Paradoxical Puma Prohibits Pyruvate Pumps to Prime Pathology
Authors: Douglas R. Green
Journal: Cancer Cell (2019)

PUMA is a pro-apoptotic Bcl-2 family protein that can act as a tumor suppressor or oncogene in different cancers. In this issue, Kim et al. show that PUMA, independent of its apoptotic function, enforces glycolytic metabolism by inhibiting the transport of pyruvate into the mitochondria, promoting hepatocellular carcinoma. PUMA is a pro-apoptotic Bcl-2 family protein that can act as a tumor suppressor or oncogene in different cancers. In this issue, Kim et al. show that PUMA, independent of its apoptotic function, enforces glycolytic metabolism by inhibiting the transport of pyruvate into the mitochondria, promoting hepatocellular carcinoma. Peter Piper picked a peck of pickled peppers.—Anonymous To paraphrase the late Stan Korsmeyer, if the control of apoptosis is the "night job" of the Bcl-2 family proteins, what are their "day jobs"? The Bcl-2 family is largely defined by this night job of apoptotic regulation and the presence of one or more of the four Bcl-2 Homology (BH) regions. The largest subfamily is made up of pro-apoptotic proteins that carry only a BH3 region and hence are called BH3-only proteins. The BH3 region, itself, is generally sufficient to carry out the night job function of BH3-only proteins, and therefore, we can wonder whether these molecules have additional functions (day jobs). Prominent among the BH3-only proteins is p53-upregulated mediator of apoptosis (PUMA), and a study in this issue (Kim et al., 2019Kim J. Yu L. Chen W. Xu Y. Wu M. Todorova D. Tang Q. Feng B. Jiang L. He J. et al.Wild-type p53 promotes cancer metabolic switch by inducing PUMA-dependent suppression of oxidative phosphorylation.Cancer Cell. 2019; 35 (this issue): 191-203Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) sheds light on a novel day job for PUMA in regulating metabolism in hepatocytes and hepatocellular carcinomas. As its name implies, PUMA is induced by p53 (and also by p73), and it is required for DNA damage-induced apoptosis in thymocytes, fibroblasts, and hematopoietic stem cells. Its expression is also induced in p53-independent ways, such as by Foxo3A upon growth factor deprivation, and C/EBP homologous protein and E2F1 following endoplasmic reticulum stress, and upon overexpression of c-Myc (Hikisz and Kiliańska, 2012Hikisz P. Kiliańska Z.M. 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). Once expressed, the BH3 of PUMA binds to the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1, and A1/Bfl, neutralizing them, and it can also directly activate the pro-apoptotic effectors Bax and Bak to cause mitochondrial outer membrane permeabilization (MOMP) and apoptosis (Green, 2018Green D.R. Cell Death: Apoptosis and Other Means to an End. Cold Spring Harbor Press, 2018Google Scholar). In addition to the BH3, PUMA possesses a mitochondrial localizing sequence that directs it to the mitochondrial outer membrane, facilitating its pro-apoptotic effects. Given these pro-apoptotic functions, it is probably not surprising that the PUMA gene is often deleted (e.g., in head and neck cancers) or silenced (e.g., in B cell lymphomas), although mutations in the coding region of PUMA have not been observed in cancers. In contrast, robust PUMA protein expression is often observed in some cancers, including colorectal carcinoma (CRC) and hepatocellular carcinoma (HCC). While deletion of the PUMA gene accelerated Myc-induced B cell lymphomagenesis, ablation of PUMA actually prevented radiation-induced thymomagenesis and chemically induced HCC in mice (Labi et al., 2010Labi V. Erlacher M. Krumschnabel G. Manzl C. Tzankov A. Pinon J. Egle A. Villunger A. Apoptosis of leukocytes triggered by acute DNA damage promotes lymphoma formation.Genes Dev. 2010; 24: 1602-1607Crossref PubMed Scopus (86) Google Scholar, Michalak et al., 2010Michalak E.M. Vandenberg C.J. Delbridge A.R. Wu L. Scott C.L. Adams J.M. Strasser A. Apoptosis-promoted tumorigenesis: gamma-irradiation-induced thymic lymphomagenesis requires Puma-driven leukocyte death.Genes Dev. 2010; 24: 1608-1613Crossref PubMed Scopus (101) Google Scholar, Qiu et al., 2011Qiu W. Wang X. Leibowitz B. Yang W. Zhang L. Yu J. PUMA-mediated apoptosis drives chemical hepatocarcinogenesis in mice.Hepatology. 2011; 54: 1249-1258Crossref PubMed Scopus (72) Google Scholar). These observations present the "PUMA paradox" in cancer: why is a pro-apoptotic protein and bona fide tumor suppressor not only expressed in some cancers, but apparently even required for their generation? Convincing attempts to explain this paradox suggest that apoptosis, such as that induced by DNA damage-induced p53 and PUMA expression, is important in driving compensatory proliferation of precursor cells, thus expanding mutant clones that become transformed upon acquisition of subsequent mutations. This idea is supported by the observation that p53 functions to suppress radiation-induced thymomagenesis, not at the initial, apoptotic phase but later as cells transform (Christophorou et al., 2006Christophorou M.A. Ringshausen I. Finch A.J. Swigart L.B. Evan G.I. The pathological response to DNA damage does not contribute to p53-mediated tumour suppression.Nature. 2006; 443: 214-217Crossref PubMed Scopus (337) Google Scholar). Indeed, induction of thymocyte apoptosis by glucocorticoids promoted radiation-induced thymomas in PUMA-deficient animals (Michalak et al., 2010Michalak E.M. Vandenberg C.J. Delbridge A.R. Wu L. Scott C.L. Adams J.M. Strasser A. Apoptosis-promoted tumorigenesis: gamma-irradiation-induced thymic lymphomagenesis requires Puma-driven leukocyte death.Genes Dev. 2010; 24: 1608-1613Crossref PubMed Scopus (101) Google Scholar). It is logical to conclude that similar apoptosis and compensatory proliferation functions in chemically induced HCC (Qiu et al., 2011Qiu W. Wang X. Leibowitz B. Yang W. Zhang L. Yu J. PUMA-mediated apoptosis drives chemical hepatocarcinogenesis in mice.Hepatology. 2011; 54: 1249-1258Crossref PubMed Scopus (72) Google Scholar). The above idea, however, does not explain why expression of this potent pro-apoptotic protein is maintained in HCC and other cancers. The observations by Kim et al. suggest a different explanation. Unlike many cancers, HCCs often express wild-type p53. They found that several HCC cell lines require p53 for their optimal proliferation and tumor formation and that p53 sustains the metabolism of these cells in their usual glycolytic state in which most pyruvate is converted to lactate. Silencing p53 shifted this metabolism to mitochondrial pyruvate uptake and OXPHOS-dependent ATP production. Similarly, they found that hepatocytes in animals carrying a constitutively active p53 displayed decreased mitochondrial pyruvate and pyruvate-dependent ATP production. While p53 can control glycolysis and OXPHOS by several different mechanisms (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 (700) Google Scholar), they found that these effects of p53 in hepatocytes and HCC cell lines were dependent on PUMA. They found that PUMA directly disrupts the function of the mitochondrial pyruvate carrier (MPC) to promote OXPHOS via entry of pyruvate into the mitochondrial TCA cycle. MPC is composed of MPC1 and MPC2, both of which are required for pyruvate transport into mitochondria. Levels of MPC1 and MPC2 are frequently reduced in many cancers and cancer cell lines, and low expression of one or both correlates with poor prognosis in CRC, non-small cell lung cancer, and kidney clear cell carcinoma (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 (246) Google Scholar). Enforced expression of these components in CRC lines shifts glycolysis to OXPHOS and reduces growth in vivo (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 (246) Google Scholar). Unlike the above cancers, expression of MPC does not appear to correlate with prognosis in HCC patients, while Kim et al. found that high PUMA expression in HCC corresponded to poor outcomes. They found that PUMA binds specifically to MPC2, disrupting its interaction with MPC1 in vitro and in cells, and thus PUMA may serve the same function as loss of MPC expression in maintaining high levels of glycolysis in HCC. Kim et al. further found that modification of PUMA is important for its metabolic activity, requiring IKKβ-mediated phosphorylation of serine 96 and 104. In contrast, cytokine-driven IKK complex activation in lymphoid cells induces phosphorylation of PUMA on S10, which targets it for proteasomal degradation (Sandow et al., 2012Sandow J.J. Jabbour A.M. Condina M.R. Daunt C.P. Stomski F.C. Green B.D. Riffkin C.D. Hoffmann P. Guthridge M.A. Silke J. et al.Cytokine receptor signaling activates an IKK-dependent phosphorylation of PUMA to prevent cell death.Cell Death Differ. 2012; 19: 633-641Crossref PubMed Scopus (27) Google Scholar). Clearly, the IKKβ-mediated phosphorylation of PUMA on S96 and S104 observed by Kim et al. does not destabilize PUMA, and it is unclear why IKKβ is active, and why S10 phosphorylation does not appear to occur in the HCC cells. The region of interaction with MPC2 in PUMA is between aa92–137, and, thus, it is likely that the modification promotes interaction either directly or with a protein that interacts with both. Nevertheless, it is unlikely that phosphorylation of PUMA on S96 and S104 affects its pro-apoptotic function. The function of PUMA to disrupt the interaction of MCP2 with MCP1 does not appear to require an intact BH3 region, and as PUMA is intrinsically unstructured, the BH3 should be available for interaction with other Bcl-2 proteins. However, the BH3 of PUMA stretches from W133 (h0) to L148 (h4) (h0–4 are hydrophobic residues that interact with the BH groove of anti-apoptotic Bcl-2 proteins [Green, 2018Green D.R. Cell Death: Apoptosis and Other Means to an End. Cold Spring Harbor Press, 2018Google Scholar]), and this partially overlaps the region of MCP2 interaction (aa92–137), which presumably is exposed, even when PUMA is bound via its BH3. (Kim et al. did not formally show that aa133–137 are required for interaction with MCP2). The persistent expression of PUMA does not result in apoptosis in HCC cells, and therefore it is likely that it is complexed with anti-apoptotic proteins at the mitochondrial outer membrane to prevent its apoptotic effects, while permitting its inhibition of MPC function (Figure 1). The effects of enforced anti-apoptotic Bcl-2 expression on the effects of PUMA-mediated MPC inhibition will be interesting to explore. The apparent requirement for PUMA to regulate HCC metabolism and promote optimal growth presents a potential opportunity for therapy. Assuming that anti-apoptotic Bcl-2 function is important for preventing PUMA-induced apoptosis, disruption of such function, even modestly, may promote such apoptosis. BH3 mimetic drugs that target Bcl-2, Bcl-xL, and Mcl-1 are in clinical use (either as approved therapies or in clinical trials), although, to date, their application to HCC has not been evident. Again, it will be useful to determine what, if any, effect such blockade will have on the function of PUMA in HCC. Wild-Type p53 Promotes Cancer Metabolic Switch by Inducing PUMA-Dependent Suppression of Oxidative PhosphorylationKim et al.Cancer CellJanuary 31, 2019In BriefHepatocellular carcinomas (HCCs) often retain the wild-type p53. Kim et al. find that p53 is important for the growth of HCC cells and that p53-regulated PUMA reduces mitochondrial pyruvate uptake and increases glycolysis in HCC, suggesting caution when designing cancer treatment strategies that activate p53. Full-Text PDF Open Archive