Wenjing Du

p53 regulates biosynthesis through direct inactivation of glucose-6-phosphate dehydrogenase

Cancer cells consume large quantities of glucose and primarily use glycolysis for ATP production, even in the presence of adequate oxygen. This metabolic signature (aerobic glycolysis or the Warburg effect) enables cancer cells to direct glucose to biosynthesis, supporting their rapid growth and proliferation. However, both causes of the Warburg effect and its connection to biosynthesis are not well understood. Here we show that the tumour suppressor p53, the most frequently mutated gene in human tumours, inhibits the pentose phosphate pathway (PPP). Through the PPP, p53 suppresses glucose consumption, NADPH production and biosynthesis. The p53 protein binds to glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the PPP, and prevents the formation of the active dimer. Tumour-associated p53 mutants lack the G6PD-inhibitory activity. Therefore, enhanced PPP glucose flux due to p53 inactivation may increase glucose consumption and direct glucose towards biosynthesis in tumour cells.

Reciprocal regulation of p53 and malic enzymes modulates metabolism and senescence

Cellular senescence both protects multicellular organisms from cancer and contributes to their ageing. The pre-eminent tumour suppressor p53 has an important role in the induction and maintenance of senescence, but how it carries out this function remains poorly understood. In addition, although increasing evidence supports the idea that metabolic changes underlie many cell-fate decisions and p53-mediated tumour suppression, few connections between metabolic enzymes and senescence have been established. Here we describe a new mechanism by which p53 links these functions. We show that p53 represses the expression of the tricarboxylic-acid-cycle-associated malic enzymes ME1 and ME2 in human and mouse cells. Both malic enzymes are important for NADPH production, lipogenesis and glutamine metabolism, but ME2 has a more profound effect. Through the inhibition of malic enzymes, p53 regulates cell metabolism and proliferation. Downregulation of ME1 and ME2 reciprocally activates p53 through distinct MDM2- and AMP-activated protein kinase-mediated mechanisms in a feed-forward manner, bolstering this pathway and enhancing p53 activation. Downregulation of ME1 and ME2 also modulates the outcome of p53 activation, leading to strong induction of senescence, but not apoptosis, whereas enforced expression of either malic enzyme suppresses senescence. Our findings define physiological functions of malic enzymes, demonstrate a positive-feedback mechanism that sustains p53 activation, and reveal a connection between metabolism and senescence mediated by p53.

The Bad Guy Cooperates with Good Cop p53: Bad Is Transcriptionally Up-Regulated by p53 and Forms a Bad/p53 Complex at the Mitochondria To Induce Apoptosis

ABSTRACT Although the regulation of several Bcl-2 family molecules, including Puma, Noxa, Bax, and Bid, by p53 has been studied intensively, the interplay between Bad (Bcl-2 antagonist of cell death) and p53 has not yet been reported thus far. Here, we report that p53 activates Bad transcription and expression through binding to a short conserved sequence located approximately 6.6 kb upstream of the translation start point. We also demonstrate that Bad physically interacts with cytoplasmic p53, thereby preventing p53 from entering the nucleus and resulting in reduced transcription of Bad. Moreover, Bad is able to direct p53 to the mitochondria and forms a p53/Bad complex at the mitochondria. Two lines of evidences support this hypothesis: first, when mitochondria purified from p53-deficient H1299 cells are incubated with p53 and either wild-type (wt) Bad or mutant Bad (this mutant binds p53 yet is unable to migrate to mitochondria), p53 can be detected only in mitochondria incubated with wt Bad and not in those incubated with mutant Bad; second, knockdown of Bad expression reduces mitochondrial localization of p53. The mitochondrial p53/Bad complex promotes apoptosis via activation and oligomerization of Bak. Elimination of Bad expression by RNA interference notably attenuates apoptosis induced by etoposide. Hence, our collective data provide the first evidence that Bad plays dual roles in both p53 transcription-dependent and -independent pathways.

TAp73 enhances the pentose phosphate pathway and supports cell proliferation

TAp73 is a structural homologue of the pre-eminent tumour suppressor p53. However, unlike p53, TAp73 is rarely mutated, and instead is frequently overexpressed in human tumours. It remains unclear whether TAp73 affords an advantage to tumour cells and if so, what the underlying mechanism is. Here we show that TAp73 supports the proliferation of human and mouse tumour cells. TAp73 activates the expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP). By stimulating G6PD, TAp73 increases PPP flux and directs glucose to the production of NADPH and ribose, for the synthesis of macromolecules and detoxification of reactive oxygen species (ROS). The growth defect of TAp73-deficient cells can be rescued by either enforced G6PD expression or the presence of nucleosides plus an ROS scavenger. These findings establish a critical role for TAp73 in regulating metabolism, and connect TAp73 and the PPP to oncogenic cell growth.

Livin promotes Smac/DIABLO degradation by ubiquitin–proteasome pathway

Livin, a member of the inhibitor of apoptosis protein (IAP) family, encodes a protein containing a single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING finger domain. It has been reported that Livin directly interacts with caspase-3 and -7 in vitro and caspase-9 in vivo via its BIR domain and is negatively regulated by Smac/DIABLO. Nonetheless, the detailed mechanism underlying its antiapoptotic function has not yet been fully characterized. In this report, we provide, for the first time, the evidence that Livin can act as an E3 ubiquitin ligase for targeting the degradation of Smac/DIABLO. Both BIR domain and RING finger domain of Livin are required for this degradation in vitro and in vivo. We also demonstrate that Livin is an unstable protein with a half-life of less than 4 h in living cells. The RING domain of Livin promotes its auto-ubiquitination, whereas the BIR domain is likely to display degradation-inhibitory activity. Mutation in the Livin BIR domain greatly enhances its instability and nullifies its binding to Smac/DIABLO, resulting in a reduced antiapoptosis inhibition. Our findings provide a novel function of Livin: it exhibits E3 ubiquitin ligase activity to degrade the pivotal apoptotic regulator Smac/DIABLO through the ubiquitin–proteasome pathway.

Siva1 suppresses epithelial–mesenchymal transition and metastasis of tumor cells by inhibiting stathmin and stabilizing microtubules

Epithelial–mesenchymal transition (EMT) enables epithelial cells to acquire motility and invasiveness that are characteristic of mesenchymal cells. It plays an important role in development and tumor cell metastasis. However, the mechanisms of EMT and their dysfunction in cancer cells are still not well understood. Here we report that Siva1 interacts with stathmin, a microtubule destabilizer. Siva1 inhibits stathmins activity directly as well as indirectly through Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of stathmin at Ser16. Via the inhibition of stathmin, Siva1 enhances the formation of microtubules and impedes focal adhesion assembly, cell migration, and EMT. Low levels of Siva1 and Ser16-phosphorylated stathmin correlate with high metastatic states of human breast cancer cells. In mouse models, knockdown of Siva1 promotes cancer dissemination, whereas overexpression of Siva1 inhibits it. These results suggest that microtubule dynamics are critical for EMT. Furthermore, they reveal an important role for Siva1 in suppressing cell migration and EMT and indicate that down-regulation of Siva1 may contribute to tumor cell metastasis.

Puma * Mcl-1 interaction is not sufficient to prevent rapid degradation of Mcl-1

Although Puma (p53 upregulated modulator of apoptosis) was known as a principal mediator of cell death in response to diverse apoptotic signals, the molecular mechanism underlying its proapoptotic regulation remains largely uncharacterized. Here we reported that myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the Bcl-2 family with a rapid turnover rate, interacts with Puma. The Puma/Mcl-1 interaction was verified by both yeast two-hybrid assay and co-immunoprecipation studies. Their binding sites were mapped to BH3 (Bcl-2 homology) domain of Puma and BH1 domain of Mcl-1, respectively. Mcl-1 and Puma was shown to colocalize at the mitochondria by immunostaining. The level of Mcl-1 was increased when coexpressed with Puma, indicating Puma is able to stabilize Mcl-1. Puma binding to Mcl-1 via its BH3 domain is the prerequisite for this effect, which is further supported by the finding that Puma mutant lacking BH3 domain no longer promotes Mcl-1 protein stability. This Puma-enhanced Mcl-1 stabilization was validated in vivo under nonoverexpression conditions. We also showed that BH1 domain is essential for Mcl-1 to inhibit Puma-induced apoptosis, since Mcl-1 mutant lacking BH1 domain completely abrogates its protective function. In addition, we concluded that binding of Puma to BH1 domain of Mcl-1 is necessary, but not sufficient to prevent rapid degradation of Mcl-1. In addition to PEST (proline, glutamic acid, serine, and threonine) and BH1 domain, some additional degradation signal is expected to reside in the C-terminal region of Mcl-1. In conclusion, our results provide the first evidence that the interaction between Mcl-1 and Puma may represent a novel mechanism by which Mcl-1 prevents apoptosis by increasing its stability through binding to Puma.

Suppression of p53 activity by Siva1.

The tumor suppressor p53 induces potent anti-proliferative responses in stressed cells; in unstressed cells this ability of p53 is restrained by Hdm2. Expression of Hdm2 is also induced by p53, thereby establishing feedback inhibition. Regulation of the p53–Hdm2 interaction and the feedback inhibition of p53 are not well understood. Here, we show that the p53–Hdm2 interaction in unstressed cells is promoted by Siva1, which, like Hdm2, is the product of a p53 target gene. Siva1 binds to both p53 and Hdm2 through distinct regions and enhances Hdm2-mediated p53 ubiquitination and degradation. Siva1 strongly inhibits p53-mediated gene expression and apoptosis. In xenograft mouse models, downregulation of Siva1 markedly inhibits tumor formation because of the activation of p53. On DNA damage, the interactions of Siva1 with both p53 and Hdm2 are diminished. The function of Siva1 seems to be related to its ability to form a homo-oligomer as the oligomerization defective splicing variant Siva2 fails to de-stabilize p53. These results identify Siva1 as an important adaptor promoting p53 degradation through Hdm2. Siva1 may be part of the negative feedback loop that inhibits p53 activity at the end of a non-lethal stress response.

p53 and Bad： remote strangers become close friends

The transcription factor p53 is a key regulator of the DNA damage response to genotoxic stress in higher eukaryotes. Mutation/inactivation of p53 has been implicated in the pathogenesis of many tumor types 1. p53 responds to cellular insults, such as DNA damage or hypoxia, either by arresting the cell cycle so that DNA damage could be repaired, or by triggering apoptosis if DNA repair is futile. Transcriptional activation of apoptosis-related genes by p53 is critical for the induction of programmed cell death. In search of target genes of p53 involved in cell-cycle arrest and/or apoptosis, numerous p53-inducible genes have been identified over the past 15 years. Among them are genes such as Bax 2, Puma 3, Noxa 4, Bid 5, p21waf1 6, and many others. These apoptosis-related factors function in different cell organelles including the cytosolic membrane, the mitochondria and the cytosol. However, none of these candidate effectors alone can be accountable for the complicated mechanisms underlying the p53 transcription-dependent apoptotic signaling pathways. Earlier work implicated that Bad may contribute to p53-induced mitochondria-mediated apoptosis, yet no known molecular mechanism had been ascribed to this observation. Our recent study published in Molecular and Cellular Biology revealed that a functional p53-binding element exists at the human bad genomic locus, residing roughly 6.6 kb upstream of the Bad translation start codon 7. The fact that this p53-binding element is relatively far away from the Bad promoter could be the reason why it has not been recognized as a p53-binding element until today. Interestingly enough, we found that a similar p53-binding region is well conserved approximately 7 kb upstream of the murine bad locus (our unpublished data).

Siva1 inhibits p53 function by acting as an ARF E3 ubiquitin ligase

The tumour suppressor alternative reading frame (ARF) is one of the most frequently mutated proteins in human cancer. It has been well established that ARF is able to stabilize and activate p53 by directly inhibiting Mdm2. ARF-mediated p53 activation in response to oncogenic stress is thought to be an important determinant of protection against cancer. However, little is known regarding the control of ARF in cells. Here, we show that Siva1 is a specific E3 ubiquitin ligase of ARF. Siva1 physically interacts with ARF both in vitro and in vivo. Through direct interaction, Siva1 promotes the ubiquitination and degradation of ARF, which in turn affects the stability of p53. Functionally, Siva1 regulates cell cycle progression and cell proliferation in an ARF/p53-dependent manner. Our results uncover a novel regulatory mechanism for the control of ARF stability, thereby revealing an important function of Siva1 in the regulation of the ARF-Mdm2-p53 pathway.