriksson S.E. Bianchi J. Wiman K.G. Targeting mutant p53 for efficient cancer therapy.Nat. Rev. Cancer. 2017; 18: 89-102Crossref PubMed Scopus (497) Google Scholar, Xu, 2008Xu Y. Induction of genetic instability by gain-of-function p53 cancer mutants.Oncogene. 2008; 27: 3501-3507Crossref PubMed Scopus (50) Google Scholar). The inactivation of p53 activity can be achieved through multiple mechanisms, including overexpression of the p53 inhibitor MDM2/MDM4 or disruption of pathways required for WTp53 activation (Bykov et al., 2017Bykov V.J.N. Eriksson S.E. Bianchi J. Wiman K.G. Targeting mutant p53 for efficient cancer therapy.Nat. Rev. Cancer. 2017; 18: 89-102Crossref PubMed Scopus (497) Google Scholar). However, the frequency of p53 mutation remains low in certain types of human cancers such as hepatocellular carcinoma (HCC) (Soussi and Wiman, 2007Soussi T. Wiman K.G. Shaping genetic alterations in human cancer: the p53 mutation paradigm.Cancer Cell. 2007; 12: 303-312Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Considering that genetic instability is a hallmark of human cancers, and cancer cells are under intensive selection for pro-survival genetic mutations (Hanahan and Weinberg, 2011Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (43102) Google Scholar), it remains a paradox why the somatic mutation of TP53 is not selected for in these cancers. Therefore, in contrast to the general assumption that WTp53 functions as a tumor suppressor, we hypothesize that WTp53 might play oncogenic roles in promoting tumorigenesis of human cancers harboring WTp53. To examine the roles of WTp53 in human cancer cells, we silenced the expression of WTp53 in human cancer cells with the tetracycline-inducible p53 short hairpin RNA (shRNA) system. The knockdown (KD) of WTp53 was achieved after doxycycline (Doxy, 1 μg/mL) treatment for 4 days, leading to the downregulation of the p53 target gene PUMA in some human cancer cells (Figures S1A and S1B). In contrast to the general assumption that WTp53 suppresses tumorigenesis, the KD of p53 inhibited the proliferation of four HCC cell lines harboring WTp53 (HepG2, SK-Hep1, BEL7404, and SMAC7721 cells) but had no apparent impact on other human cancer cell lines harboring WTp53 (Figure 1A). Considering the importance of glycolysis in cancer cell proliferation (Hanahan and Weinberg, 2011Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (43102) Google Scholar), to understand the basis of the differential impact of WTp53 KD on the proliferation of various human cancer cell lines, we analyzed the impact of WTp53 KD on glycolysis. The KD of WTp53 decreased glycolysis in the four HCC cell lines but not other human cancer cell lines, supporting the notion that the impaired proliferation was due to the inhibition of glycolysis in these HCC cells after p53 depletion (Figures 1B and 1C). Consistent data were obtained using distinct p53 shRNAs, ruling out the possibility of off-target effects (Figures S2A–S2C). To further understand how WTp53 promotes glycolysis in these HCC cells, we analyzed the pyruvate-driven oxidative phosphorylation (OXPHOS) activity in these cells. Our data indicated that p53 KD significantly increased pyruvate-driven ATP production in HCC cells but not in other human cancer cell lines, suggesting that WTp53 suppresses mitochondrial OXPHOS in these HCC cells (Figure 1D). Because TP53 is not frequently mutated in HCC (Soussi and Wiman, 2007Soussi T. Wiman K.G. Shaping genetic alterations in human cancer: the p53 mutation paradigm.Cancer Cell. 2007; 12: 303-312Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar), we focused on two HCC cell lines (HepG2 and SK-Hep1) to further investigate the roles of WTp53 in tumorigenesis. WTp53 can be activated by RITA, a small-molecule p53 activator (Issaeva et al., 2004Issaeva N. Bozko P. Enge M. Protopopova M. Verhoef L.G.G.C. Masucci M. Pramanik A. Selivanova G. Small molecule RITA binds to p53, blocks p53–HDM-2 interaction and activates p53 function in tumors.Nat. Med. 2004; 10: 1321-1328Crossref PubMed Scopus (650) Google Scholar). Treatment of HepG2 cells with RITA (0.3 μM) modestly increased the expression of p53 target genes such as PUMA and CDKN1A, and promoted glycolysis in a p53-dependent manner (Figures 1E–1G). This dosage of RITA did not induce apoptosis of HCC cells (Figure S2D). The activation of WTp53 in SK-Hep1 cells by RITA also induced the expression of PUMA and CDKN1A as well as glycolysis (Figures S2E and S2F). Therefore, the modest activation of WTp53 in HCCs promotes cancer metabolic switch without inducing significant apoptosis. Consistent with the findings that the KD of WTp53 inhibited the cellular proliferation and glycolysis of HepG2 and SK-Hep1 cells but not those of HeLa and HCT116 cells, the KD of WTp53 greatly suppressed the in vivo tumorigenesis of HepG2 and SK-Hep1 cells but not that of HeLa and HCT116 cells (Figures 1H, 1I, and S2G–S2I). These data supported the notion that WTp53 is required for the tumorigenesis and glycolytic metabolism of HCC cells. Human cancer cells such as HeLa and HCT116 cells derived their energy primarily from glycolysis, and their OXPHOS activity had essentially been abolished via WTp53-independent mechanisms (Figures 1B, 1C, and S2I). In summary, these findings suggest that WTp53 is required to maintain cancer metabolic switch in human cancer cells with relatively higher mitochondrial OXPHOS activity. To understand the mechanism of how WTp53 suppresses mitochondrial OXPHOS, we focused on PUMA, which is a transcriptional target of p53 and encodes a mitochondrial protein required for p53-dependent apoptosis (Jeffers et al., 2003Jeffers J.R. Parganas E. Lee Y. Yang C. Wang J. Brennan J. MacLean K.H. Han J. Chittenden T. Ihle J.N. et al.PUMA is an essential mediator of p53-dependent and -independent apoptotic pathways.Cancer Cell. 2003; 4: 321-328Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar, Villunger et al., 2003Villunger A. Michalak E.M. Coultas L. Mullauer F. Bock G. Ausserlechner M.J. Adams J.M. Strasser A. p53- and drug-induced apoptotic responses mediated by BH3-only proteins PUMA and noxa.Science. 2003; 302: 1036-1038Crossref PubMed Scopus (1100) Google Scholar). In addition, experimental and clinical data suggest that PUMA is oncogenic (Hikisz and Kiliańska, 2012Hikisz P. Kiliańska Z. PUMA, a critical mediator of cell death—one decade on from its discovery.Cell. Mol. Biol. Lett. 2012; 17: 646-669Crossref PubMed Scopus (94) Google Scholar). PUMA KD in HepG2 cells greatly inhibited their growth in vitro and in vivo, supporting the notion that PUMA mediates WTp53 function in promoting tumorigenesis of HCC cells (Figures 2A and 2B). In addition, similar to the impact of WTp53 KD on OXPHOS of HepG2 cells, PUMA KD in HepG2 cells inhibited the production of glycolytic ATP and lactate (Figures 2C–2E). PUMA KD, but not p21 KD, increased the pyruvate-driven OXPHOS and pyruvate mitochondrial uptake (Figures 2F and 2G). By generating PUMA knockout (PUMA KO) HepG2 cells using the CRISPR/CAS9 approach (Figures S3A–S3C), we showed that PUMA was important for WTp53-dependent suppression of OXPHOS in HepG2 cells after RITA treatment (Figure S3D). Because the inhibition of mitochondrial pyruvate uptake inhibits OXPHOS and induces glycolysis (Schell et al., 2014Schell J.C. Olson K.A. Jiang L. Hawkins A.J. Van Vranken J.G. Xie J. Egnatchik R.A. Earl E.G. DeBerardinis R.J. Rutter J. A role for the mitochondrial pyruvate carrier as a repressor of the Warburg effect and colon cancer cell growth.Mol. Cell. 2014; 56: 400-413Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), these data indicate that PUMA mediates the roles of WTp53 in promoting metabolic switch from OXPHOS to glycolysis in some HCC cells. The silencing of PUMA appears to have a more dramatic impact on OXPHOS than silencing of WTp53, suggesting that WTp53 is only one of the inducers of PUMA in human cancer cells. To confirm the physiological relevance of this metabolic role of the p53-PUMA axis, we examined the roles of p53 and PUMA in mitochondrial pyruvate metabolism in mouse liver. Using the Trp53TSD (Thr18Ser20 mutated to Asp) knockin mouse model with constitutively active p53 (Liu et al., 2010Liu D. Ou L. Clemenson Jr., G.D. Chao C. Lutske M.E. Zambetti G.P. Gage F.H. Xu Y. PUMA is required for p53-induced depletion of adult stem cells.Nat. Cell Biol. 2010; 12: 993-998Crossref PubMed Scopus (91) Google Scholar) and Puma−/− mice (Jeffers et al., 2003Jeffers J.R. Parganas E. Lee Y. Yang C. Wang J. Brennan J. MacLean K.H. Han J. Chittenden T. Ihle J.N. et al.PUMA is an essential mediator of p53-dependent and -independent apoptotic pathways.Cancer Cell. 2003; 4: 321-328Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar), we showed that the activation of p53 in mouse liver increased the expression of Puma and Cdkn1a (Figure 2H). In addition, WTp53 suppressed mitochondrial pyruvate uptake and pyruvate-driven mitochondrial ATP production in mouse hepatocytes in a PUMA-dependent manner (Figure 2I). Therefore, the p53-PUMA pathway plays an evolutionarily conserved and physiological role in inhibiting mitochondrial pyruvate uptake and OXPHOS. Mitochondrial pyruvate carrier (MPC), a mitochondrial membrane complex composed of MPC1 and MPC2 proteins, is important to transport pyruvate into mitochondria (Herzig et al., 2012Herzig S. Raemy E. Montessuit S. Veuthey J.L. Zamboni N. Westermann B. Kunji E.R. Martinou J.C. Identification and functional expression of the mitochondrial pyruvate carrier.Science. 2012; 337: 93-96Crossref PubMed Scopus (469) Google Scholar). To elucidate the mechanism of how PUMA inhibits mitochondrial pyruvate uptake, we hypothesized that PUMA, also a mitochondrial membrane protein (Wilfling et al., 2012Wilfling F. Weber A. Potthoff S. Vogtle F.N. Meisinger C. Paschen S.A. Hacker G. BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax.Cell Death Differ. 2012; 19: 1328-1336Crossref PubMed Scopus (54) Google Scholar), might inhibit the function of MPC through protein-protein interaction. Using co-immunoprecipitation (CO-IP), we demonstrated that the overexpressed PUMA isoforms α and β interacted with MPC (Figure 3A). Consistent with this finding, the interaction between the endogenous MPC and PUMA was confirmed in HepG2 cells with CO-IP (Figure 3B). To further validate the interaction between MPC and PUMA in live cells, we employed fluorescence resonance energy transfer (FRET) assay in live cells co-expressing eGFP-tagged MPC2 and mCherry-tagged PUMA or various deletion mutants of PUMA. The data demonstrated that the full-length PUMA and two PUMA deletion mutants (del92 and delBH3) interacted with MPC2 in live cells, but PUMA deletion mutant (del137) failed to interact with MPC2, indicating that the PUMA domain between amino acids (aa) 92 and 137 is required for the interaction with MPC2 (Figure 3C). To examine the interaction between endogenous PUMA and MPC in HepG2 cells, we used confocal immunofluorescence analysis to show close colocalization of the endogenous PUMA and MPC1 in HepG2 cells (Figure S4A). In addition, using primary antibodies against PUMA and MPC1 followed by Cy5/Cy3 conjugated secondary antibodies, we showed significant FRET efficiency (>10%) between endogenous PUMA and MPC1, indicating significant interaction between the endogenous PUMA and MPC1 (Figures S4B and S4C). Since the oligomerization of MPC1 and MPC2 is required for the function of MPC to transport pyruvate into mitochondria (Herzig et al., 2012Herzig S. Raemy E. Montessuit S. Veuthey J.L. Zamboni N. Westermann B. Kunji E.R. Martinou J.C. Identification and functional expression of the mitochondrial pyruvate carrier.Science. 2012; 337: 93-96Crossref PubMed Scopus (469) Google Scholar), we examined the impact of PUMA on the oligomerization of MPC1 and MPC2. PUMA KO in HepG2 cells increased the hetero-oligomerization of MPC1 and MPC2 as well as the homo-oligomerization of MPC2 (Figure 3D). In support of this conclusion, using FRET assay, we demonstrated that PUMA inhibited both the homo-oligomerization of MPC2 and the hetero-oligomerization of MPC1 and MPC2 in live 293 cells (Figure 3E). As predicted from the PUMA-MPC2 interaction data, when ectopically expressed in PUMA KO cells at the endogenous levels that did not induce apoptosis, both PUMA and PUMA mutant (delBH3) effectively suppressed MPC-dependent pyruvate uptake into the mitochondria and rescued the proliferation defects of PUMA KO cells (Figures S4D–S4G). Therefore, PUMA inhibits the mitochondrial pyruvate uptake by disrupting the oligomerization and function of the MPC complex. To further understand the mechanism regulating the interaction between PUMA and MPC, we focused on the central domain (aa 92–137) of PUMA that is required for the interaction between MPC and PUMA. There are three potential phosphorylation sites at Ser10, Ser96. and Ser106 of PUMA (Fricker et al., 2010Fricker M. O'Prey J. Tolkovsky A.M. Ryan K.M. Phosphorylation of PUMA modulates its apoptotic function by regulating protein stability.Cell Death Dis. 2010; 1: e59Crossref PubMed Scopus (55) Google Scholar), two of which are located within the region of PUMA required for its interaction with MPC. Using the phosphorylation site prediction software GPS (3.538 score in high threshold cutoff) (Xue et al., 2005Xue Y. Zhou F. Zhu M. Ahmed K. Chen G. Yao X. GPS: a comprehensive www server for phosphorylation sites prediction.Nucleic Acids Res. 2005; 33: W184-W187Crossref PubMed Scopus (201) Google Scholar), we predicted that S96 and S106 could be the phosphorylation sites of IκB kinase β (IKKβ). In support of this notion, we showed that the inhibition of IKKβ kinase activity with specific inhibitors prevented the Ser/Thr phosphorylation of PUMA after tumor necrosis factor alpha (TNF-α) stimulation (Figure 4A). In addition, the IKKβ-mediated phosphorylation of the phosphorylation site mutant (S96/106A) of PUMA, denoted MT PUMA, was significantly reduced compared with that of WT PUMA after TNF-α stimulation (Figure 4B). Together, these data indicate that IKKβ phosphorylates PUMA at S96 and S106. While it has been reported that the phosphorylation of PUMA at S10 regulates its apoptotic function (Fricker et al., 2010Fricker M. O'Prey J. Tolkovsky A.M. Ryan K.M. Phosphorylation of PUMA modulates its apoptotic function by regulating protein stability.Cell Death Dis. 2010; 1: e59Crossref PubMed Scopus (55) Google Scholar), the phosphorylation of PUMA at S96 and S106 did not affect its protein stability and apoptotic activity (Figures S5A–S5D). To elucidate the roles of IKKβ in PUMA-MPC interaction, we examined the impact of the increased IKKβ expression on the oligomerization of MPC1 and MPC2. The increased expression of IKKβ promoted the interaction between PUMA and MPC and decreased the oligomerization of MPC1 and MPC2, suggesting that phosphorylation of PUMA at S96/106 by IKKβ promotes the interaction between PUMA and MPC (Figure 4C). The IKKβ pathway is oncogenic and drives the cancer metabolic switch from