Cancer cells exhibit metabolic dependencies that distinguish them from their normal counterparts1. Among these addictions is an increased utilization of the amino acid glutamine (Gln) to fuel anabolic processes2. Indeed, the spectrum of Gln-dependent tumors and the mechanisms whereby Gln supports cancer metabolism remain areas of active investigation. Here we report the identification of a non-canonical pathway of Gln utilization in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for tumor growth. While most cells utilize glutamate dehydrogenase (GLUD1) to convert Gln-derived glutamate (Glu) into α-ketoglutarate in the mitochondria to fuel the tricarboxylic acid (TCA) cycle, PDAC relies on a distinct pathway to fuel the TCA cycle such that Gln-derived aspartate is transported into the cytoplasm where it can be converted into oxaloacetate (OAA) by aspartate transaminase (GOT1). Subsequently, this OAA is converted into malate and then pyruvate, ostensibly increasing the NADPH/NADP+ ratio which can potentially maintain the cellular redox state. Importantly, PDAC cells are strongly dependent on this series of reactions, as Gln deprivation or genetic inhibition of any enzyme in this pathway leads to an increase in reactive oxygen species and a reduction in reduced glutathione. Moreover, knockdown of any component enzyme in this series of reactions also results in a pronounced suppression of PDAC growth in vitro and in vivo. Furthermore, we establish that the reprogramming of Gln metabolism is mediated by oncogenic Kras, the signature genetic alteration in PDAC, via the transcriptional upregulation and repression of key metabolic enzymes in this pathway. The essentiality of this pathway in PDAC and the fact that it is dispensable in normal cells may provide novel therapeutic approaches to treat these refractory tumors.
Aberrant Skp2 signaling has been implicated as a driving event in tumorigenesis. Although the underlying molecular mechanisms remain elusive, cytoplasmic Skp2 correlates with more aggressive forms of breast and prostate cancers. Here, we report that Skp2 is acetylated by p300 at K68 and K71, which is a process that can be antagonized by the SIRT3 deacetylase. Inactivation of SIRT3 leads to elevated Skp2 acetylation, which leads to increased Skp2 stability through impairment of the Cdh1-mediated proteolysis pathway. As a result, Skp2 oncogenic function is increased, whereby cells expressing an acetylation-mimetic mutant display enhanced cellular proliferation and tumorigenesis in vivo. Moreover, acetylation of Skp2 in the nuclear localization signal (NLS) promotes its cytoplasmic retention, and cytoplasmic Skp2 enhances cellular migration through ubiquitination and destruction of E-cadherin. Thus, our study identifies an acetylation-dependent regulatory mechanism governing Skp2 oncogenic function and provides insight into how cytoplasmic Skp2 controls cellular migration.
Tumors exhibit metabolic reprogramming characterized by increased cellular reactive oxygen species (ROS) and the preferential use of glucose, as first published by Otto Warburg in 1956, referred to as the “Warburg effect.” However, the mechanism(s) linking these processes remain largely elusive. Murine tumors lacking Sirt3 exhibit abnormally high levels of ROS that directly induce genomic instability and increase HIF-1α protein levels. The subsequent transcription of HIFα-dependent target genes results in cellular metabolic reprogramming and increased cellular glucose consumption. In addition, agents that scavenge ROS or reverse the Warburg effect prevent the transformation and malignant phenotype observed in cells lacking Sirt3. Thus, mice lacking Sirt3 provide a model mechanistically connecting aberrant ROS, the Warburg effect, and carcinogenesis.
Sirt3; Warburg; Metabolism; Mitochondria; ROS; MnSOD; HIF-1α; Acetylation; Acetylome; Cancer; Metabolic Reprogramming
Decremental loss of PTEN results in cancer susceptibility and tumor progression. In turn this raises the possibility that PTEN elevation might be an attractive option for cancer prevention and therapy. We have generated several transgenic mouse lines with variably elevated PTEN expression levels, taking advantage of BAC (Bacterial Artificial Chromosome)-mediated transgenesis. Super-PTEN mutants are viable and show reduced body size due to decreased cell number, with no effect on cell size. Unexpectedly, PTEN elevation at the organism level results in healthy metabolism characterized by increased energy expenditure and reduced body fat accumulation. Cells derived from these mice show reduced glucose and glutamine uptake, increased mitochondrial oxidative phosphorylation, and are resistant to oncogenic transformation. Mechanistically we find that PTEN elevation orchestrates this metabolic switch by regulating PI3K-dependent and independent pathways, and negatively impacts two of the most pronounced metabolic features of tumor cells: glutaminolysis and the Warburg effect.
Calorie restriction (CR) extends lifespan and ameliorates age-related pathologies in most species studied; yet the mechanisms underlying these effects remain unclear. Using mouse skeletal muscle as a model, we show that CR acts in part by enhancing the function of tissue-specific stem cells. Even short-term CR significantly enhanced stem cell availability and activity in the muscle of young and old animals, in concert with an increase in mitochondrial abundance and induction of conserved metabolic and longevity regulators. Moreover, CR enhanced endogenous muscle repair and CR initiated in either donor or recipient animals improved the contribution of donor cells to regenerating muscle following transplant. These studies indicate that metabolic factors play a critical role in regulating stem cell function and that this regulation can influence the efficacy of recovery from injury and the engraftment of transplanted cells.
Ras-driven tumors are often refractory to conventional therapies. Here we identify a promising targeted therapeutic strategy for two Ras-driven cancers: Nf1-deficient malignancies and KRas/p53-mutant lung cancer. We show that agents that enhance proteotoxic stress, including the HSP90 inhibitor IPI-504, induce tumor regression in aggressive mouse models, but only when combined with rapamycin. These agents synergize by promoting irresolvable ER stress, resulting in catastrophic ER and mitochondrial damage. This process is fueled by oxidative stress, which is caused by IPI-504-dependent production of reactive oxygen species, and the rapamycin-dependent suppression of glutathione, an important endogenous antioxidant. Notably, the mechanism by which these agents cooperate reveals a therapeutic paradigm that can be expanded to develop additional combinations.
Cancer cells exhibit an aberrant metabolism that facilitates more efficient
production of biomass and hence tumor growth and progression. However, the genetic
cues modulating this metabolic switch remain largely undetermined. We identified a
metabolic function for the promyelocytic leukemia (PML) gene,
uncovering an unexpected role for this bona fide tumor suppressor in breast cancer
cell survival. We found that PML acted as both a negative regulator
of PPARγ coactivator 1A (PGC1A) acetylation and a potent activator of
PPAR signaling and fatty acid oxidation. We further showed that PML
promoted ATP production and inhibited anoikis. Importantly, PML
expression allowed luminal filling in 3D basement membrane breast culture models, an
effect that was reverted by the pharmacological inhibition of fatty acid oxidation.
Additionally, immunohistochemical analysis of breast cancer biopsies revealed that
PML was overexpressed in a subset of breast cancers and enriched in triple-negative
cases. Indeed, PML expression in breast cancer correlated strikingly
with reduced time to recurrence, a gene signature of poor prognosis, and activated
PPAR signaling. These findings have important therapeutic implications, as
PML and its key role in fatty acid oxidation metabolism are
amenable to pharmacological suppression, a potential future mode of cancer prevention
Tumor cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of oxygen. This metabolic reprogramming, known as the Warburg effect, provides tumor cells with the substrates required for biomass generation. Here, we show that the mitochondrial NAD-dependent deacetylase SIRT3 is a crucial regulator of the Warburg effect. Mechanistically, SIRT3 mediates metabolic reprogramming by destabilizing hypoxia-inducible factor-1α (HIF1α), a transcription factor that controls glycolytic gene expression. SIRT3 loss increases reactive oxygen species production, leading to HIF1α stabilization. SIRT3 expression is reduced in human breast cancers, and its loss correlates with the upregulation of HIF1α target genes. Finally, we find that SIRT3 overexpression represses glycolysis and proliferation in breast cancer cells, providing a metabolic mechanism for tumor suppression.
Sirtuins are a highly conserved family of proteins whose activity can extend lifespan in model organisms such as yeast, worms, and flies. Mammals contain seven sirtuins (SIRT1-7) that modulate distinct metabolic and stress response pathways. Three sirtuins, SIRT3, SIRT4 and SIRT5, are located in the mitochondrion, a dynamic organelle that functions as the primary site of oxidative metabolism and plays critical roles in apoptosis and intracellular signaling. Recent findings have shed light on how the mitochondrial sirtuins function in the control of basic mitochondrial biology, including energy production, metabolism, apoptosis, and intracellular signaling.
Aging is the outcome of a balance between damage and repair. The rate of aging and the appearance of age-related pathology are modulated by stress response and repair pathways that gradually decline, including the proteostasis and DNA damage repair networks and mitochondrial respiratory metabolism. Highly conserved insulin/IGF-1, TOR, and sirtuin signaling pathways in turn, control these critical cellular responses. The coordinated action of these signaling pathways maintains cellular and organismal homeostasis in the face of external perturbations, such as changes in nutrient availability, temperature and oxygen level, as well as internal perturbations, such as protein misfolding and DNA damage. Studies in model organisms suggest that changes in signaling can augment these critical stress response systems, increasing lifespan and reducing age-related pathology. The systems biology of stress response signaling thus provides a new approach to the understanding and potential treatment of age-related diseases.
aging; DNA damage; mitochondria; stress response; proteostasis; autophagy; insulin; TOR; sirtuin
Sirtuins (SIRT1-7) are a family of NAD-dependent deacetylases and/or ADP-ribosyltransferases that are involved in metabolism, stress responses and longevity. SIRT3 is localized to mitochondria, where it deacetylates and activates a number of enzymes involved in fuel oxidation and energy production.
In this study, we performed a proteomic screen to identify SIRT3 interacting proteins and identified several subunits of complex II and V of the electron transport chain. Two subunits of complex II (also known as succinate dehydrogenase, or SDH), SDHA and SDHB, interacted specifically with SIRT3. Using mass spectrometry, we identified 13 acetylation sites on SDHA, including six novel acetylated residues. SDHA is hyperacetylated in SIRT3 KO mice and SIRT3 directly deacetylates SDHA in a NAD-dependent manner. Finally, we found that SIRT3 regulates SDH activity both in cells and in murine brown adipose tissue.
Our study identifies SDHA as a binding partner and substrate for SIRT3 deacetylase activity. SIRT3 loss results in decreased SDH enzyme activity, suggesting that SIRT3 may be an important physiological regulator of SDH activity.
Aging is accompanied by a decline in the healthy function of multiple organ systems, leading to increased incidence and mortality from diseases such as type II diabetes mellitus, neurodegenerative diseases, cancer, and cardiovascular disease. Historically, researchers have focused on investigating individual pathways in isolated organs as a strategy to identify the root cause of a disease, with hopes of designing better drugs. Studies of aging in yeast led to the discovery of a family of conserved enzymes known as the sirtuins, which affect multiple pathways that increase the life span and the overall health of organisms. Since the discovery of the first known mammalian sirtuin, SIRT1, 10 years ago, there have been major advances in our understanding of the enzymology of sirtuins, their regulation, and their ability to broadly improve mammalian physiology and health span. This review summarizes and discusses the advances of the past decade and the challenges that will confront the field in the coming years.
chromatin; metabolism; deacetylase; cancer; cardiovascular; inflammation
Sirtuins are NAD-dependent protein deacetylases that connect metabolism and aging. In mammals, there are seven sirtuins (SIRT1-7), three of which are associated with mitochondria. Here we show that SIRT5 localizes in the mitochondrial matrix and interacts with carbamoyl phosphate synthetase 1 (CPS1), an enzyme, catalyzing the initial step of the urea cycle for ammonia detoxification and disposal. SIRT5 deacetylates CPS1 and up-regulates its activity. During fasting, NAD in liver mitochondria increases, thereby triggering SIRT5 deacetylation of CPS1 and adaptation to the increase in amino acid catabolism. Indeed, SIRT5 KO mice fail to up-regulate CPS1 activity and show elevated blood ammonia during fasting. Similar effects occur during long-term calorie restriction or a high protein diet. These findings demonstrate SIRT5 plays a pivotal role in ammonia detoxification and disposal by activating CPS1.
Cytosolic internalization is a requirement for the toxicity of secretory ribonucleases. Here, we investigate the mechanism of internalization of Onconase® (ONC), a toxic protein, and ribonuclease A (RNase A), a nontoxic homolog. Microscopy studies indicate that both ribonucleases readily bind to the cell surface and are internalized via acidic vesicles. Blocking dynamin-dependent endocytosis prevents transferrin internalization, but does not hinder RNase A internalization. ONC and G88R RNase A, which is a toxic variant, demonstrate enhanced cytotoxicity in the absence of clathrin- and dynamin-mediated endocytosis. The cytosolic entry of ribonucleases does not require an acidic environment or transport to the ER, and likely occurs from endosomes. Thus, common proteins—secretory ribonuclease—enter the cytosol by a pathway that is distinct from that of other known toxins.
dynamin; endocytosis; endosomes; ribonuclease; toxin
Kras is commonly mutated in colon cancers, but mutations in Nras are rare. We have used genetically engineered mice to determine whether and how these related oncogenes regulate homeostasis and tumorigenesis in the colon. Expression of K-RasG12D in the colonic epithelium stimulated hyperproliferation in a Mek-dependent manner. N-RasG12D did not alter the growth properties of the epithelium, but was able to confer resistance to apoptosis. In the context of an Apc-mutant colonic tumor, activation of K-Ras led to defects in terminal differentiation and expansion of putative stem cells within the tumor epithelium. This K-Ras tumor phenotype was associated with attenuated signaling through the MAPK pathway, and human colon cancer cells expressing mutant K-Ras were hypersensitive to inhibition of Raf, but not Mek. These studies demonstrate clear phenotypic differences between mutant Kras and Nras, and suggest that the oncogenic phenotype of mutant K-Ras might be mediated by noncanonical signaling through Ras effector pathways.
Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.
Onconase® (ONC) is a homolog of RNase A that is in clinical trials as a cancer chemotherapeutic agent. The toxicity of ONC and RNase A variants relies on their ability to evade the cytosolic ribonuclease inhibitor protein (RI) and degrade cellular RNA. We find that these ribonucleases are more toxic for more rapidly growing cells. The enhanced cytotoxicity does not arise from variation in the endogenous level of RI, which is virtually constant. Overproduction of RI diminishes the potency of toxic RNase A variants, but has no effect on the cytotoxicity of ONC. Thus, RI constrains the cytotoxicity of RNase A. These data provide new insights for the development of an optimal ribonuclease-based cancer chemotherapy.