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Summary Mdm2 E3 ubiquitin ligase-mediated p53 degradation is generally accepted as the major mechanism for p53 regulation; nevertheless, the in vivo significance of this function has not been unequivocally established. Here, we have generated an Mdm2 Y487A knockin mouse; Mdm2 Y487A mutation inactivates Mdm2 E3 ligase function without affecting its ability to bind its homolog MdmX. Unexpectedly, Mdm2 Y487A/Y487A mice were viable and developed normally into adulthood. While disruption of Mdm2 E3 ligase function resulted in p53 accumulation, p53 transcriptional activity remained low; however, exposure to sublethal stress resulted in hyperactive p53 and p53-dependent mortality in Mdm2 Y487A/Y487A mice. These findings reveal a potentially dispensable nature for Mdm2 E3 ligase function in p53 regulation, providing insight that may affect how this pathway is targeted therapeutically.

Classically, p53 is considered to be an overarching tumor suppressor gene, important in its role as a transcription factor for a number of genes critical for cell cycle arrest, apoptosis, and senescence. More recently, the scope of p53 function has been further broadened, with evidence emerging that supports essential roles for p53 in reproduction and metabolism. The homologous proteins Mdm2 and MdmX function as the primary negative regulators of p53 stability and activity. Canonically, Mdm2 is thought to regulate p53 through 2 mechanisms: 1) through directly binding the p53 transactivation domain, suppressing p53 activity, and 2) through functioning as an E3 ubiquitin ligase capable of ubiquitinating p53, targeting it for nuclear export and degradation. MdmX similarly functions to bind the p53 transactivation domain; however, it is not characterized to harbor any intrinsic E3 ubiquitin ligase activity. Despite extensive study, the advent of a number of mouse models has brought to light the necessity of studying the p53 pathway at physiological levels and emphasized the major differences that can exist between in vitro and in vivo analysis. While many questions remain, a focus on the use of in vivo models in p53 study is providing a clearer view of how this pathway is regulated, with a newfound emphasis on the role of the Mdm2:MdmX heterodimer, and with that a better understanding of how this pathway could be better manipulated for therapeutic gains.

In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. To systematically identify genes that modulate cancer cell survival under stresses, we performed genome-wide shRNA screens under hypoxia or lactic acidosis. We discovered that genetic depletion of acetyl-CoA carboxylase (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Additionally, the loss of ACLY or ACC1 reduced levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate is paradoxically increased under hypoxia when ACC1 or ACLY are depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulate the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future., Author Summary During the development of most solid tumors, there are characteristic physiological differences in the tumor that result from tumor cells outgrowing their local blood supply. Two of these physiological differences, or “stresses,” that occur in the tumor are low oxygen levels (hypoxia) and an accumulation of lactic acidic (lactic acidosis). Cancer cells experiencing hypoxia and lactic acidosis tend to be more resistant to chemo- and radio-therapy and metastasize more readily. Therefore, it is important to understand how tumor cells adapt to and survive these stresses. We used a large scale screening experiment in order to find which genes and proteins are involved in tumor cell adaptation and survival under hypoxia or lactic acidosis. We found that inhibiting either of two genes involved in lipid synthesis allowed tumor cells to survive hypoxia. This occurred because silencing these genes led to an increase in the metabolite α-ketoglutarate, which repressed a transcription factor that contributed to cell death under hypoxia. This research specifically advances our understanding of how tumor cells survive hypoxia and lactic acidosis and more broadly enhances our understanding of the cellular biology of solid tumors.

Mitochondrial outer membrane permeabilization (MOMP) is a critical control point during apoptosis that results in the release of pro-apoptotic mitochondrial contents such as cytochrome c. MOMP is largely controlled by Bcl-2 family proteins such as Bax, which under various apoptotic stresses becomes activated and oligomerizes on the outer mitochondrial membrane. Bax oligomerization helps promote the diffusion of the mitochondrial contents into the cytoplasm activating the caspase cascade. In turn, Bax is regulated primarily by anti-apoptotic Bcl-2 proteins including Bcl-xL, which was recently shown to prevent Bax from accumulating at the mitochondria. However, the exact mechanisms by which Bcl-xL regulates Bax and thereby MOMP remain partially understood. In this study, we show that the small CHCH-domain-containing protein CHCHD2 binds to Bcl-xL and inhibits the mitochondrial accumulation and oligomerization of Bax. Our data show that in response to apoptotic stimuli, mitochondrial CHCHD2 decreases prior to MOMP. Furthermore, when CHCHD2 is absent from the mitochondria, the ability of Bcl-xL to inhibit Bax activation and to prevent apoptosis is attenuated, which results in increases in Bax oligomerization, MOMP and apoptosis. Collectively, our findings establish CHCHD2, a previously uncharacterized small mitochondrial protein with no known homology to the Bcl-2 family, as one of the negative regulators of mitochondria-mediated apoptosis.

The tumor suppressor p53 has recently been shown to regulate energy metabolism through multiple mechanisms. However, the in vivo signaling pathways related to p53-mediated metabolic regulation remain largely uncharacterized. By using mice bearing a single amino acid substitution at cysteine residue 305 of mouse double minute 2 (Mdm2(C305F)), which renders Mdm2 deficient in binding ribosomal proteins (RPs) RPL11 and RPL5, we show that the RP-Mdm2-p53 signaling pathway is critical for sensing nutrient deprivation and maintaining liver lipid homeostasis. Although the Mdm2(C305F) mutation does not significantly affect growth and development in mice, this mutation promotes fat accumulation under normal feeding conditions and hepatosteatosis under acute fasting conditions. We show that nutrient deprivation inhibits rRNA biosynthesis, increases RP-Mdm2 interaction, and induces p53-mediated transactivation of malonyl-CoA decarboxylase (MCD), which catalyzes the degradation of malonyl-CoA to acetyl-CoA, thus modulating lipid partitioning. Fasted Mdm2(C305F) mice demonstrate attenuated MCD induction and enhanced malonyl-CoA accumulation in addition to decreased oxidative respiration and increased fatty acid accumulation in the liver. Thus, the RP-Mdm2-p53 pathway appears to function as an endogenous sensor responsible for stimulating fatty acid oxidation in response to nutrient depletion.

The Mdm2 proto-oncoprotein is the primary negative regulator for p53. While it is believed that Mdm2 degradation is regulated via its own E3 ubiquitin ligase activity, recent development of knock-in mouse models demonstrates that Mdm2 E3 ligase function is dispensable for self-degradation in vivo. Here, we show that the anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase for Mdm2 degradation. We demonstrate that APC2, a scaffold subunit of APC/C, binds to Mdm2 and is required for Mdm2 polyubiquitination and proteasomal degradation. Downregulation of APC2 by RNAi results in transcription-independent accumulation of Mdm2 and attenuation of stress-induced p53 stabilization, leading to decreased senescence and increased cell survival. Furthermore, APC2 expression is frequently downregulated in human cancers; in tumor cell lines, APC2 downregulation correlates with Mdm2 overexpression. Our study shows the regulation of Mdm2 by the E3 ubiquitin ligase APC/C and has important therapeutic implications for tumors with Mdm2 overexpression.

The TP53 tumor suppressor gene and its protein product p53, play a critical role in the cellular stress response. Upon exposure to stressors, such as DNA damage, oncogene activation, or perturbations to the ribosome biogenesis, p53 is activated and is capable of inducing cell cycle arrest, apoptosis, or senescence via the transactivation of genes such as p21, Noxa, Bax, Puma, and GADD45. While p53 expression, and activity, is integral to the cellular stress response, overexpression of p53 is inhibitory to embryonic development, illustrating the importance of proper p53 regulation. Growing evidence has implicated the nucleolus, as well as nucleolar proteins, in the regulation of both p53 and its primary negative regulator Mdm2. The role of nucleolar proteins, including ARF, B23/NPM, and ribosomal proteins, in mediating the p53 stress response serves to link the cell cycle progression and protein synthesis, and suggests p53 is a common regulator capable of coordinating these processes to best maintain the genetic integrity of the cell.

The transcription factor p53 plays a critical role in maintaining homeostasis as it relates to cellular growth, proliferation, and metabolism. In an effort to identify novel p53 target genes, a microarray approach was utilized to identify DHRS3 (also known as retSDR1) as a robust candidate gene. DHRS3 is a highly conserved member of the short chain alcohol dehydrogenase/reductase superfamily with a reported role in lipid and retinoid metabolism. Here, we demonstrate that DHRS3 is an endoplasmic reticulum (ER) protein that is shuttled to the ER via an N-terminal endoplasmic reticulum targeting signal. One important function of the ER is synthesis of neutral lipids that are packaged into lipid droplets whose biogenesis occurs from ER-derived membranes. DHRS3 is enriched at focal points of lipid droplet budding where it also localizes to the phospholipid monolayer of ER-derived lipid droplets. p53 promotes lipid droplet accumulation in a manner consistent with DHRS3 enrichment in the ER. As a p53 target gene, the observations of Dhrs3 location and potential function provide novel insight into an unexpected role for p53 in lipid droplet dynamics with implications in cancer cell metabolism and obesity.