Mitochondrial Substrates in Cancer: Drivers or Passengers? Bj¨orn Kruspig, Boris Zhivotovsky, Vladimir Gogvadze PII: DOI: Reference:
S1567-7249(14)00122-6 doi: 10.1016/j.mito.2014.08.007 MITOCH 956
To appear in:
Mitochondrion
Please cite this article as: Kruspig, Bj¨orn, Zhivotovsky, Boris, Gogvadze, Vladimir, Mitochondrial Substrates in Cancer: Drivers or Passengers?, Mitochondrion (2014), doi: 10.1016/j.mito.2014.08.007
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Mitochondrial Substrates in Cancer: Drivers or Passengers?
Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210,
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Björn Kruspig1, Boris Zhivotovsky1,2, and Vladimir Gogvadze1,2,*
MV Lomonosov Moscow State University, 119991 Moscow, Russia
*Correspondence to:
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Vladimir Gogvadze,
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171 77 Stockholm, Sweden
Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, 171 77 Stockholm, Sweden. Tel.: +46 8 524 87515 Fax: +46 8 329041
E-mail: [email protected]
Running title: Mitochondrial substrates in cancer
ACCEPTED MANUSCRIPT Abstract The majority of cancers demonstrate various tumor-specific metabolic aberrations, such as
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increased glycolysis even under aerobic conditions (Warburg effect), whereas mitochondrial
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metabolic activity and their contribution to cellular energy production are restrained. One of the most important mechanisms for this metabolic switch is the alteration in the abundance,
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utilization, and localization of various mitochondrial substrates. Numerous lines of evidence
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connect disturbances in mitochondrial metabolic pathways with tumorigenesis and provide an
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intriguing rationale for utilizing mitochondria as targets for anticancer therapy.
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Keywords: mitochondria; metabolism; Warburg Effect; cancer; cell death; therapy.
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ACCEPTED MANUSCRIPT Introduction For decades, the role of mitochondria in cancer was vastly underestimated by the research
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community, which focused primarily on cancer genetics and ignored the seminal findings by the
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German biochemist and Nobel Laureate Otto Warburg, who hypothesized as early as 1927 the importance of these organelles for tumorigenesis (Warburg et al., 1927). Discoveries in recent
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decades have clearly confirmed his hypothesis, showing that disturbances of mitochondrial
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functions are not only key features of cancer, but also of other, mainly neurodegenerative, disorders, e.g., Parkinson’s, Alzheimer’s, and many other diseases. The importance of
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mitochondria under pathological conditions stems from their central role in many vital physiological processes in the cell, including ATP production, calcium homeostasis control, and
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reactive oxygen species (ROS) production, as well as in the execution and regulation of different cell death modalities. Not only have mitochondria come to the researcher’s focus because of
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their role in various pathologies, but they are also regarded as promising targets for therapeutic interventions in these diseases.
Not only do mitochondria facilitate energy production in the form of ATP, but the majority of metabolic pathways are also directly or indirectly linked to these organelles. The driving force for most mitochondrial functions is the mitochondrial membrane potential, which is mandatory for ATP production, mitochondrial calcium accumulation, and other physiological pathways. The proton gradient is built up by proton pumps in the complexes of the electron transport chain and is fueled by electrons, which are provided by the reducing agents NADH and FADH2. These reducing equivalents arise from the citric acid or tricarboxylic acid (TCA) cycle, which is the most important cellular metabolic network for oxidation of various energy sources, such as glucose, glutamine, and lipids. Many of the countless catabolic metabolic pathways
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ACCEPTED MANUSCRIPT merge at the level of the TCA cycle, fueling both energy production and anabolic processes by providing building blocks for the synthesis of amino acids and other important cellular
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components. The mitochondrial metabolic network comprises a large number of enzymes, as
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well as their specific substrates, which are located in the mitochondrial matrix, integrated into the inner- or outer mitochondrial membrane, intermembrane space or even in the cytosol, linked to
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the mitochondria via transporters and specific pumps. This network is highly tunable and
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flexible, allowing the cell to adjust to different intra- and extracellular conditions, such as nutrient starvation, hypoxia, or other forms of cellular stress. One of the most important
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regulatory mechanisms for adjusting metabolic pathways is the modulation of the availability of mitochondrial substrates, which serves as a sensor for specific cellular conditions, and at the
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same time as a feedback loop for fine-tuning the enzymatic activities. Metabolic dysregulation in cancer has long been regarded as a mere by-product of
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tumorigenesis to support tumor growth and survival. As mentioned above, originating from the findings of Otto Warburg, and after a rediscovery in recent years, it became more and more apparent that metabolic changes in cancer cells are not only a consequence of malignant transformation, but seem to be essential for this process and are regarded as a crucial hallmark of cancer (Hanahan and Weinberg, 2011). Most cells utilize glucose as their main energy source, which is metabolized after its uptake in a set of glycolytic enzymatic reactions to form pyruvate. In normal cells, under normoxic conditions, most of the ATP is produced via oxidative phosphorylation (OXPHOS) in the mitochondria, whereas in cancer cells there is a shift towards glycolysis and suppression of mitochondrial function. This metabolic shift is mainly mediated by changes in the fate of pyruvate, the end-product of glycolysis. In normal cells, pyruvate is directed to the mitochondria
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ACCEPTED MANUSCRIPT where it is metabolized by pyruvate dehydrogenase (PDH) to enter the TCA cycle and fuel OXPHOS. In contrast, in most tumor cells, the activity of PDH is suppressed, causing a reduced
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flow of pyruvate to the mitochondria, and a decrease in OXPHOS. Under these conditions pyruvate is mainly converted to lactate by lactate dehydrogenase (LDH). Named after Otto
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Warburg, the “Warburg Effect” can be found in the majority of tumors. The reliance of tumor
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cells on glucose is even used for diagnostic purposes in fluorodeoxyglucose (18F-FDG) positron
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emission tomography (PET) and therapeutically by utilizing the non-metabolizable glucose analog 2-deoxyglucose to block glycolysis and cancer growth. Glycolysis, despite being less
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efficient in terms of ATP yield per glucose molecule when compared to OXPHOS-driven energy production (2 vs. 36 molecules of ATP), renders tumor cells more resistant to oxygen-
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deprivation as a result of excessive growth or high metabolic activity and poor oxygen supply. It provides the cell with resources to sustain proliferation. For a long time, it was assumed that
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these metabolic changes are caused by defects in mitochondria but were, in fact, later found to be based on specific metabolic regulatory signaling. Thus, the phosphoinositide 3-kinase (PI3K) (Jiang et al., 2001) and mammalian target of rapamycin (mTOR) (Wouters and Koritzinsky, 2008) pathways were shown to induce a pseudo-hypoxic state independently of oxygen by stabilization of the hypoxia-inducible factor 1α (HIF1α), which causes a strong increase in glycolysis known as the Warburg phenotype. Ultimately, different utilization, abundance, and localization of mitochondrial substrates might lead to tumor-specific adaptations of cellular metabolic pathways. The aim of this article is to give an overview of the current knowledge about the role of mitochondrial substrates in cancer development and their utilization as targets for potential therapeutic approaches.
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ACCEPTED MANUSCRIPT Pyruvate The metabolic intermediate and glycolytic end-product pyruvate is a crucial switch between
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aerobic and anaerobic metabolism, and also serves as an important precursor for glucose, amino
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acid, and lipid synthesis. The fate of pyruvate is mainly determined by its subcellular localization. The main cytosolic catabolic reaction is mediated by LDH, reducing pyruvate to L-
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lactate, and at the same time producing one molecule of ATP and regenerating NAD+, a critical
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cofactor for glycolysis function at the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level (Icard et al., 2012). In contrast, when metabolized in the mitochondrial matrix, pyruvate is
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oxidized to fuel the TCA cycle and OXPHOS. Under normoxic conditions, the majority of pyruvate is directed towards the mitochondria by an active transport through the inner
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mitochondrial membrane, mediated by the mitochondrial pyruvate carrier (MPC) (Halestrap, 1975), thereby linking the cytosolic glycolytic pathway with the mitochondrial TCA cycle.
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Within the mitochondrial matrix, PDH, a second key enzyme for this process, catalyzes the conversion to acetyl-CoA, NADH, and carbon dioxide. Acetyl-CoA thereafter enters the TCA cycle and ultimately fuels OXPHOS. As mentioned earlier, a characteristic of a majority of cancers is the switch from oxidative, mitochondrial carbon metabolism to a reductive, cytosolic production of lactate, even under normoxic conditions. This Warburg Effect is mediated in principle by a change of pyruvate distribution in the cell, mainly driven by the activation of HIF1α. This transcription factor controls the expression of a large number of genes that encode proteins related to adaptation to hypoxia, such as those involved in the stimulation of angiogenesis and increased glycolysis, but has also been found to be upregulated due to intratumoral hypoxia or prooncogenic aberrant cell signaling (Semenza, 2010). HIF1α further induces the transcriptional
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ACCEPTED MANUSCRIPT upregulation of several critical proteins involved in pyruvate metabolism, such as pyruvate dehydrogenase kinase 1 (PDK1), monocarboxylate transporter 4 (MCT4), and lactate
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dehydrogenase A (LDHA) (Denko, 2008; Schodel et al., 2011). PDK1, the largest of the four
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PDK isoforms, was shown to phosphorylate the E1 subunit of the PDH complex at all three known phosphorylation sites (Ser-264, Ser-271, and Ser-203), thereby causing a marked
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decrease in its enzymatic activity (Kato et al., 2008). The concurrent increase in cytosolic
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pyruvate means a higher abundance of substrate for the LDH enzyme, which is met by HIF1αmediated transcriptional upregulation of LDHA, and leads to increased lactate production. As an
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adaptation, MCT4 levels are elevated in the plasma membrane under hypoxic conditions, enabling cells to pump out the excess lactate. This increased export of lactate leads to
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acidification of the extracellular environment, which was proposed to select for highly malignant cancer cells and even protect tumor cells from chemotherapy because cellular uptake of weakly
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basic drugs, such as anthracyclines or vinca alkaloids, is significantly reduced under these conditions (De Milito and Fais, 2005). Thus, it was suggested that the low pH surrounding tumors represents a "niche engineering" strategy, supporting malignant tumor growth and invasion into surrounding tissue (Estrella et al., 2013). A very important role in deregulation of pyruvate metabolism in cancer plays pyruvate kinase isoenzyme M2 (PKM2), since knockdown of this isoform was shown to decrease glycolysis and inhibit cell proliferation in a panel of cancer cells (Christofk et al., 2008). Pyruvate kinase (PK) mediates the last step of glycolysis, producing pyruvate and ATP from phosphoenolpyruvate and ADP. The two PK genes, PKLR and PKM2, give rise to four different isozymes, in which PKLR codes for the two splice forms PKL and PKR, and PKM2 for PKM1 and PKM2 (Noguchi et al., 1986; Noguchi et al., 1987). The PK enzyme is active as a tetramer;
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ACCEPTED MANUSCRIPT however, due to alternative splicing, PKM2 exclusively features the ability to form inactive monomers, less-active dimers, and highly active tetramers, making it a very potent switch for
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reprogramming cell metabolism. Several regulatory mechanisms have been described for PK
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activity, including an allosteric feed-forward stimulation by fructose-1,6-bisphosphate (Jurica et al., 1998; Waygood and Sanwal, 1974), and a negative feedback loop by ATP inhibition
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(Waygood and Sanwal, 1972).
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PKM2 is the most prevalent PK isoform in embryonic tissue, and is later replaced in adult tissue by PKL in liver, PKLR in erythrocytes, and PKM1 in muscle tissue and the brain. In many
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cancers, such as liver, colon, renal, and lung carcinomas, PKM2 levels were found to be upregulated (reviewed in (Wong et al., 2014)). This upregulation is controlled by the
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PI3/AKT/mTOR signaling pathway, and mediated transcriptionally by HIF1α and by c-Mycheterogenous nuclear ribonucleoproteins (hnRNPs)-dependent gene splicing (Chen et al., 2010;
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Sun et al., 2011). Interestingly, PKM2 was shown to form a positive feedback regulation with HIF1α, being a direct transcriptional target of HIF1α and at the same time promoting HIF1αmediated transactivation and reprogramming of glucose metabolism. Hydroxylation of PKM2 by prolyl hydroxylase 3 (PHD3) facilitates a direct interaction with HIF1α and a promotion of transcriptional transactivation of HIF1α target genes by increasing the HIF1α binding to target promoters and recruitment of the p300 co-activator (Luo et al., 2011). Since PKM2 and PHD3 are HIF1α targets, the described mechanism acts as a positive feedback loop, sustaining the expression of glycolytic genes and thereby facilitating the Warburg Effect. Additionally, these important findings highlight the non-metabolic functions of PKM2 in the nucleus, which will be discussed later. Transcriptional upregulation of PKM2 under normoxic conditions, induced by
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ACCEPTED MANUSCRIPT Epidermal growth factor receptor (EGFR) signaling, was further shown to facilitate anaerobic glycolysis (Yang et al., 2012).
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The findings and the relevance of increased PKM2 levels in cancer were recently
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challenged by a work by Bluemlein and colleagues, who could not detect any specific upregulation of PKM2 levels when comparing normal vs. tumor tissue by using proteomic
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approaches. Alternatively, the authors suggest a post-translational regulation of PKM2 in cancer
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(Bluemlein et al., 2011). Indeed, several specific post-translational modifications, such as phosphorylation, acetylation, hydroxylation, sumoylation, and oxidation, were shown to
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influence the balance between metabolically active homotetrameric and inactive dimeric PKM2 in favor of the latter in most cancer cells (Wong et al., 2014). The decrease in PK activity as an
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outcome of this interconversion leads to a halt in the glycolytic pathway and accumulation of glycolytic intermediates, as well as inhibition of mitochondrial carbon fluxes. The resulting
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abundance of glycolytic intermediates leads to a redirection to other, mainly anabolic, pathways, such as the pentose pathway, thereby meeting the need of highly proliferating cancer cells for nucleotide and amino acid precursors. As mentioned earlier, PKM2 not only regulates aerobic glycolysis in the cytoplasm, but was also shown to have non-glycolytic functions by being translocated to the nucleus (Hoshino et al., 2007; Stetak et al., 2007) and acting as a transcriptional co-activator for HIF1α, as well as Oct-4, β-catenin, and Stat3 (Gao et al., 2012; Lee et al., 2008; Luo et al., 2011; Yang et al., 2011). Nuclear translocation of PKM2 and binding of phosphorylated β-catenin, in response to EGFR signaling, results in transcriptional transactivation of β-catenin targets such as cyclin D and c-Myc (Yang et al., 2011). Non-metabolic functions of PKM2 are mainly based on its kinase activity, and in a recent report the nuclear transport mechanism was described in detail. Wang
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ACCEPTED MANUSCRIPT and co-workers revealed that Jumonji C (JmjC)-domain-containing dioxygenase directly binds to PKM2 and thereby hinders the formation of its tetrameric, metabolically active form and is
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involved in the nuclear translocation of dimeric PKM2, as well as its transactivation of HIF1α,
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further demonstrating the crucial role of PKM2 in the tumor’s adaptive response to hypoxic stress (Wang et al., 2014).
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Thus, pyruvate is directly and indirectly involved in many adaptive mechanisms in
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tumors, especially in response to oxygen-related stress, in which the subcellular localization and the metabolic fate are key factors for its pro-oncogenic function in tumorigenesis. Given its
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important role in tumor-specific pathways, pyruvate metabolism was explored as a potential therapeutic target. The alkylating agent and pyruvate analog 3-bromopyruvate (3BP) has been
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demonstrated to cause ATP depletion and cell death in multiple tumor models (Geschwind et al., 2002; Ko et al., 2001) without or with only very minimal systemic toxicity. Molecular targets
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were identified to be hexokinase (Ko et al., 2001), as well as the glycolytic enzyme GAPDH (Ganapathy-Kanniappan et al., 2009) and 3BP was further shown to potentiate platinum-induced toxicity and cause production of ROS (Ihrlund et al., 2008). The tumor specificity of this small molecule is based on the overexpression in many tumors of MCT-1, the primary transporter responsible for 3BP uptake (Matsumoto et al., 2013; Thangaraju et al., 2009).
Glutamine, α-Ketoglutarate, and D-2-hydroxyglutarate Besides being the most abundant amino acid in blood and necessary as a supplement for almost all cell lines growing in culture, glutamine was shown to be of utmost importance for tumor metabolism, to the extent that cancer cells are literally addicted to it (Eagle, 1955; Wise and Thompson, 2010). Although being by definition not an essential amino acid, a majority of
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ACCEPTED MANUSCRIPT rapidly proliferating cells, including cancer cells, need glutamine for efficient proliferation due to their high demand that cannot be met by the intracellular production by glutamine synthetase. It
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should be noted that not all tumors require an exogenous supply of glutamine for their growth, as
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demonstrated by different responses to glutamine deprivation in lung as well as breast cancer cell lines (Kung et al., 2011; van den Heuvel et al., 2012). The independence of these cells of
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glutamine was shown to be mainly mediated by the presence of pyruvate carboxylase activity to
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fuel anaplerosis (Cheng et al., 2011). Another link between glutamine metabolism and tumorigenesis was discovered by several reports showing that the oncogene c-Myc induces a
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transcriptional program leading to increased glutaminolysis and dependency on glutamine as a carbon source (Gao et al., 2009; Wise et al., 2008; Yuneva et al., 2007).
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Glutamine is, after glucose, the second most important carbon-based energy source and precursor for several anabolic pathways, in particular, as a nitrogen donor for proteins and
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nucleotides. After transport into the cell by specific importers, the following metabolic steps can be categorized in reactions either utilizing the γ-nitrogen of glutamine for nucleotide or hexosamine synthesis or using the α-nitrogen or carbon backbone for fueling the TCA cycle. The first step for the latter category is mediated by glutaminases and comprises the removal of the amide group to form glutamate. Currently, four different isozymes for glutaminases have been identified, encoded by two different paralogous genes, GLS and GLS2, which are located on chromosome 23 and 12, respectively, and are believed to have been derived by gene duplication. Each gene gives rise to two isoforms: GLS encodes for the kidney-type glutaminase (KGA) (Shapiro et al., 1991) and glutaminase C (GAC) (Elgadi et al., 1999), determined by alternative splicing, whereas GLS2 codes for the liver-type glutaminase (LGA) (short) (Smith and Watford,
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ACCEPTED MANUSCRIPT 1990) and GAB (long) (de la Rosa et al., 2009; Gomez-Fabre et al., 2000), mediated by alternative transcription initiation and promoter usage (Aledo et al., 2000).
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The role of glutaminases in cancer is isoform-specific; GLS is proposed to have
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oncogenic function, whereas GLS1 seems to act as a tumor suppressor. GLS was found to be upregulated in, for example, breast cancer and its inhibition or silencing resulted in a profound
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decrease in tumor growth (Cheng et al., 2011; Lobo et al., 2000; Wang et al., 2010). The
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mechanism underlying this oncogenic expression of GLS was demonstrated to be dependent on the oncogenic transcription factor c-Myc, mediated by a suppression of miR-23a/b (Gao et al.,
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2009). c-Myc was further found to induce the upregulation of the glutamine importer SLC1A5, thereby stimulating glutamine catabolism and cell proliferation. Recently, another, c-Myc-
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independent mechanism was revealed: ErbB2 signaling leads to elevated GLS expression by activation of NF‐kB in ErbB2-positive breast cancer cells (Qie et al., 2013). In contrast, GLS2 is
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speculated to be a tumor suppressor in cells due to it being a p53 target, linking the common loss of p53 with the metabolic shift of tumors from mitochondrial OXPHOS towards glycolysis (Hu et al., 2010; Suzuki et al., 2010). Further strengthening this view are findings documenting the loss of the GAB GLS2 isoform in highly malignant glioblastomas and anaplastic astrocytomas in the brain, whereas artificial reintroduction of GLS2 led to decreased tumor growth and migration (Szeliga et al., 2009; Szeliga et al., 2005). Glutamate, the product of glutaminase enzyme activity, serves multiple roles in the cell. It might act as a precursor of the most important cellular antioxidant, glutathione, as well as a donor for amino groups for non-essential amino acids, such as serine, glycine, aspartate, and alanine. During oxidative glutaminolysis, glutamate is transformed to the TCA cycle intermediate α-ketoglutarate (αKG) by removal of the amine group. This reaction is either
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ACCEPTED MANUSCRIPT catalyzed by transaminases (such as glutamate pyruvate transaminase or glutamate oxaloacetate transaminase) for production of non-essential amino acids, or via oxidative deamination by
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glutamate dehydrogenase (GDH). When supply of glucose is abundant, the transamination
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pathway prevails, whereas under conditions of limited glucose, the GDH-mediated conversion is necessary to fuel carbon to the TCA cycle (Yang et al., 2009). In the latter case, glutamine, via
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αKG and the TCA cycle, serves both as an energy supply providing reducing equivalents and as
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a major precursor for anabolic processes.
Metabolized αKG can either be mediated by oxidative or reductive pathways (Holleran et
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al., 1995). αKG can be oxidized by α-ketoglutarate dehydrogenase (αKGDH) with formation of succinate, thereby fueling the TCA cycle for energy production. However, the reductive pathway
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is catalyzed by isocitrate dehydrogenase (IDH), carboxylating αKG to isocitrate, and finally citrate, which is both a TCA cycle substrate and an important precursor for cytosolic fatty acid
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synthesis. The reductive pathway is favored by cells under hypoxic conditions, enabling them to compensate for the drop in glucose-derived citrate production by using glutamine carboxylation to maintain citrate levels, and thereby sustaining lipid metabolism and cell proliferation (Metallo et al., 2012; Mullen et al., 2012; Scott et al., 2011; Wise et al., 2011). Recently, a mechanism for the hypoxic switch to reductive glutamine metabolism was proposed, showing a HIF1αdependent proteasomal degradation of the αKGDH subunit OGDH2, thereby causing a severe drop of αKGDH activity and a redirection of αKG to metabolization by IDH (Sun and Denko, 2014). The authors also demonstrated that this metabolic switch supports maintenance of lipid synthesis and is essential for hypoxic growth. Three different IDH isoforms exist in humans: the NAD+-dependent IDH3 as part of the TCA cycle, and the NADP+-dependent isoforms IDH1 and IDH2. IDH1 and IDH2 were shown
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ACCEPTED MANUSCRIPT to play an intriguing role in cancer: heterozygous mutations were identified for both isozymes and can be found in various malignancies, such as glioma, acute myeloid leukemia (AML),
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chondrosarcoma, cholangiocarcinoma, and angioimmunoblastic T-cell lymphoma (Duncan et al.,
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2012; Mardis et al., 2009; Parsons et al., 2008; Ward et al., 2010; Yan et al., 2009). These missense mutations in specific arginine residues of the enzyme active site have severe
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consequences, leading to formation of the oncometabolite D-2-hydroxyglutarate (D2HG) instead
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of αKG (Dang et al., 2009). This so far poorly studied metabolite can accumulate up to millimolar levels in the cell and is associated with epigenetic changes, impairment of defense
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mechanisms against ROS, and changes in redox homeostasis. The underlying mechanism was shown to be mainly mediated by D2HG acting as a competitive inhibitor of αKG-dependent
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dioxygenases, such as the ten-eleven translocation (TET) family of dioxygenases, JmjC-domaincontaining histone demethylases, prolyl hydroxylases (PHD) and lysyl hydroxylases (LHD)
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(Rose et al., 2011; Xu et al., 2011). How exactly these modulations of enzyme activities contribute to tumorigenesis is not fully understood, but are proposed to be based on epigenetic modifications, resulting in differential expression of genes involved in cell proliferation (TET proteins, JmjC demethylases), and changes in HIF1α levels (PHD), as well as aberrant extracellular matrix structures induced by changes in collagen synthesis (LHD) (Duncan et al., 2012; Lee et al., 2007; Lu et al., 2012; Turcan et al., 2012). Especially for glioblastoma, these findings have led to clinical relevance, since 60–80% of all astrocytomas, oligodendrogliomas, and oligoastrocytomas of WHO grade II and III harbor IDH1 mutations and are therefore regularly checked for D2HG as a marker for diagnostic and prognostic purposes (Hartmann et al., 2009). Furthermore, D2HG levels are measured in serum/plasma of AML (Pollyea et al., 2013) and in glioma patients using non-invasive proton magnetic resonance spectroscopy (MRS)
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development of specific small-molecule inhibitors with the aim of reducing D2HG levels and
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inhibiting tumorigenesis. Inhibitors were developed against mutant IDH1 R132H in glioma, as well as targeted to IDH2 R140Q in AML, resulting in decreased D2HG levels and inhibition of
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cell growth, and were proven to be successful in a glioma xenograft model for IDH1 R132H
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(Kim and DeBerardinis, 2013; Rohle et al., 2013; Wang et al., 2013). Nevertheless, it should be mentioned that their effect was mainly cytostatic and not cytotoxic, making them promising
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agents for a co-treatment setting with other chemotherapeutic drugs. Glutamine metabolism in tumors is clinically utilized via several therapeutic approaches,
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using either amino acid analogs or inhibitors directed against different steps of glutamine metabolism, such as glutamine import and glutaminase enzymatic activity. Therapeutic success
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of these approaches has shown large variation because in addition to targeting the glutamine pathway only in a non-selective manner, amino acid analogs, such as acivicin and azaserine, induced severe side effects such as gastrointestinal toxicity and neurotoxicity (Ahluwalia et al., 1990). Therefore, a more direct approach was developed by inhibiting the cellular glutamine transporter, sodium-dependent neutral amino acid transporter type 2 (ASCT2), by L-γ-glutamylp-nitroanilide (GPNA), which was shown to reduce cell proliferation in lung cancer (Hassanein et al., 2013). GPNA treatment had a striking side effect as concurrent uptake of the glycolytic inhibitor 3BP was significantly increased due to GPNA-mediated stabilization of monocarboxylate transporter (MCT1), making GPNA a promising adjuvant for 3-BP therapy (Cardaci et al., 2012). Other strategies focus on suppression of glutaminases by employing two different specific inhibitors, compound 968 and Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-
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ACCEPTED MANUSCRIPT yl)ethyl sulfide (BPTES), which both exhibit an anti-proliferative effect in cellular and xenograft models (Le et al., 2012; Wang et al., 2010).
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Radiolabeled amino acids, which have been used for imaging applications in humans for decades, are becoming more and more attractive for diagnostic purposes in cancer. Since not all
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tumors depend entirely on glucose as an energy source, 18F-FDG-based diagnostic PET scans are
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not applicable in some cancer types. In particular, tumors with active Myc/MycN, such as
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MycN-amplified neuroblastoma (Qing et al., 2012), have been shown to rely on glutamine and are thereby potentially suitable for glutamine-based PET, which is currently under development
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(Krasikova et al., 2011; Qu et al., 2012; Qu et al., 2011). Thus, glutamine-based PET could be of
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great value in complementing existing metabolic imaging techniques.
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Citrate
Citrate plays a central role in cellular metabolism as a substrate of the mitochondrial TCA cycle
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and also as a crucial switch between metabolic pathways, including OXPHOS, glycolysis, fatty acid synthesis, and gluconeogenesis. Citrate is formed in the mitochondria either by the canonical reaction (from acetyl-CoA and oxaloacetate catalyzed by citrate synthase), or from αKG via a reversed TCA cycle mediated by IDH. Within the mitochondria, the primary function of citrate is to fuel the TCA cycle, and thus contribute to cellular ATP production via OXPHOS. In addition, citrate may act as an allosteric regulator of several metabolic enzymes. Citrate was shown to repress the TCA cycle by inhibition of PDH and SDH activities (Taylor and Halperin, 1973)(Hillar et al., 1975), whereas after its cytosolic translocation via the specific citrate carrier (CIC) (Bisaccia et al., 1989; Kramer and Palmieri, 1989), it acts as a glycolytic inhibitor. Thus, in normal cells, citrate, together with ATP, plays a pivotal role in the mediation of the Pasteur effect (Salas et al., 1965) via attenuation of the enzymatic activity of phosphofructokinase 1 16
ACCEPTED MANUSCRIPT (PFK1). A similar effect was demonstrated for 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase (PFK2) (Chesney, 2006). In addition to its role in regulating catabolic processes,
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cytosolic citrate stimulates anabolic reactions such as lipid synthesis by enhancing acetyl-CoA
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carboxylase activity and by serving as a source for acetyl-CoA itself via cleavage by ATP-citrate lyase (ACLY), ultimately inducing fatty acid synthesis. ACLY was further shown to be
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important for metabolic modulation mediated by oncogenic PI3K/AKT signaling (Bauer et al.,
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2005), and its knockdown caused tumor suppression and cell differentiation (Hanai et al., 2013; Hatzivassiliou et al., 2005). The level of citrate in the cytosol is determined by the mitochondrial
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export rate and can be modulated by differential expression of the CIC gene. Several regulatory transcription factors, such as Sp1 (Iacobazzi et al., 2008) or NF-κB, can induce or inhibit CIC
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expression, thus linking CIC and cytosolic citrate levels with pro-inflammatory pathways (Infantino et al., 2011). In tumor cells, CIC was found to be transcriptionally upregulated and its
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inhibition has anti-tumor activity, potentially opening a therapeutic window for specific CIC inhibitors such as 1,2,3-benzene-tricarboxylate (BTA) or siRNA (Catalina-Rodriguez et al., 2012). Furthermore, citrate-mediated changes of fructose-2,6-bisphosphate (F2,6P) levels significantly influence tumor cell proliferation. Inhibition of PFK2 suppressed cell proliferation, whereas overexpression of PFK3 stimulated proliferation (Yalcin et al., 2009a; Yalcin et al., 2009b). Hence, changing cytosolic citrate levels is a powerful tool for modulation of cellular energy metabolism and important anabolic processes. Recently, another cancer-related role of citrate was proposed, suggesting the potential usage of citrate for therapeutic approaches in cancer. Citrate was shown to either induce cell death directly in various tumor cell lines (Lu et al., 2011; Yousefi et al., 2004) or facilitate toxic effects of conventional anticancer drugs such as cisplatin in malignant pleural mesothelioma
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ACCEPTED MANUSCRIPT cells (Zhang et al., 2009). These toxic effects were explained by the ability of citrate to chelate calcium and suppress glycolysis, and thereby reduce cellular ATP levels, cellular growth, and
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cellular capacity to repair DNA damage induced by cisplatin. A reduction of induced myeloid
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leukemia cell differentiation protein (Mcl-1) level has been reported to be a consequence of treating cells with citrate, causing loss of viability and ultimately cell death (Lincet et al., 2013).
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Even in a clinical setting, the anticancer effect of citrate has been demonstrated. Two
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cancer patients suffering from medullary thyroid cancer or primary peritoneal mesothelioma were reported to have improved in their health condition after oral administration of citrate as a
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sole anticancer treatment (Bucay, 2011; Halabe Bucay, 2009). An alternative mechanism of citrate-induced toxicity was proposed showing activation of initiator caspases by citrate in a set
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of neuroblastoma cell lines. Sensitivity of cells to citrate was dependent on the expression of caspase-8 (Kruspig et al., 2012b). This member of the initiator family of caspases can be
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activated by the so-called "induced proximity model" via adaptor-mediated clustering of zymogens (Salvesen and Dixit, 1999). Strikingly, in a study using a cell-free system, citrate was able to induce both dimerization and activation of caspase-8. The underlying mechanism was proposed to be the kosmotropic feature of citrate (Boatright et al., 2003; Pop et al., 2007). Kosmotropes are salts that promote and stabilize water–water interactions, and thereby are able to stabilize intermolecular interactions in macromolecules such as proteins. This kosmotropic property of citrate might also be involved in the activation of apical caspases in vivo. Similar to caspase-8, caspase-2 can also be stabilized and activated by citrate (Kruspig et al., 2012b). After conversion to acetyl-CoA, citrate was shown to affect caspase activation via another mechanism, i.e., modulation of protein N--acetylation. Acetyl-CoA is a key cofactor for N--acetylation and changes in its cytosolic abundance affect the activity of N-
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ACCEPTED MANUSCRIPT acetyltransferase protein complexes (NatA, NatB, NatC, NatD, and NatE), and consequently the post-transcriptional modification and activation of target proteins, including pro-apoptotic
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proteins such as caspase-2, -3, and -9 (Yi et al., 2007). This exemplifies the tight interplay
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between metabolism and apoptosis that has been shown for several metabolic pathways (Nutt et al., 2009; Nutt et al., 2005; Schafer et al., 2009; Vaughn and Deshmukh, 2008). Strikingly, this
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interconnection between apoptotic pathways and cellular metabolism is not unidirectional, since
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apoptotic protein Bcl-xL was shown to influence regulation of the mitochondrial membrane potential (Gottlieb et al., 2000; Vander Heiden et al., 1999). Overexpression of this anti-
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apoptotic Bcl-2 family member, a common feature in tumors in order to avoid apoptosis, was further shown to significantly decrease the level of cytosolic citrate, leading to an inhibition of
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N--acetylation of caspases and thereby contributing to the anti-apoptotic function of Bcl-xL (Yi et al., 2011). Citrate demonstrated a synergistic effect with ABT-737, a small-molecule inhibitor
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of anti-apoptotic members of the Bcl-2 family that is currently being tested in clinical trials. In combination, citrate and ABT-737 strongly inhibited the expression of Mcl-1 and thereby caused prominent apoptotic cell death (Lincet et al., 2013). The underlying mechanism of citratemediated downregulation of this anti-apoptotic protein is unclear as yet, but the authors speculated that citrate could cause a drop in the cellular ATP level by inhibition of glycolysis, leading to AMPK pathway activation, which in turn could lead to a GSK-3-mediated phosphorylation and consequent proteasomal degradation of Mcl-1. Summarizing the current knowledge of citrate’s role in cancer, it is an important metabolic switch involved in tumor-related adaptations and has the potential to be used as an anticancer agent, and thus citrate is a very attractive metabolite and it will be of great importance
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ACCEPTED MANUSCRIPT to further elucidate how the different molecular features of citrate orchestrate its overall effect on
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cancer cells.
Succinate
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Succinate provides electrons to the mitochondrial respiratory chain via Complex II, also known
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as succinate dehydrogenase (SDH) or succinate:ubiquinone oxidoreductase (SQR). This complex
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represents a unique structure, as it is the only enzyme that participates in both the TCA cycle and the electron transport chain (ETC). Reflecting its dual role, the four subunits of Complex II are
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divided according to their functional contribution, into the SDH part, consisting of SDHA and SDHB, and the SQR part, comprising SDHC and SDHD (Sun et al., 2005). Unlike all other
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mitochondrial respiratory complexes, Complex II is entirely nuclear encoded, and therefore lacks
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any contribution from the mitochondrial genome for its expression. Localized in the inner
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mitochondrial membrane, facing the matrix, the SDH part of Complex II catalyzes the oxidation of succinate to fumarate and the concurrent production of FADH2. The subunits SDHC and SDHD form the intramembranous SQR part of Complex II and mediate the transfer of electrons from the first catalytic step to reduce ubiquinone to ubiquinol, and subsequently to the respiratory Complex III. Other structural abnormalities further highlight the special role of Complex II within the respiratory chain. In contrast to Complexes I, III, and IV, which form a so-called respirasome, Complex II resides relatively separated from this supramolecular complex (Lenaz and Genova, 2009; Schagger and Pfeiffer, 2000). It was established that the complexes responsible for the leakage of electrons are Complex I and III. For many years, Complex II was not considered as a source of ROS. However, various recent publications focused attention on Complex II as an important site of ROS production in the form of superoxide (Gleason et al., 2011; Ishii et al., 2005; Moreno-Sanchez et al., 2013; Quinlan et al., 2012), and as a direct 20
ACCEPTED MANUSCRIPT consequence was also linked to ROS-mediated execution of apoptotic cell death (Ricci et al., 2003). First, overexpression of SDHC induced cell death and cells lacking this subunit of
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Complex II were much less sensitive towards several chemotherapeutic agents, such as cisplatin,
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doxorubicin, and etoposide (Albayrak et al., 2003). Later on, this effect was proposed to be mediated by a specific disintegration of Complex II, triggered by pH changes after treatment
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with apoptotic stimuli, causing a dissociation of SDHA/SDHB subunits, which functionally
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represent the SDH fraction of the complex. This results in an impairment of the SQR activity of the membrane-bound SDHC/SDHD subunits, while leaving the SDH activity of the dissociated
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SDHA/SDHB part intact, which in turn was shown to excessively produce superoxide, ultimately causing cell death (Lemarie et al., 2011). Based on these findings, Complex II was suggested to
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act as a cell death sensor, responding to acidification upon toxic stimuli by its disassembly and thereby further facilitating ROS-mediated apoptosis (Grimm, 2013). Reflecting this crucial
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function in cell death, it is of no surprise that Complex II was also found to be a tumor suppressor in various tumors. The first direct connection to cancer was established in familial paraganglioma and pheochromocytoma cases, which were found to carry germline mutations in SDHB, SDHC, and SDHD (Astuti et al., 2001a; Astuti et al., 2001b; Baysal et al., 2000; Niemann and Muller, 2000). Later, mutations were also reported in renal cell carcinoma and gastrointestinal stromal tumors (GIST) (Miettinen et al., 2011; Ricketts et al., 2008). Besides its role in cell death, another mechanism was discovered explaining how Complex II mutations can facilitate tumorigenesis. Early reports linking SDH mutations with familial cancer cases revealed a concomitant activation of hypoxia-inducible genes (Baysal, 2003), which was shown to be due to cytosolic accumulation of succinate, a result of impaired SDH function, ultimately leading to a stabilization and activation of HIF1α (Selak et al., 2005). Under normoxic conditions, this master
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ACCEPTED MANUSCRIPT regulator of hypoxic adaptation is targeted for proteasomal degradation by a process involving the von Hippel-Lindau (VHL) ubiquitin-ligase and HIF-PHD, which serve as intracellular
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oxygen-sensors since oxygen is a crucial cofactor for their enzymatic activity. Besides oxygen,
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this enzyme requires several other cofactors, including αKG, which is metabolized to succinate during this reaction. Elevated cytosolic succinate levels cause impairment of the 2-oxoglutarate
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(2-OG)-dependent HIF1-PHD activity by product inhibition, creating a so-called pseudo-hypoxic
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state due to HIF1α stabilization, independent of cellular oxygen levels. Mutations in SDH subunits can lead to activation of an oncogenic signaling pathway, leading to HIF1α stabilization
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and concomitant expression of genes involved in glycolysis, angiogenesis, and metastasis, and thereby facilitating tumorigenesis. Interestingly, cytosolic succinate levels were also shown to be
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elevated by inflammatory processes independently of SDH mutations. Under these conditions,
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succinate is derived from glutamine-dependent anaplerosis, as well as the γ-aminobutyric acid
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(GABA) shunt, and was shown to serve as an inflammatory signal, as well as leading to HIF1α activation (Tannahill et al., 2013). Hence, the tumorigenic effect of inflammatory processes could be in part due to an increase in cytosolic succinate levels. Succinate and Complex II are not only associated with tumorigenesis, but also utilized as targets for anticancer therapy. Therapeutic approaches vary depending on the type of tumor and their specific characteristics. Despite only representing a small fraction of tumors, as mentioned earlier, certain familial cancers carry SDH mutations that were shown to be important for tumorigenesis. As a promising new approach for treatment of these cancers, cell-permeable αKG derivatives were successfully tested in both cellular and xenograft models. The underlying rationale is that αKG, a cofactor for PHD enzymes, restores enzymatic activity of these
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ACCEPTED MANUSCRIPT hydroxylases, causing HIF1α destabilization and thereby reverses the pseudo-hypoxic condition and pro-tumorigenic effect (MacKenzie et al., 2007; Tennant et al., 2009).
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For the vast majority of tumors, which lack mutations in the Complex II subunits, different chemotherapeutic drugs targeting Complex II have been tested. The redox-silent
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vitamin E analog -tocopheryl succinate (α-TOS) was shown as early as 1982 to have antiproliferative effects in mouse melanoma cells (Prasad and Edwards-Prasad, 1982), and
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furthermore, proved to be effective in a large variety of experimental cancers such as colon, breast, and neuroblastoma, in both in vitro and in vivo settings (Neuzil et al., 2001b; Stapelberg
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et al., 2004; Swettenham et al., 2005). The underlying mechanism for apoptosis induction by αTOS was investigated in several studies, providing different models for its mode of action, but
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without finding the specific molecular target of α-TOS. In a study by Dong and colleagues, this target was identified to be the ubiquinone (UbQ) binding site of Complex II, and the authors
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provided evidence for the involvement of superoxide production in this process (Dong et al., 2008). Later on, this model was extended by findings that α-TOS additionally triggers an increase in cellular calcium levels, which in combination with ROS production leads to mitochondrial destabilization and cell death (Gogvadze et al., 2010). This compound is particularly attractive as a potential anticancer drug, as it was shown to be selective for cancers, while being non-toxic to normal cells (Neuzil et al., 2001a), and to kill cancer cells irrespective of their p53 or MycN status (Kruspig et al., 2012a). The cancer-specificity of α-TOS is thought to be based on a higher antioxidant capacity and elevated activity of intracellular esterases in non-malignant cells, which can cleave and thereby detoxify α-TOS into its two non-toxic components, i.e., antioxidant α-tocopherol and succinate (Carini et al., 1990; Fariss et al., 2001; Neuzil and Massa, 2005). A large effort has been made to improve the molecular properties of α-
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ACCEPTED MANUSCRIPT TOS by structural modifications of this compound, leading to a more specific accumulation in the mitochondria and a higher efficacy (Kovarova et al., 2014; Prochazka et al., 2013). It should
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be taken into account that despite its well-documented toxic effect, when used at low
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concentrations in a co-treatment setting with conventional anticancer drugs, α-TOS was shown to either sensitize cells to cell death, in the case of combination with etoposide, or to protect cells,
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when treated together with cisplatin (Kruspig et al., 2013). Thus, α-TOS should be used in
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combined treatment with conventionally used anticancer drugs cautiously. Recent observations demonstrate that targeting Complex II can modulate the cellular
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response to anticancer treatment. Stimulation of Complex II activity by supplying exogenous succinate protected cells from cisplatin-induced apoptosis as assessed by caspase-3-like activity,
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release of cytochrome c, or Poly (ADP-ribose) polymerase (PARP) cleavage (Kruspig et al., 2013). Succinate usually shows poor passage through the plasma membrane, but when applied at
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high concentrations it can slowly penetrate into the cell. This effect of succinate is apparently based on the ability of this substrate to monopolize the respiratory chain of mitochondria (Krebs et al., 1961). One of the consequences of this monopolization is an even greater reduction of mitochondrial pyridine nucleotides than that achieved by Complex I substrates (Chance and Hollunger, 1961). The redox state of mitochondrial pyridine nucleotides is involved in the regulation of various processes, such as the detoxification of hydrogen peroxide and organic hydroperoxides via the glutathione-dependent defense system (Sies and Summer, 1975), or the induction of mitochondrial permeability transition (MPT) (Costantini et al., 1996). Indeed, mitochondria oxidizing succinate can accumulate 3–4-times more Ca2+ before the onset of MPT as compared to mitochondria oxidizing NAD-dependent substrates (Kondrashova et al., 1982). Moreover, oxidation of succinate makes mitochondria resistant to oxidative stress; in the
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ACCEPTED MANUSCRIPT absence, as well as in the presence, of the oxidant t-butylhydroperoxide, mitochondria retained more Ca2+ with succinate than with -hydroxybutyrate as the respiratory substrate (Gogvadze et
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al., 1996). In contrast, inhibition of Complex II by thenoyltrifluoroacetone (TTFA) markedly
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stimulated cell death induced by low doses of cisplatin (unpublished data).
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Lately, similar to IDH mutations, mutations in SDH and in fumarate hydratase (FH) were reported, leading to accumulation of succinate or fumarate, respectively, and concomitant
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inhibition of several α-KG-dependent dioxygenases by product inhibition (Xiao et al., 2012). As discussed earlier, this class of enzymes, which includes histone demethylases and DNA
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hydroxylases, plays an important role in hydroxylation reactions of several different substrates in the cell, such as proteins and DNA. As a result of SDH mutations, the accumulation of succinate
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was linked to a hypermethylated phenotype in GIST, as well as paraganglioma (Killian et al., 2013; Letouze et al., 2013). The severity of these epigenetic modifications, leading to gene
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silencing, correlates with the aggressiveness of certain tumor subtypes, because the highly malignant SDHB-mutated tumors were also shown to have the most pronounced hypermethylated state (Letouze et al., 2013). These findings highlight a new and interesting interplay between the TCA cycle and tumor-specific epigenetic changes, and further represent a potential therapeutic target in these malignancies. Clinically relevant are the findings that patients harboring mutations in SDHB and SDHD also had a higher probability of increased succinate levels in plasma (Hobert et al., 2012), which suggests that this could be explored for diagnostic purposes, thereby providing a more cost-effective and easy method of screening for cancers harboring SDH mutations.
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ACCEPTED MANUSCRIPT Concluding remarks Changes in the abundance, localization, and metabolism of mitochondrial substrates are a crucial
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part of the specific metabolic aberrations found in the majority of cancers. Moreover, it has
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become clear that some metabolites by themselves can directly drive tumorigenesis, exemplified by succinate and its ability to induce pseudo-hypoxic conditions and alter gene expression by
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DNA hypermethylation in cancer cells harboring SDH mutations. In addition, mitochondrial
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substrates are directly or indirectly being utilized for clinical approaches, such as diagnosis or anticancer therapy. Although several chemotherapeutical interventions targeting different aspects
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of mitochondrial metabolism are already being tested either in laboratories or in clinical trials, future research should focus on tackling the shortcomings of conventional anticancer therapy and
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providing a better understanding of the role of mitochondrial metabolism in cancer biology.
Acknowledgements
The work in the author’s laboratories was supported by grants from the Swedish and Stockholm Cancer Societies, the Swedish Childhood Cancer Foundation, the Swedish Research Council, the Russian Science Foundation, and the Russian Foundation for Basic Research.
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ACCEPTED MANUSCRIPT Figure 1: Cellular glucose and glutamine metabolism. Overview of cellular glucose and glutamine metabolism, as well as the mitochondrial
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tricarboxylic acid cycle. Metabolic enzymes that are discussed within this review are indicated.
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Figure adapted from BioCarta.
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Figure 2: Role of succinate in cancer metabolism and tumorigenesis, and potential
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chemotherapeutic interventions.
Under physiological conditions (black), succinate, as a substrate of the TCA cycle, is oxidized to
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fumarate by the SDHA subunit of Complex II/SDH, and electrons are transferred further via
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subunit SDHB to the ubiquinone binding site formed by subunits SDHC and SDHD. Ubiquinone is reduced to ubiquinol, and subsequently relays the electrons to Complex III of the respiratory
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chain. Specific drugs (blue) may be used to inhibit this process at different subunits of the SDH complex. 3-NPA and malonate bind and block the SDHA subunit and thereby prevent oxidation of succinate. α-TOS and thenoyltrifluoroacetone (TTFA) bind and inhibit the ubiquinone binding site, which may lead to leakage of electrons and the formation of ROS in the form of superoxide radicals. Mutations (red) in different subunits of SDH are found in several cancers and can cause ROS formation and accumulation of succinate. Increased cytosolic succinate levels may induce HIF1α stabilization by product inhibition of HIF-1-PHD, resulting in concomitant pseudohypoxic conditions, and thus induction of angiogenesis, glycolysis, and metastasis. Further, accumulation of succinate in the cytosol may lead to DNA hypermethylation by inhibition of αKG-dependent dioxygenases. Exogenous (green) supply of succinate can induce a reversed electron flow, monopolization of the respiratory chain, and an increase in the pool of pyridine
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ACCEPTED MANUSCRIPT nucleotides, and thereby protect mitochondria from oxidative stress and OMM permeabilization.
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Figure adapted from BioCarta and David Goodsell & RCSB Protein Data Bank.
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Figure 3: Effects of citrate treatment on cellular metabolism and mechanisms of citrateinduced toxicity.
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Citrate treatment may cause suppression of the TCA cycle by inhibition of PDH and SDH
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activity, as well as glycolysis, by attenuating PFK1. Increased cytosolic citrate can enforce fatty acid synthesis after conversion to acetyl-CoA by ACLY. Dimerization and activation of apical
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caspase-2/-8, induced by the kosmotropic feature of citrate, as well post-transcriptional
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adapted from BioCarta.
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modification by N--acetylation of caspase-2/-3/-9 can facilitate apoptotic cell death. Figure
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References
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Ahluwalia, G.S., Grem, J.L., Hao, Z., Cooney, D.A., 1990. Metabolism and action of amino acid analog anti-cancer agents. Pharmacology & therapeutics 46, 243-271. Albayrak, T., Scherhammer, V., Schoenfeld, N., Braziulis, E., Mund, T., Bauer, M.K.A., Scheffler, I.E., Grimm, S., 2003. The tumor suppressor cybL, a component of the respiratory chain, mediates apoptosis induction. Mol Biol Cell 14, 3082-3096. Aledo, J.C., Gomez-Fabre, P.M., Olalla, L., Marquez, J., 2000. Identification of two human glutaminase loci and tissue-specific expression of the two related genes. Mammalian genome : official journal of the International Mammalian Genome Society 11, 1107-1110. Andronesi, O.C., Kim, G.S., Gerstner, E., Batchelor, T., Tzika, A.A., Fantin, V.R., Vander Heiden, M.G., Sorensen, A.G., 2012. Detection of 2-hydroxyglutarate in IDH-mutated glioma patients by in vivo spectral-editing and 2D correlation magnetic resonance spectroscopy. Science translational medicine 4, 116ra114. Astuti, D., Douglas, F., Lennard, T.W., Aligianis, I.A., Woodward, E.R., Evans, D.G., Eng, C., Latif, F., Maher, E.R., 2001a. Germline SDHD mutation in familial phaeochromocytoma. Lancet 357, 1181-1182. Astuti, D., Latif, F., Dallol, A., Dahia, P.L., Douglas, F., George, E., Skoldberg, F., Husebye, E.S., Eng, C., Maher, E.R., 2001b. Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. American journal of human genetics 69, 49-54. Bauer, D.E., Hatzivassiliou, G., Zhao, F., Andreadis, C., Thompson, C.B., 2005. ATP citrate lyase is an important component of cell growth and transformation. Oncogene 24, 6314-6322. Baysal, B.E., 2003. On the association of succinate dehydrogenase mutations with hereditary paraganglioma. Trends in endocrinology and metabolism: TEM 14, 453-459. Baysal, B.E., Ferrell, R.E., Willett-Brozick, J.E., Lawrence, E.C., Myssiorek, D., Bosch, A., van der Mey, A., Taschner, P.E., Rubinstein, W.S., Myers, E.N., Richard, C.W., 3rd, Cornelisse, C.J., Devilee, P., Devlin, B., 2000. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 287, 848-851. Bisaccia, F., De Palma, A., Palmieri, F., 1989. Identification and purification of the tricarboxylate carrier from rat liver mitochondria. Biochimica et biophysica acta 977, 171-176. Bluemlein, K., Gruning, N.M., Feichtinger, R.G., Lehrach, H., Kofler, B., Ralser, M., 2011. No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis. Oncotarget 2, 393-400. Boatright, K.M., Renatus, M., Scott, F.L., Sperandio, S., Shin, H., Pedersen, I.M., Ricci, J.E., Edris, W.A., Sutherlin, D.P., Green, D.R., Salvesen, G.S., 2003. A unified model for apical caspase activation. Molecular cell 11, 529-541. Bucay, A.H., 2011. Clinical report: a patient with primary peritoneal mesothelioma that has improved after taking citric acid orally. Clinics and research in hepatology and gastroenterology 35, 241. Cardaci, S., Rizza, S., Filomeni, G., Bernardini, R., Bertocchi, F., Mattei, M., Paci, M., Rotilio, G., Ciriolo, M.R., 2012. Glutamine deprivation enhances antitumor activity of 3-bromopyruvate through the stabilization of monocarboxylate transporter-1. Cancer research 72, 4526-4536. Carini, R., Poli, G., Dianzani, M.U., Maddix, S.P., Slater, T.F., Cheeseman, K.H., 1990. Comparative evaluation of the antioxidant activity of alpha-tocopherol, alpha-tocopherol polyethylene glycol 1000 succinate and alpha-tocopherol succinate in isolated hepatocytes and liver microsomal suspensions. Biochemical pharmacology 39, 1597-1601. 29
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Catalina-Rodriguez, O., Kolukula, V.K., Tomita, Y., Preet, A., Palmieri, F., Wellstein, A., Byers, S., Giaccia, A.J., Glasgow, E., Albanese, C., Avantaggiati, M.L., 2012. The mitochondrial citrate transporter, CIC, is essential for mitochondrial homeostasis. Oncotarget 3, 1220-1235. Chance, B., Hollunger, G., 1961. The interaction of energy and electron transfer reactions in mitochondria. I. General properties and nature of the products of succinate-linked reduction of pyridine nucleotide. The Journal of biological chemistry 236, 1534-1543. Chen, M., Zhang, J., Manley, J.L., 2010. Turning on a fuel switch of cancer: hnRNP proteins regulate alternative splicing of pyruvate kinase mRNA. Cancer research 70, 8977-8980. Cheng, T., Sudderth, J., Yang, C., Mullen, A.R., Jin, E.S., Mates, J.M., DeBerardinis, R.J., 2011. Pyruvate carboxylase is required for glutamine-independent growth of tumor cells. Proceedings of the National Academy of Sciences of the United States of America 108, 8674-8679. Chesney, J., 2006. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and tumor cell glycolysis. Current opinion in clinical nutrition and metabolic care 9, 535-539. Choi, C., Ganji, S.K., DeBerardinis, R.J., Hatanpaa, K.J., Rakheja, D., Kovacs, Z., Yang, X.L., Mashimo, T., Raisanen, J.M., Marin-Valencia, I., Pascual, J.M., Madden, C.J., Mickey, B.E., Malloy, C.R., Bachoo, R.M., Maher, E.A., 2012. 2-hydroxyglutarate detection by magnetic resonance spectroscopy in IDH-mutated patients with gliomas. Nature medicine 18, 624-629. Christofk, H.R., Vander Heiden, M.G., Harris, M.H., Ramanathan, A., Gerszten, R.E., Wei, R., Fleming, M.D., Schreiber, S.L., Cantley, L.C., 2008. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452, 230-233. Costantini, P., Chernyak, B.V., Petronilli, V., Bernardi, P., 1996. Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. The Journal of biological chemistry 271, 6746-6751. Dang, L., White, D.W., Gross, S., Bennett, B.D., Bittinger, M.A., Driggers, E.M., Fantin, V.R., Jang, H.G., Jin, S., Keenan, M.C., Marks, K.M., Prins, R.M., Ward, P.S., Yen, K.E., Liau, L.M., Rabinowitz, J.D., Cantley, L.C., Thompson, C.B., Vander Heiden, M.G., Su, S.M., 2009. Cancer-associated IDH1 mutations produce 2hydroxyglutarate. Nature 462, 739-744. de la Rosa, V., Campos-Sandoval, J.A., Martin-Rufian, M., Cardona, C., Mates, J.M., Segura, J.A., Alonso, F.J., Marquez, J., 2009. A novel glutaminase isoform in mammalian tissues. Neurochemistry international 55, 76-84. De Milito, A., Fais, S., 2005. Tumor acidity, chemoresistance and proton pump inhibitors. Future oncology 1, 779-786. Denko, N.C., 2008. Hypoxia, HIF1 and glucose metabolism in the solid tumour. Nature reviews. Cancer 8, 705-713. Dong, L.F., Low, P., Dyason, J.C., Wang, X.F., Prochazka, L., Witting, P.K., Freeman, R., Swettenham, E., Valis, K., Liu, J., Zobalova, R., Turanek, J., Spitz, D.R., Domann, F.E., Scheffler, I.E., Ralph, S.J., Neuzil, J., 2008. Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II. Oncogene 27, 4324-4335. Duncan, C.G., Barwick, B.G., Jin, G., Rago, C., Kapoor-Vazirani, P., Powell, D.R., Chi, J.T., Bigner, D.D., Vertino, P.M., Yan, H., 2012. A heterozygous IDH1R132H/WT mutation induces genome-wide alterations in DNA methylation. Genome research 22, 2339-2355. Eagle, H., 1955. Nutrition needs of mammalian cells in tissue culture. Science 122, 501-514. Elgadi, K.M., Meguid, R.A., Qian, M., Souba, W.W., Abcouwer, S.F., 1999. Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alternative splicing. Physiological genomics 1, 51-62. Elkhaled, A., Jalbert, L.E., Phillips, J.J., Yoshihara, H.A., Parvataneni, R., Srinivasan, R., Bourne, G., Berger, M.S., Chang, S.M., Cha, S., Nelson, S.J., 2012. Magnetic resonance of 2-hydroxyglutarate in IDH1mutated low-grade gliomas. Science translational medicine 4, 116ra115. 30
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Estrella, V., Chen, T., Lloyd, M., Wojtkowiak, J., Cornnell, H.H., Ibrahim-Hashim, A., Bailey, K., Balagurunathan, Y., Rothberg, J.M., Sloane, B.F., Johnson, J., Gatenby, R.A., Gillies, R.J., 2013. Acidity generated by the tumor microenvironment drives local invasion. Cancer research 73, 1524-1535. Fariss, M.W., Nicholls-Grzemski, F.A., Tirmenstein, M.A., Zhang, J.G., 2001. Enhanced antioxidant and cytoprotective abilities of vitamin E succinate is associated with a rapid uptake advantage in rat hepatocytes and mitochondria. Free radical biology & medicine 31, 530-541. Ganapathy-Kanniappan, S., Geschwind, J.F., Kunjithapatham, R., Buijs, M., Vossen, J.A., Tchernyshyov, I., Cole, R.N., Syed, L.H., Rao, P.P., Ota, S., Vali, M., 2009. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is pyruvylated during 3-bromopyruvate mediated cancer cell death. Anticancer research 29, 4909-4918. Gao, P., Tchernyshyov, I., Chang, T.C., Lee, Y.S., Kita, K., Ochi, T., Zeller, K.I., De Marzo, A.M., Van Eyk, J.E., Mendell, J.T., Dang, C.V., 2009. c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Nature 458, 762-765. Gao, X., Wang, H., Yang, J.J., Liu, X., Liu, Z.R., 2012. Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Molecular cell 45, 598-609. Geschwind, J.F., Ko, Y.H., Torbenson, M.S., Magee, C., Pedersen, P.L., 2002. Novel therapy for liver cancer: direct intraarterial injection of a potent inhibitor of ATP production. Cancer research 62, 39093913. Gleason, C., Huang, S., Thatcher, L.F., Foley, R.C., Anderson, C.R., Carroll, A.J., Millar, A.H., Singh, K.B., 2011. Mitochondrial complex II has a key role in mitochondrial-derived reactive oxygen species influence on plant stress gene regulation and defense. Proceedings of the National Academy of Sciences of the United States of America 108, 10768-10773. Gogvadze, V., Norberg, E., Orrenius, S., Zhivotovsky, B., 2010. Involvement of Ca2+ and ROS in alphatocopheryl succinate-induced mitochondrial permeabilization. International journal of cancer. Journal international du cancer 127, 1823-1832. Gogvadze, V., Schweizer, M., Richter, C., 1996. Control of the pyridine nucleotide-linked Ca2+ release from mitochondria by respiratory substrates. Cell calcium 19, 521-526. Gomez-Fabre, P.M., Aledo, J.C., Del Castillo-Olivares, A., Alonso, F.J., Nunez De Castro, I., Campos, J.A., Marquez, J., 2000. Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase. The Biochemical journal 345 Pt 2, 365-375. Gottlieb, E., Vander Heiden, M.G., Thompson, C.B., 2000. Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis. Molecular and cellular biology 20, 5680-5689. Grimm, S., 2013. Respiratory chain complex II as general sensor for apoptosis. Bba-Bioenergetics 1827, 565-572. Halabe Bucay, A., 2009. Hypothesis proved...citric acid (citrate) does improve cancer: a case of a patient suffering from medullary thyroid cancer. Medical hypotheses 73, 271. Halestrap, A.P., 1975. The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. The Biochemical journal 148, 85-96. Hanahan, D., Weinberg, R.A., 2011. Hallmarks of cancer: the next generation. Cell 144, 646-674. Hanai, J.I., Doro, N., Seth, P., Sukhatme, V.P., 2013. ATP citrate lyase knockdown impacts cancer stem cells in vitro. Cell death & disease 4, e696. Hartmann, C., Meyer, J., Balss, J., Capper, D., Mueller, W., Christians, A., Felsberg, J., Wolter, M., Mawrin, C., Wick, W., Weller, M., Herold-Mende, C., Unterberg, A., Jeuken, J.W., Wesseling, P., Reifenberger, G., von Deimling, A., 2009. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta neuropathologica 118, 469-474.
31
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Hassanein, M., Hoeksema, M.D., Shiota, M., Qian, J., Harris, B.K., Chen, H., Clark, J.E., Alborn, W.E., Eisenberg, R., Massion, P.P., 2013. SLC1A5 mediates glutamine transport required for lung cancer cell growth and survival. Clinical cancer research : an official journal of the American Association for Cancer Research 19, 560-570. Hatzivassiliou, G., Zhao, F., Bauer, D.E., Andreadis, C., Shaw, A.N., Dhanak, D., Hingorani, S.R., Tuveson, D.A., Thompson, C.B., 2005. ATP citrate lyase inhibition can suppress tumor cell growth. Cancer cell 8, 311-321. Hillar, M., Lott, V., Lennox, B., 1975. Correlation of the effects of citric acid cycle metabolites on succinate oxidation by rat liver mitochondria and submitochondrial particles. Journal of bioenergetics 7, 1-16. Hobert, J.A., Mester, J.L., Moline, J., Eng, C., 2012. Elevated plasma succinate in PTEN, SDHB, and SDHD mutation-positive individuals. Genetics in medicine : official journal of the American College of Medical Genetics 14, 616-619. Holleran, A.L., Briscoe, D.A., Fiskum, G., Kelleher, J.K., 1995. Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation. Molecular and cellular biochemistry 152, 95-101. Hoshino, A., Hirst, J.A., Fujii, H., 2007. Regulation of cell proliferation by interleukin-3-induced nuclear translocation of pyruvate kinase. The Journal of biological chemistry 282, 17706-17711. Hu, W., Zhang, C., Wu, R., Sun, Y., Levine, A., Feng, Z., 2010. Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function. Proceedings of the National Academy of Sciences of the United States of America 107, 7455-7460. Iacobazzi, V., Infantino, V., Palmieri, F., 2008. Epigenetic mechanisms and Sp1 regulate mitochondrial citrate carrier gene expression. Biochemical and biophysical research communications 376, 15-20. Icard, P., Poulain, L., Lincet, H., 2012. Understanding the central role of citrate in the metabolism of cancer cells. Biochimica et biophysica acta 1825, 111-116. Ihrlund, L.S., Hernlund, E., Khan, O., Shoshan, M.C., 2008. 3-Bromopyruvate as inhibitor of tumour cell energy metabolism and chemopotentiator of platinum drugs. Molecular oncology 2, 94-101. Infantino, V., Convertini, P., Cucci, L., Panaro, M.A., Di Noia, M.A., Calvello, R., Palmieri, F., Iacobazzi, V., 2011. The mitochondrial citrate carrier: a new player in inflammation. The Biochemical journal 438, 433436. Ishii, T., Yasuda, K., Akatsuka, A., Hino, O., Hartman, P.S., Ishii, N., 2005. A mutation in the SDHC gene of complex II increases oxidative stress, resulting in apoptosis and tumorigenesis. Cancer research 65, 203209. Jiang, B.H., Jiang, G., Zheng, J.Z., Lu, Z., Hunter, T., Vogt, P.K., 2001. Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research 12, 363-369. Jurica, M.S., Mesecar, A., Heath, P.J., Shi, W., Nowak, T., Stoddard, B.L., 1998. The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate. Structure 6, 195-210. Kato, M., Wynn, R.M., Chuang, J.L., Tso, S.C., Machius, M., Li, J., Chuang, D.T., 2008. Structural basis for inactivation of the human pyruvate dehydrogenase complex by phosphorylation: role of disordered phosphorylation loops. Structure 16, 1849-1859. Killian, J.K., Kim, S.Y., Miettinen, M., Smith, C., Merino, M., Tsokos, M., Quezado, M., Smith, W.I., Jr., Jahromi, M.S., Xekouki, P., Szarek, E., Walker, R.L., Lasota, J., Raffeld, M., Klotzle, B., Wang, Z., Jones, L., Zhu, Y., Wang, Y., Waterfall, J.J., O'Sullivan, M.J., Bibikova, M., Pacak, K., Stratakis, C., Janeway, K.A., Schiffman, J.D., Fan, J.B., Helman, L., Meltzer, P.S., 2013. Succinate dehydrogenase mutation underlies global epigenomic divergence in gastrointestinal stromal tumor. Cancer discovery 3, 648-657. Kim, J., DeBerardinis, R.J., 2013. Cancer. Silencing a metabolic oncogene. Science 340, 558-559.
32
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Ko, Y.H., Pedersen, P.L., Geschwind, J.F., 2001. Glucose catabolism in the rabbit VX2 tumor model for liver cancer: characterization and targeting hexokinase. Cancer letters 173, 83-91. Kondrashova, M.N., Gogvadze, V.G., Medvedev, B.I., Babsky, A.M., 1982. Succinic acid oxidation as the only energy support of intensive Ca2+ uptake by mitochondria. Biochemical and biophysical research communications 109, 376-381. Kovarova, J., Bajzikova, M., Vondrusova, M., Stursa, J., Goodwin, J., Nguyen, M., Zobalova, R., Pesdar, E.A., Truksa, J., Tomasetti, M., Dong, L.F., Neuzil, J., 2014. Mitochondrial targeting of alpha-tocopheryl succinate enhances its anti-mesothelioma efficacy. Redox report : communications in free radical research 19, 16-25. Kramer, R., Palmieri, F., 1989. Molecular aspects of isolated and reconstituted carrier proteins from animal mitochondria. Biochimica et biophysica acta 974, 1-23. Krasikova, R.N., Kuznetsova, O.F., Fedorova, O.S., Belokon, Y.N., Maleev, V.I., Mu, L., Ametamey, S., Schubiger, P.A., Friebe, M., Berndt, M., Koglin, N., Mueller, A., Graham, K., Lehmann, L., Dinkelborg, L.M., 2011. 4-[18F]fluoroglutamic acid (BAY 85-8050), a new amino acid radiotracer for PET imaging of tumors: synthesis and in vitro characterization. Journal of medicinal chemistry 54, 406-410. Krebs, H.A., Eggleston, L.V., D'Alessandro, A., 1961. The effect of succinate and amytal on the reduction of acetoacetate in animal tissues. The Biochemical journal 79, 537-549. Kruspig, B., Nilchian, A., Bejarano, I., Orrenius, S., Zhivotovsky, B., Gogvadze, V., 2012a. Targeting mitochondria by alpha-tocopheryl succinate kills neuroblastoma cells irrespective of MycN oncogene expression. Cellular and Molecular Life Sciences 69, 2091-2099. Kruspig, B., Nilchian, A., Orrenius, S., Zhivotovsky, B., Gogvadze, V., 2012b. Citrate kills tumor cells through activation of apical caspases. Cellular and Molecular Life Sciences 69, 4229-4237. Kruspig, B., Zhivotovsky, B., Gogvadze, V., 2013. Contrasting effects of alpha-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis. Mitochondrion 13, 533-538. Kung, H.N., Marks, J.R., Chi, J.T., 2011. Glutamine synthetase is a genetic determinant of cell typespecific glutamine independence in breast epithelia. PLoS genetics 7, e1002229. Le, A., Lane, A.N., Hamaker, M., Bose, S., Gouw, A., Barbi, J., Tsukamoto, T., Rojas, C.J., Slusher, B.S., Zhang, H., Zimmerman, L.J., Liebler, D.C., Slebos, R.J., Lorkiewicz, P.K., Higashi, R.M., Fan, T.W., Dang, C.V., 2012. Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells. Cell metabolism 15, 110-121. Lee, B.H., Tothova, Z., Levine, R.L., Anderson, K., Buza-Vidas, N., Cullen, D.E., McDowell, E.P., Adelsperger, J., Frohling, S., Huntly, B.J., Beran, M., Jacobsen, S.E., Gilliland, D.G., 2007. FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia. Cancer cell 12, 367-380. Lee, J., Kim, H.K., Han, Y.M., Kim, J., 2008. Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. The international journal of biochemistry & cell biology 40, 1043-1054. Lemarie, A., Huc, L., Pazarentzos, E., Mahul-Mellier, A.L., Grimm, S., 2011. Specific disintegration of complex II succinate: ubiquinone oxidoreductase links pH changes to oxidative stress for apoptosis induction. Cell death and differentiation 18, 338-349. Lenaz, G., Genova, M.L., 2009. Mobility and function of coenzyme Q (ubiquinone) in the mitochondrial respiratory chain. Biochimica et biophysica acta 1787, 563-573. Letouze, E., Martinelli, C., Loriot, C., Burnichon, N., Abermil, N., Ottolenghi, C., Janin, M., Menara, M., Nguyen, A.T., Benit, P., Buffet, A., Marcaillou, C., Bertherat, J., Amar, L., Rustin, P., De Reynies, A., Gimenez-Roqueplo, A.P., Favier, J., 2013. SDH mutations establish a hypermethylator phenotype in paraganglioma. Cancer cell 23, 739-752.
33
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Lincet, H., Kafara, P., Giffard, F., Abeilard-Lemoisson, E., Duval, M., Louis, M.H., Poulain, L., Icard, P., 2013. Inhibition of Mcl-1 expression by citrate enhances the effect of Bcl-xL inhibitors on human ovarian carcinoma cells. Journal of ovarian research 6, 72. Lobo, C., Ruiz-Bellido, M.A., Aledo, J.C., Marquez, J., Nunez De Castro, I., Alonso, F.J., 2000. Inhibition of glutaminase expression by antisense mRNA decreases growth and tumourigenicity of tumour cells. The Biochemical journal 348 Pt 2, 257-261. Lu, C., Ward, P.S., Kapoor, G.S., Rohle, D., Turcan, S., Abdel-Wahab, O., Edwards, C.R., Khanin, R., Figueroa, M.E., Melnick, A., Wellen, K.E., O'Rourke, D.M., Berger, S.L., Chan, T.A., Levine, R.L., Mellinghoff, I.K., Thompson, C.B., 2012. IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature 483, 474-478. Lu, Y., Zhang, X., Zhang, H., Lan, J., Huang, G., Varin, E., Lincet, H., Poulain, L., Icard, P., 2011. Citrate induces apoptotic cell death: a promising way to treat gastric carcinoma? Anticancer research 31, 797805. Luo, W., Hu, H., Chang, R., Zhong, J., Knabel, M., O'Meally, R., Cole, R.N., Pandey, A., Semenza, G.L., 2011. Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell 145, 732744. MacKenzie, E.D., Selak, M.A., Tennant, D.A., Payne, L.J., Crosby, S., Frederiksen, C.M., Watson, D.G., Gottlieb, E., 2007. Cell-permeating alpha-ketoglutarate derivatives alleviate pseudohypoxia in succinate dehydrogenase-deficient cells. Molecular and cellular biology 27, 3282-3289. Mardis, E.R., Ding, L., Dooling, D.J., Larson, D.E., McLellan, M.D., Chen, K., Koboldt, D.C., Fulton, R.S., Delehaunty, K.D., McGrath, S.D., Fulton, L.A., Locke, D.P., Magrini, V.J., Abbott, R.M., Vickery, T.L., Reed, J.S., Robinson, J.S., Wylie, T., Smith, S.M., Carmichael, L., Eldred, J.M., Harris, C.C., Walker, J., Peck, J.B., Du, F., Dukes, A.F., Sanderson, G.E., Brummett, A.M., Clark, E., McMichael, J.F., Meyer, R.J., Schindler, J.K., Pohl, C.S., Wallis, J.W., Shi, X., Lin, L., Schmidt, H., Tang, Y., Haipek, C., Wiechert, M.E., Ivy, J.V., Kalicki, J., Elliott, G., Ries, R.E., Payton, J.E., Westervelt, P., Tomasson, M.H., Watson, M.A., Baty, J., Heath, S., Shannon, W.D., Nagarajan, R., Link, D.C., Walter, M.J., Graubert, T.A., DiPersio, J.F., Wilson, R.K., Ley, T.J., 2009. Recurring mutations found by sequencing an acute myeloid leukemia genome. The New England journal of medicine 361, 1058-1066. Matsumoto, S., Saito, K., Yasui, H., Morris, H.D., Munasinghe, J.P., Lizak, M., Merkle, H., ArdenkjaerLarsen, J.H., Choudhuri, R., Devasahayam, N., Subramanian, S., Koretsky, A.P., Mitchell, J.B., Krishna, M.C., 2013. EPR oxygen imaging and hyperpolarized 13C MRI of pyruvate metabolism as noninvasive biomarkers of tumor treatment response to a glycolysis inhibitor 3-bromopyruvate. Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine 69, 1443-1450. Metallo, C.M., Gameiro, P.A., Bell, E.L., Mattaini, K.R., Yang, J., Hiller, K., Jewell, C.M., Johnson, Z.R., Irvine, D.J., Guarente, L., Kelleher, J.K., Vander Heiden, M.G., Iliopoulos, O., Stephanopoulos, G., 2012. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 481, 380-384. Miettinen, M., Wang, Z.F., Sarlomo-Rikala, M., Osuch, C., Rutkowski, P., Lasota, J., 2011. Succinate dehydrogenase-deficient GISTs: a clinicopathologic, immunohistochemical, and molecular genetic study of 66 gastric GISTs with predilection to young age. The American journal of surgical pathology 35, 17121721. Moreno-Sanchez, R., Hernandez-Esquivel, L., Rivero-Segura, N.A., Marin-Hernandez, A., Neuzil, J., Ralph, S.J., Rodriguez-Enriquez, S., 2013. Reactive oxygen species are generated by the respiratory complex II-evidence for lack of contribution of the reverse electron flow in complex I. The FEBS journal 280, 927938. Mullen, A.R., Wheaton, W.W., Jin, E.S., Chen, P.H., Sullivan, L.B., Cheng, T., Yang, Y., Linehan, W.M., Chandel, N.S., DeBerardinis, R.J., 2012. Reductive carboxylation supports growth in tumour cells with defective mitochondria. Nature 481, 385-388. 34
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Neuzil, J., Massa, H., 2005. Hepatic processing determines dual activity of alpha-tocopheryl succinate: a novel paradigm for a shift in biological activity due to pro-vitamin-to-vitamin conversion. Biochemical and biophysical research communications 327, 1024-1027. Neuzil, J., Weber, T., Gellert, N., Weber, C., 2001a. Selective cancer cell killing by alpha-tocopheryl succinate. British journal of cancer 84, 87-89. Neuzil, J., Weber, T., Schroder, A., Lu, M., Ostermann, G., Gellert, N., Mayne, G.C., Olejnicka, B., NegreSalvayre, A., Sticha, M., Coffey, R.J., Weber, C., 2001b. Induction of cancer cell apoptosis by alphatocopheryl succinate: molecular pathways and structural requirements. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 15, 403-415. Niemann, S., Muller, U., 2000. Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nature genetics 26, 268-270. Noguchi, T., Inoue, H., Tanaka, T., 1986. The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. The Journal of biological chemistry 261, 13807-13812. Noguchi, T., Yamada, K., Inoue, H., Matsuda, T., Tanaka, T., 1987. The L- and R-type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters. The Journal of biological chemistry 262, 14366-14371. Nutt, L.K., Buchakjian, M.R., Gan, E., Darbandi, R., Yoon, S.Y., Wu, J.Q., Miyamoto, Y.J., Gibbons, J.A., Andersen, J.L., Freel, C.D., Tang, W., He, C., Kurokawa, M., Wang, Y., Margolis, S.S., Fissore, R.A., Kornbluth, S., 2009. Metabolic control of oocyte apoptosis mediated by 14-3-3zeta-regulated dephosphorylation of caspase-2. Developmental cell 16, 856-866. Nutt, L.K., Margolis, S.S., Jensen, M., Herman, C.E., Dunphy, W.G., Rathmell, J.C., Kornbluth, S., 2005. Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2. Cell 123, 89-103. Parsons, D.W., Jones, S., Zhang, X., Lin, J.C., Leary, R.J., Angenendt, P., Mankoo, P., Carter, H., Siu, I.M., Gallia, G.L., Olivi, A., McLendon, R., Rasheed, B.A., Keir, S., Nikolskaya, T., Nikolsky, Y., Busam, D.A., Tekleab, H., Diaz, L.A., Jr., Hartigan, J., Smith, D.R., Strausberg, R.L., Marie, S.K., Shinjo, S.M., Yan, H., Riggins, G.J., Bigner, D.D., Karchin, R., Papadopoulos, N., Parmigiani, G., Vogelstein, B., Velculescu, V.E., Kinzler, K.W., 2008. An integrated genomic analysis of human glioblastoma multiforme. Science 321, 1807-1812. Pollyea, D.A., Kohrt, H.E., Zhang, B., Zehnder, J., Schenkein, D., Fantin, V., Straley, K., Vasanthakumar, A., Abdel-Wahab, O., Levine, R., Godley, L.A., Medeiros, B.C., 2013. 2-Hydroxyglutarate in IDH mutant acute myeloid leukemia: predicting patient responses, minimal residual disease and correlations with methylcytosine and hydroxymethylcytosine levels. Leukemia & lymphoma 54, 408-410. Pop, C., Fitzgerald, P., Green, D.R., Salvesen, G.S., 2007. Role of proteolysis in caspase-8 activation and stabilization. Biochemistry 46, 4398-4407. Pope, W.B., Prins, R.M., Albert Thomas, M., Nagarajan, R., Yen, K.E., Bittinger, M.A., Salamon, N., Chou, A.P., Yong, W.H., Soto, H., Wilson, N., Driggers, E., Jang, H.G., Su, S.M., Schenkein, D.P., Lai, A., Cloughesy, T.F., Kornblum, H.I., Wu, H., Fantin, V.R., Liau, L.M., 2012. Non-invasive detection of 2hydroxyglutarate and other metabolites in IDH1 mutant glioma patients using magnetic resonance spectroscopy. Journal of neuro-oncology 107, 197-205. Prasad, K.N., Edwards-Prasad, J., 1982. Effects of tocopherol (vitamin E) acid succinate on morphological alterations and growth inhibition in melanoma cells in culture. Cancer research 42, 550-555. Prochazka, L., Koudelka, S., Dong, L.F., Stursa, J., Goodwin, J., Neca, J., Slavik, J., Ciganek, M., Masek, J., Kluckova, K., Nguyen, M., Turanek, J., Neuzil, J., 2013. Mitochondrial targeting overcomes ABCA1dependent resistance of lung carcinoma to alpha-tocopheryl succinate. Apoptosis : an international journal on programmed cell death 18, 286-299.
35
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Qie, S., Chu, C., Li, W., Wang, C., Sang, N., 2013. ErbB2 activation upregulates glutaminase 1 expression which promotes breast cancer cell proliferation. Journal of cellular biochemistry. Qing, G., Li, B., Vu, A., Skuli, N., Walton, Z.E., Liu, X., Mayes, P.A., Wise, D.R., Thompson, C.B., Maris, J.M., Hogarty, M.D., Simon, M.C., 2012. ATF4 regulates MYC-mediated neuroblastoma cell death upon glutamine deprivation. Cancer cell 22, 631-644. Qu, W., Oya, S., Lieberman, B.P., Ploessl, K., Wang, L., Wise, D.R., Divgi, C.R., Chodosh, L.A., Thompson, C.B., Kung, H.F., 2012. Preparation and characterization of L-[5-11C]-glutamine for metabolic imaging of tumors. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 53, 98-105. Qu, W., Zha, Z., Ploessl, K., Lieberman, B.P., Zhu, L., Wise, D.R., Thompson, C.B., Kung, H.F., 2011. Synthesis of optically pure 4-fluoro-glutamines as potential metabolic imaging agents for tumors. Journal of the American Chemical Society 133, 1122-1133. Quinlan, C.L., Orr, A.L., Perevoshchikova, I.V., Treberg, J.R., Ackrell, B.A., Brand, M.D., 2012. Mitochondrial complex II can generate reactive oxygen species at high rates in both the forward and reverse reactions. The Journal of biological chemistry 287, 27255-27264. Ricci, J.E., Gottlieb, R.A., Green, D.R., 2003. Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis. The Journal of cell biology 160, 65-75. Ricketts, C., Woodward, E.R., Killick, P., Morris, M.R., Astuti, D., Latif, F., Maher, E.R., 2008. Germline SDHB mutations and familial renal cell carcinoma. Journal of the National Cancer Institute 100, 12601262. Rohle, D., Popovici-Muller, J., Palaskas, N., Turcan, S., Grommes, C., Campos, C., Tsoi, J., Clark, O., Oldrini, B., Komisopoulou, E., Kunii, K., Pedraza, A., Schalm, S., Silverman, L., Miller, A., Wang, F., Yang, H., Chen, Y., Kernytsky, A., Rosenblum, M.K., Liu, W., Biller, S.A., Su, S.M., Brennan, C.W., Chan, T.A., Graeber, T.G., Yen, K.E., Mellinghoff, I.K., 2013. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science 340, 626-630. Rose, N.R., McDonough, M.A., King, O.N., Kawamura, A., Schofield, C.J., 2011. Inhibition of 2oxoglutarate dependent oxygenases. Chemical Society reviews 40, 4364-4397. Salas, M.L., Vinuela, E., Salas, M., Sols, A., 1965. Citrate Inhibition of Phosphofructokinase and the Pasteur Effect. Biochemical and biophysical research communications 19, 371-376. Salvesen, G.S., Dixit, V.M., 1999. Caspase activation: the induced-proximity model. Proceedings of the National Academy of Sciences of the United States of America 96, 10964-10967. Schafer, Z.T., Grassian, A.R., Song, L., Jiang, Z., Gerhart-Hines, Z., Irie, H.Y., Gao, S., Puigserver, P., Brugge, J.S., 2009. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 461, 109-113. Schagger, H., Pfeiffer, K., 2000. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. The EMBO journal 19, 1777-1783. Schodel, J., Oikonomopoulos, S., Ragoussis, J., Pugh, C.W., Ratcliffe, P.J., Mole, D.R., 2011. Highresolution genome-wide mapping of HIF-binding sites by ChIP-seq. Blood 117, e207-217. Scott, D.A., Richardson, A.D., Filipp, F.V., Knutzen, C.A., Chiang, G.G., Ronai, Z.A., Osterman, A.L., Smith, J.W., 2011. Comparative metabolic flux profiling of melanoma cell lines: beyond the Warburg effect. The Journal of biological chemistry 286, 42626-42634. Selak, M.A., Armour, S.M., MacKenzie, E.D., Boulahbel, H., Watson, D.G., Mansfield, K.D., Pan, Y., Simon, M.C., Thompson, C.B., Gottlieb, E., 2005. Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer cell 7, 77-85. Semenza, G.L., 2010. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 29, 625-634. Shapiro, R.A., Farrell, L., Srinivasan, M., Curthoys, N.P., 1991. Isolation, characterization, and in vitro expression of a cDNA that encodes the kidney isoenzyme of the mitochondrial glutaminase. The Journal of biological chemistry 266, 18792-18796. 36
ACCEPTED MANUSCRIPT
AC CE P
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D
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RI
PT
Sies, H., Summer, K.H., 1975. Hydroperoxide-metabolizing systems in rat liver. European journal of biochemistry / FEBS 57, 503-512. Smith, E.M., Watford, M., 1990. Molecular cloning of a cDNA for rat hepatic glutaminase. Sequence similarity to kidney-type glutaminase. The Journal of biological chemistry 265, 10631-10636. Stapelberg, M., Tomasetti, M., Alleva, R., Gellert, N., Procopio, A., Neuzil, J., 2004. alpha-Tocopheryl succinate inhibits proliferation of mesothelioma cells by selective down-regulation of fibroblast growth factor receptors. Biochemical and biophysical research communications 318, 636-641. Stetak, A., Veress, R., Ovadi, J., Csermely, P., Keri, G., Ullrich, A., 2007. Nuclear translocation of the tumor marker pyruvate kinase M2 induces programmed cell death. Cancer research 67, 1602-1608. Sun, F., Huo, X., Zhai, Y., Wang, A., Xu, J., Su, D., Bartlam, M., Rao, Z., 2005. Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121, 1043-1057. Sun, Q., Chen, X., Ma, J., Peng, H., Wang, F., Zha, X., Wang, Y., Jing, Y., Yang, H., Chen, R., Chang, L., Zhang, Y., Goto, J., Onda, H., Chen, T., Wang, M.R., Lu, Y., You, H., Kwiatkowski, D., Zhang, H., 2011. Mammalian target of rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth. Proceedings of the National Academy of Sciences of the United States of America 108, 4129-4134. Sun, R.C., Denko, N.C., 2014. Hypoxic Regulation of Glutamine Metabolism through HIF1 and SIAH2 Supports Lipid Synthesis that Is Necessary for Tumor Growth. Cell metabolism 19, 285-292. Suzuki, S., Tanaka, T., Poyurovsky, M.V., Nagano, H., Mayama, T., Ohkubo, S., Lokshin, M., Hosokawa, H., Nakayama, T., Suzuki, Y., Sugano, S., Sato, E., Nagao, T., Yokote, K., Tatsuno, I., Prives, C., 2010. Phosphate-activated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive oxygen species. Proceedings of the National Academy of Sciences of the United States of America 107, 7461-7466. Swettenham, E., Witting, P.K., Salvatore, B.A., Neuzil, J., 2005. Alpha-tocopheryl succinate selectively induces apoptosis in neuroblastoma cells: potential therapy of malignancies of the nervous system? Journal of neurochemistry 94, 1448-1456. Szeliga, M., Obara-Michlewska, M., Matyja, E., Lazarczyk, M., Lobo, C., Hilgier, W., Alonso, F.J., Marquez, J., Albrecht, J., 2009. Transfection with liver-type glutaminase cDNA alters gene expression and reduces survival, migration and proliferation of T98G glioma cells. Glia 57, 1014-1023. Szeliga, M., Sidoryk, M., Matyja, E., Kowalczyk, P., Albrecht, J., 2005. Lack of expression of the liver-type glutaminase (LGA) mRNA in human malignant gliomas. Neuroscience letters 374, 171-173. Tannahill, G.M., Curtis, A.M., Adamik, J., Palsson-McDermott, E.M., McGettrick, A.F., Goel, G., Frezza, C., Bernard, N.J., Kelly, B., Foley, N.H., Zheng, L., Gardet, A., Tong, Z., Jany, S.S., Corr, S.C., Haneklaus, M., Caffrey, B.E., Pierce, K., Walmsley, S., Beasley, F.C., Cummins, E., Nizet, V., Whyte, M., Taylor, C.T., Lin, H., Masters, S.L., Gottlieb, E., Kelly, V.P., Clish, C., Auron, P.E., Xavier, R.J., O'Neill, L.A., 2013. Succinate is an inflammatory signal that induces IL-1beta through HIF-1alpha. Nature 496, 238-242. Taylor, W.M., Halperin, M.L., 1973. Regulation of pyruvate dehydrogenase in muscle. Inhibition by citrate. The Journal of biological chemistry 248, 6080-6083. Tennant, D.A., Frezza, C., MacKenzie, E.D., Nguyen, Q.D., Zheng, L., Selak, M.A., Roberts, D.L., Dive, C., Watson, D.G., Aboagye, E.O., Gottlieb, E., 2009. Reactivating HIF prolyl hydroxylases under hypoxia results in metabolic catastrophe and cell death. Oncogene 28, 4009-4021. Thangaraju, M., Karunakaran, S.K., Itagaki, S., Gopal, E., Elangovan, S., Prasad, P.D., Ganapathy, V., 2009. Transport by SLC5A8 with subsequent inhibition of histone deacetylase 1 (HDAC1) and HDAC3 underlies the antitumor activity of 3-bromopyruvate. Cancer 115, 4655-4666. Turcan, S., Rohle, D., Goenka, A., Walsh, L.A., Fang, F., Yilmaz, E., Campos, C., Fabius, A.W., Lu, C., Ward, P.S., Thompson, C.B., Kaufman, A., Guryanova, O., Levine, R., Heguy, A., Viale, A., Morris, L.G., Huse, J.T., Mellinghoff, I.K., Chan, T.A., 2012. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 483, 479-483. 37
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RI
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van den Heuvel, A.P., Jing, J., Wooster, R.F., Bachman, K.E., 2012. Analysis of glutamine dependency in non-small cell lung cancer: GLS1 splice variant GAC is essential for cancer cell growth. Cancer biology & therapy 13, 1185-1194. Vander Heiden, M.G., Chandel, N.S., Schumacker, P.T., Thompson, C.B., 1999. Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange. Molecular cell 3, 159-167. Vaughn, A.E., Deshmukh, M., 2008. Glucose metabolism inhibits apoptosis in neurons and cancer cells by redox inactivation of cytochrome c. Nature cell biology 10, 1477-1483. Wang, F., Travins, J., DeLaBarre, B., Penard-Lacronique, V., Schalm, S., Hansen, E., Straley, K., Kernytsky, A., Liu, W., Gliser, C., Yang, H., Gross, S., Artin, E., Saada, V., Mylonas, E., Quivoron, C., Popovici-Muller, J., Saunders, J.O., Salituro, F.G., Yan, S., Murray, S., Wei, W., Gao, Y., Dang, L., Dorsch, M., Agresta, S., Schenkein, D.P., Biller, S.A., Su, S.M., de Botton, S., Yen, K.E., 2013. Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation. Science 340, 622-626. Wang, H.J., Hsieh, Y.J., Cheng, W.C., Lin, C.P., Lin, Y.S., Yang, S.F., Chen, C.C., Izumiya, Y., Yu, J.S., Kung, H.J., Wang, W.C., 2014. JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1alphamediated glucose metabolism. Proceedings of the National Academy of Sciences of the United States of America 111, 279-284. Wang, J.B., Erickson, J.W., Fuji, R., Ramachandran, S., Gao, P., Dinavahi, R., Wilson, K.F., Ambrosio, A.L., Dias, S.M., Dang, C.V., Cerione, R.A., 2010. Targeting mitochondrial glutaminase activity inhibits oncogenic transformation. Cancer cell 18, 207-219. Warburg, O., Wind, F., Negelein, E., 1927. The Metabolism of Tumors in the Body. The Journal of general physiology 8, 519-530. Ward, P.S., Patel, J., Wise, D.R., Abdel-Wahab, O., Bennett, B.D., Coller, H.A., Cross, J.R., Fantin, V.R., Hedvat, C.V., Perl, A.E., Rabinowitz, J.D., Carroll, M., Su, S.M., Sharp, K.A., Levine, R.L., Thompson, C.B., 2010. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer cell 17, 225-234. Waygood, E.B., Sanwal, B.D., 1972. Regulation of pyruvate kinases by succinyl coenzyme A. Biochemical and biophysical research communications 48, 402-407. Waygood, E.B., Sanwal, B.D., 1974. The control of pyruvate kinases of Escherichia coli. I. Physicochemical and regulatory properties of the enzyme activated by fructose 1,6-diphosphate. The Journal of biological chemistry 249, 265-274. Wise, D.R., DeBerardinis, R.J., Mancuso, A., Sayed, N., Zhang, X.Y., Pfeiffer, H.K., Nissim, I., Daikhin, E., Yudkoff, M., McMahon, S.B., Thompson, C.B., 2008. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proceedings of the National Academy of Sciences of the United States of America 105, 18782-18787. Wise, D.R., Thompson, C.B., 2010. Glutamine addiction: a new therapeutic target in cancer. Trends in biochemical sciences 35, 427-433. Wise, D.R., Ward, P.S., Shay, J.E., Cross, J.R., Gruber, J.J., Sachdeva, U.M., Platt, J.M., DeMatteo, R.G., Simon, M.C., Thompson, C.B., 2011. Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of alpha-ketoglutarate to citrate to support cell growth and viability. Proceedings of the National Academy of Sciences of the United States of America 108, 19611-19616. Wong, N., Ojo, D., Yan, J., Tang, D., 2014. PKM2 contributes to cancer metabolism. Cancer letters. Wouters, B.G., Koritzinsky, M., 2008. Hypoxia signalling through mTOR and the unfolded protein response in cancer. Nature reviews. Cancer 8, 851-864. Xiao, M., Yang, H., Xu, W., Ma, S., Lin, H., Zhu, H., Liu, L., Liu, Y., Yang, C., Xu, Y., Zhao, S., Ye, D., Xiong, Y., Guan, K.L., 2012. Inhibition of alpha-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors. Genes & development 26, 1326-1338. 38
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RI
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Xu, W., Yang, H., Liu, Y., Yang, Y., Wang, P., Kim, S.H., Ito, S., Yang, C., Wang, P., Xiao, M.T., Liu, L.X., Jiang, W.Q., Liu, J., Zhang, J.Y., Wang, B., Frye, S., Zhang, Y., Xu, Y.H., Lei, Q.Y., Guan, K.L., Zhao, S.M., Xiong, Y., 2011. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutaratedependent dioxygenases. Cancer cell 19, 17-30. Yalcin, A., Clem, B.F., Simmons, A., Lane, A., Nelson, K., Clem, A.L., Brock, E., Siow, D., Wattenberg, B., Telang, S., Chesney, J., 2009a. Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases. The Journal of biological chemistry 284, 24223-24232. Yalcin, A., Telang, S., Clem, B., Chesney, J., 2009b. Regulation of glucose metabolism by 6phosphofructo-2-kinase/fructose-2,6-bisphosphatases in cancer. Experimental and molecular pathology 86, 174-179. Yan, H., Parsons, D.W., Jin, G., McLendon, R., Rasheed, B.A., Yuan, W., Kos, I., Batinic-Haberle, I., Jones, S., Riggins, G.J., Friedman, H., Friedman, A., Reardon, D., Herndon, J., Kinzler, K.W., Velculescu, V.E., Vogelstein, B., Bigner, D.D., 2009. IDH1 and IDH2 mutations in gliomas. The New England journal of medicine 360, 765-773. Yang, C., Sudderth, J., Dang, T., Bachoo, R.M., McDonald, J.G., DeBerardinis, R.J., 2009. Glioblastoma cells require glutamate dehydrogenase to survive impairments of glucose metabolism or Akt signaling. Cancer research 69, 7986-7993. Yang, W., Xia, Y., Cao, Y., Zheng, Y., Bu, W., Zhang, L., You, M.J., Koh, M.Y., Cote, G., Aldape, K., Li, Y., Verma, I.M., Chiao, P.J., Lu, Z., 2012. EGFR-induced and PKCepsilon monoubiquitylation-dependent NFkappaB activation upregulates PKM2 expression and promotes tumorigenesis. Molecular cell 48, 771784. Yang, W., Xia, Y., Ji, H., Zheng, Y., Liang, J., Huang, W., Gao, X., Aldape, K., Lu, Z., 2011. Nuclear PKM2 regulates beta-catenin transactivation upon EGFR activation. Nature 480, 118-122. Yi, C.H., Pan, H., Seebacher, J., Jang, I.H., Hyberts, S.G., Heffron, G.J., Vander Heiden, M.G., Yang, R., Li, F., Locasale, J.W., Sharfi, H., Zhai, B., Rodriguez-Mias, R., Luithardt, H., Cantley, L.C., Daley, G.Q., Asara, J.M., Gygi, S.P., Wagner, G., Liu, C.F., Yuan, J., 2011. Metabolic regulation of protein N-alpha-acetylation by Bcl-xL promotes cell survival. Cell 146, 607-620. Yi, C.H., Sogah, D.K., Boyce, M., Degterev, A., Christofferson, D.E., Yuan, J., 2007. A genome-wide RNAi screen reveals multiple regulators of caspase activation. The Journal of cell biology 179, 619-626. Yousefi, S., Owens, J.W., Cesario, T.C., 2004. Citrate shows specific, dose-dependent lympholytic activity in neoplastic cell lines. Leukemia & lymphoma 45, 1657-1665. Yuneva, M., Zamboni, N., Oefner, P., Sachidanandam, R., Lazebnik, Y., 2007. Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells. The Journal of cell biology 178, 93-105. Zhang, X., Varin, E., Allouche, S., Lu, Y., Poulain, L., Icard, P., 2009. Effect of citrate on malignant pleural mesothelioma cells: a synergistic effect with cisplatin. Anticancer research 29, 1249-1254.
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Highlights Mitochondria play crucial role in various cell death modalities Metabolic aberrations are hallmarks of cancer Metabolic switch in tumor cells is associated with alteration in the abundance, utilization, and localization of various mitochondrial substrates Targeting mitochondria is a promising strategy to combat cancer
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S1567-7249(14)00122-6 doi: 10.1016/j.mito.2014.08.007 MITOCH 956
To appear in:
Mitochondrion
Please cite this article as: Kruspig, Bj¨orn, Zhivotovsky, Boris, Gogvadze, Vladimir, Mitochondrial Substrates in Cancer: Drivers or Passengers?, Mitochondrion (2014), doi: 10.1016/j.mito.2014.08.007
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Mitochondrial Substrates in Cancer: Drivers or Passengers?
Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210,
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Björn Kruspig1, Boris Zhivotovsky1,2, and Vladimir Gogvadze1,2,*
MV Lomonosov Moscow State University, 119991 Moscow, Russia
*Correspondence to:
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Vladimir Gogvadze,
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171 77 Stockholm, Sweden
Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, 171 77 Stockholm, Sweden. Tel.: +46 8 524 87515 Fax: +46 8 329041
E-mail: [email protected]
Running title: Mitochondrial substrates in cancer
ACCEPTED MANUSCRIPT Abstract The majority of cancers demonstrate various tumor-specific metabolic aberrations, such as
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increased glycolysis even under aerobic conditions (Warburg effect), whereas mitochondrial
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metabolic activity and their contribution to cellular energy production are restrained. One of the most important mechanisms for this metabolic switch is the alteration in the abundance,
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utilization, and localization of various mitochondrial substrates. Numerous lines of evidence
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connect disturbances in mitochondrial metabolic pathways with tumorigenesis and provide an
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intriguing rationale for utilizing mitochondria as targets for anticancer therapy.
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Keywords: mitochondria; metabolism; Warburg Effect; cancer; cell death; therapy.
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ACCEPTED MANUSCRIPT Introduction For decades, the role of mitochondria in cancer was vastly underestimated by the research
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community, which focused primarily on cancer genetics and ignored the seminal findings by the
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German biochemist and Nobel Laureate Otto Warburg, who hypothesized as early as 1927 the importance of these organelles for tumorigenesis (Warburg et al., 1927). Discoveries in recent
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decades have clearly confirmed his hypothesis, showing that disturbances of mitochondrial
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functions are not only key features of cancer, but also of other, mainly neurodegenerative, disorders, e.g., Parkinson’s, Alzheimer’s, and many other diseases. The importance of
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mitochondria under pathological conditions stems from their central role in many vital physiological processes in the cell, including ATP production, calcium homeostasis control, and
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reactive oxygen species (ROS) production, as well as in the execution and regulation of different cell death modalities. Not only have mitochondria come to the researcher’s focus because of
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their role in various pathologies, but they are also regarded as promising targets for therapeutic interventions in these diseases.
Not only do mitochondria facilitate energy production in the form of ATP, but the majority of metabolic pathways are also directly or indirectly linked to these organelles. The driving force for most mitochondrial functions is the mitochondrial membrane potential, which is mandatory for ATP production, mitochondrial calcium accumulation, and other physiological pathways. The proton gradient is built up by proton pumps in the complexes of the electron transport chain and is fueled by electrons, which are provided by the reducing agents NADH and FADH2. These reducing equivalents arise from the citric acid or tricarboxylic acid (TCA) cycle, which is the most important cellular metabolic network for oxidation of various energy sources, such as glucose, glutamine, and lipids. Many of the countless catabolic metabolic pathways
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ACCEPTED MANUSCRIPT merge at the level of the TCA cycle, fueling both energy production and anabolic processes by providing building blocks for the synthesis of amino acids and other important cellular
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components. The mitochondrial metabolic network comprises a large number of enzymes, as
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well as their specific substrates, which are located in the mitochondrial matrix, integrated into the inner- or outer mitochondrial membrane, intermembrane space or even in the cytosol, linked to
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the mitochondria via transporters and specific pumps. This network is highly tunable and
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flexible, allowing the cell to adjust to different intra- and extracellular conditions, such as nutrient starvation, hypoxia, or other forms of cellular stress. One of the most important
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regulatory mechanisms for adjusting metabolic pathways is the modulation of the availability of mitochondrial substrates, which serves as a sensor for specific cellular conditions, and at the
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same time as a feedback loop for fine-tuning the enzymatic activities. Metabolic dysregulation in cancer has long been regarded as a mere by-product of
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tumorigenesis to support tumor growth and survival. As mentioned above, originating from the findings of Otto Warburg, and after a rediscovery in recent years, it became more and more apparent that metabolic changes in cancer cells are not only a consequence of malignant transformation, but seem to be essential for this process and are regarded as a crucial hallmark of cancer (Hanahan and Weinberg, 2011). Most cells utilize glucose as their main energy source, which is metabolized after its uptake in a set of glycolytic enzymatic reactions to form pyruvate. In normal cells, under normoxic conditions, most of the ATP is produced via oxidative phosphorylation (OXPHOS) in the mitochondria, whereas in cancer cells there is a shift towards glycolysis and suppression of mitochondrial function. This metabolic shift is mainly mediated by changes in the fate of pyruvate, the end-product of glycolysis. In normal cells, pyruvate is directed to the mitochondria
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ACCEPTED MANUSCRIPT where it is metabolized by pyruvate dehydrogenase (PDH) to enter the TCA cycle and fuel OXPHOS. In contrast, in most tumor cells, the activity of PDH is suppressed, causing a reduced
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flow of pyruvate to the mitochondria, and a decrease in OXPHOS. Under these conditions pyruvate is mainly converted to lactate by lactate dehydrogenase (LDH). Named after Otto
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Warburg, the “Warburg Effect” can be found in the majority of tumors. The reliance of tumor
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cells on glucose is even used for diagnostic purposes in fluorodeoxyglucose (18F-FDG) positron
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emission tomography (PET) and therapeutically by utilizing the non-metabolizable glucose analog 2-deoxyglucose to block glycolysis and cancer growth. Glycolysis, despite being less
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efficient in terms of ATP yield per glucose molecule when compared to OXPHOS-driven energy production (2 vs. 36 molecules of ATP), renders tumor cells more resistant to oxygen-
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deprivation as a result of excessive growth or high metabolic activity and poor oxygen supply. It provides the cell with resources to sustain proliferation. For a long time, it was assumed that
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these metabolic changes are caused by defects in mitochondria but were, in fact, later found to be based on specific metabolic regulatory signaling. Thus, the phosphoinositide 3-kinase (PI3K) (Jiang et al., 2001) and mammalian target of rapamycin (mTOR) (Wouters and Koritzinsky, 2008) pathways were shown to induce a pseudo-hypoxic state independently of oxygen by stabilization of the hypoxia-inducible factor 1α (HIF1α), which causes a strong increase in glycolysis known as the Warburg phenotype. Ultimately, different utilization, abundance, and localization of mitochondrial substrates might lead to tumor-specific adaptations of cellular metabolic pathways. The aim of this article is to give an overview of the current knowledge about the role of mitochondrial substrates in cancer development and their utilization as targets for potential therapeutic approaches.
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ACCEPTED MANUSCRIPT Pyruvate The metabolic intermediate and glycolytic end-product pyruvate is a crucial switch between
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aerobic and anaerobic metabolism, and also serves as an important precursor for glucose, amino
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acid, and lipid synthesis. The fate of pyruvate is mainly determined by its subcellular localization. The main cytosolic catabolic reaction is mediated by LDH, reducing pyruvate to L-
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lactate, and at the same time producing one molecule of ATP and regenerating NAD+, a critical
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cofactor for glycolysis function at the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level (Icard et al., 2012). In contrast, when metabolized in the mitochondrial matrix, pyruvate is
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oxidized to fuel the TCA cycle and OXPHOS. Under normoxic conditions, the majority of pyruvate is directed towards the mitochondria by an active transport through the inner
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mitochondrial membrane, mediated by the mitochondrial pyruvate carrier (MPC) (Halestrap, 1975), thereby linking the cytosolic glycolytic pathway with the mitochondrial TCA cycle.
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Within the mitochondrial matrix, PDH, a second key enzyme for this process, catalyzes the conversion to acetyl-CoA, NADH, and carbon dioxide. Acetyl-CoA thereafter enters the TCA cycle and ultimately fuels OXPHOS. As mentioned earlier, a characteristic of a majority of cancers is the switch from oxidative, mitochondrial carbon metabolism to a reductive, cytosolic production of lactate, even under normoxic conditions. This Warburg Effect is mediated in principle by a change of pyruvate distribution in the cell, mainly driven by the activation of HIF1α. This transcription factor controls the expression of a large number of genes that encode proteins related to adaptation to hypoxia, such as those involved in the stimulation of angiogenesis and increased glycolysis, but has also been found to be upregulated due to intratumoral hypoxia or prooncogenic aberrant cell signaling (Semenza, 2010). HIF1α further induces the transcriptional
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ACCEPTED MANUSCRIPT upregulation of several critical proteins involved in pyruvate metabolism, such as pyruvate dehydrogenase kinase 1 (PDK1), monocarboxylate transporter 4 (MCT4), and lactate
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dehydrogenase A (LDHA) (Denko, 2008; Schodel et al., 2011). PDK1, the largest of the four
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PDK isoforms, was shown to phosphorylate the E1 subunit of the PDH complex at all three known phosphorylation sites (Ser-264, Ser-271, and Ser-203), thereby causing a marked
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decrease in its enzymatic activity (Kato et al., 2008). The concurrent increase in cytosolic
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pyruvate means a higher abundance of substrate for the LDH enzyme, which is met by HIF1αmediated transcriptional upregulation of LDHA, and leads to increased lactate production. As an
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adaptation, MCT4 levels are elevated in the plasma membrane under hypoxic conditions, enabling cells to pump out the excess lactate. This increased export of lactate leads to
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acidification of the extracellular environment, which was proposed to select for highly malignant cancer cells and even protect tumor cells from chemotherapy because cellular uptake of weakly
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basic drugs, such as anthracyclines or vinca alkaloids, is significantly reduced under these conditions (De Milito and Fais, 2005). Thus, it was suggested that the low pH surrounding tumors represents a "niche engineering" strategy, supporting malignant tumor growth and invasion into surrounding tissue (Estrella et al., 2013). A very important role in deregulation of pyruvate metabolism in cancer plays pyruvate kinase isoenzyme M2 (PKM2), since knockdown of this isoform was shown to decrease glycolysis and inhibit cell proliferation in a panel of cancer cells (Christofk et al., 2008). Pyruvate kinase (PK) mediates the last step of glycolysis, producing pyruvate and ATP from phosphoenolpyruvate and ADP. The two PK genes, PKLR and PKM2, give rise to four different isozymes, in which PKLR codes for the two splice forms PKL and PKR, and PKM2 for PKM1 and PKM2 (Noguchi et al., 1986; Noguchi et al., 1987). The PK enzyme is active as a tetramer;
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ACCEPTED MANUSCRIPT however, due to alternative splicing, PKM2 exclusively features the ability to form inactive monomers, less-active dimers, and highly active tetramers, making it a very potent switch for
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reprogramming cell metabolism. Several regulatory mechanisms have been described for PK
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activity, including an allosteric feed-forward stimulation by fructose-1,6-bisphosphate (Jurica et al., 1998; Waygood and Sanwal, 1974), and a negative feedback loop by ATP inhibition
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(Waygood and Sanwal, 1972).
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PKM2 is the most prevalent PK isoform in embryonic tissue, and is later replaced in adult tissue by PKL in liver, PKLR in erythrocytes, and PKM1 in muscle tissue and the brain. In many
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cancers, such as liver, colon, renal, and lung carcinomas, PKM2 levels were found to be upregulated (reviewed in (Wong et al., 2014)). This upregulation is controlled by the
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PI3/AKT/mTOR signaling pathway, and mediated transcriptionally by HIF1α and by c-Mycheterogenous nuclear ribonucleoproteins (hnRNPs)-dependent gene splicing (Chen et al., 2010;
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Sun et al., 2011). Interestingly, PKM2 was shown to form a positive feedback regulation with HIF1α, being a direct transcriptional target of HIF1α and at the same time promoting HIF1αmediated transactivation and reprogramming of glucose metabolism. Hydroxylation of PKM2 by prolyl hydroxylase 3 (PHD3) facilitates a direct interaction with HIF1α and a promotion of transcriptional transactivation of HIF1α target genes by increasing the HIF1α binding to target promoters and recruitment of the p300 co-activator (Luo et al., 2011). Since PKM2 and PHD3 are HIF1α targets, the described mechanism acts as a positive feedback loop, sustaining the expression of glycolytic genes and thereby facilitating the Warburg Effect. Additionally, these important findings highlight the non-metabolic functions of PKM2 in the nucleus, which will be discussed later. Transcriptional upregulation of PKM2 under normoxic conditions, induced by
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ACCEPTED MANUSCRIPT Epidermal growth factor receptor (EGFR) signaling, was further shown to facilitate anaerobic glycolysis (Yang et al., 2012).
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The findings and the relevance of increased PKM2 levels in cancer were recently
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challenged by a work by Bluemlein and colleagues, who could not detect any specific upregulation of PKM2 levels when comparing normal vs. tumor tissue by using proteomic
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approaches. Alternatively, the authors suggest a post-translational regulation of PKM2 in cancer
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(Bluemlein et al., 2011). Indeed, several specific post-translational modifications, such as phosphorylation, acetylation, hydroxylation, sumoylation, and oxidation, were shown to
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influence the balance between metabolically active homotetrameric and inactive dimeric PKM2 in favor of the latter in most cancer cells (Wong et al., 2014). The decrease in PK activity as an
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outcome of this interconversion leads to a halt in the glycolytic pathway and accumulation of glycolytic intermediates, as well as inhibition of mitochondrial carbon fluxes. The resulting
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abundance of glycolytic intermediates leads to a redirection to other, mainly anabolic, pathways, such as the pentose pathway, thereby meeting the need of highly proliferating cancer cells for nucleotide and amino acid precursors. As mentioned earlier, PKM2 not only regulates aerobic glycolysis in the cytoplasm, but was also shown to have non-glycolytic functions by being translocated to the nucleus (Hoshino et al., 2007; Stetak et al., 2007) and acting as a transcriptional co-activator for HIF1α, as well as Oct-4, β-catenin, and Stat3 (Gao et al., 2012; Lee et al., 2008; Luo et al., 2011; Yang et al., 2011). Nuclear translocation of PKM2 and binding of phosphorylated β-catenin, in response to EGFR signaling, results in transcriptional transactivation of β-catenin targets such as cyclin D and c-Myc (Yang et al., 2011). Non-metabolic functions of PKM2 are mainly based on its kinase activity, and in a recent report the nuclear transport mechanism was described in detail. Wang
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ACCEPTED MANUSCRIPT and co-workers revealed that Jumonji C (JmjC)-domain-containing dioxygenase directly binds to PKM2 and thereby hinders the formation of its tetrameric, metabolically active form and is
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involved in the nuclear translocation of dimeric PKM2, as well as its transactivation of HIF1α,
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further demonstrating the crucial role of PKM2 in the tumor’s adaptive response to hypoxic stress (Wang et al., 2014).
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Thus, pyruvate is directly and indirectly involved in many adaptive mechanisms in
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tumors, especially in response to oxygen-related stress, in which the subcellular localization and the metabolic fate are key factors for its pro-oncogenic function in tumorigenesis. Given its
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important role in tumor-specific pathways, pyruvate metabolism was explored as a potential therapeutic target. The alkylating agent and pyruvate analog 3-bromopyruvate (3BP) has been
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demonstrated to cause ATP depletion and cell death in multiple tumor models (Geschwind et al., 2002; Ko et al., 2001) without or with only very minimal systemic toxicity. Molecular targets
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were identified to be hexokinase (Ko et al., 2001), as well as the glycolytic enzyme GAPDH (Ganapathy-Kanniappan et al., 2009) and 3BP was further shown to potentiate platinum-induced toxicity and cause production of ROS (Ihrlund et al., 2008). The tumor specificity of this small molecule is based on the overexpression in many tumors of MCT-1, the primary transporter responsible for 3BP uptake (Matsumoto et al., 2013; Thangaraju et al., 2009).
Glutamine, α-Ketoglutarate, and D-2-hydroxyglutarate Besides being the most abundant amino acid in blood and necessary as a supplement for almost all cell lines growing in culture, glutamine was shown to be of utmost importance for tumor metabolism, to the extent that cancer cells are literally addicted to it (Eagle, 1955; Wise and Thompson, 2010). Although being by definition not an essential amino acid, a majority of
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ACCEPTED MANUSCRIPT rapidly proliferating cells, including cancer cells, need glutamine for efficient proliferation due to their high demand that cannot be met by the intracellular production by glutamine synthetase. It
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should be noted that not all tumors require an exogenous supply of glutamine for their growth, as
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demonstrated by different responses to glutamine deprivation in lung as well as breast cancer cell lines (Kung et al., 2011; van den Heuvel et al., 2012). The independence of these cells of
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glutamine was shown to be mainly mediated by the presence of pyruvate carboxylase activity to
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fuel anaplerosis (Cheng et al., 2011). Another link between glutamine metabolism and tumorigenesis was discovered by several reports showing that the oncogene c-Myc induces a
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transcriptional program leading to increased glutaminolysis and dependency on glutamine as a carbon source (Gao et al., 2009; Wise et al., 2008; Yuneva et al., 2007).
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Glutamine is, after glucose, the second most important carbon-based energy source and precursor for several anabolic pathways, in particular, as a nitrogen donor for proteins and
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nucleotides. After transport into the cell by specific importers, the following metabolic steps can be categorized in reactions either utilizing the γ-nitrogen of glutamine for nucleotide or hexosamine synthesis or using the α-nitrogen or carbon backbone for fueling the TCA cycle. The first step for the latter category is mediated by glutaminases and comprises the removal of the amide group to form glutamate. Currently, four different isozymes for glutaminases have been identified, encoded by two different paralogous genes, GLS and GLS2, which are located on chromosome 23 and 12, respectively, and are believed to have been derived by gene duplication. Each gene gives rise to two isoforms: GLS encodes for the kidney-type glutaminase (KGA) (Shapiro et al., 1991) and glutaminase C (GAC) (Elgadi et al., 1999), determined by alternative splicing, whereas GLS2 codes for the liver-type glutaminase (LGA) (short) (Smith and Watford,
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ACCEPTED MANUSCRIPT 1990) and GAB (long) (de la Rosa et al., 2009; Gomez-Fabre et al., 2000), mediated by alternative transcription initiation and promoter usage (Aledo et al., 2000).
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The role of glutaminases in cancer is isoform-specific; GLS is proposed to have
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oncogenic function, whereas GLS1 seems to act as a tumor suppressor. GLS was found to be upregulated in, for example, breast cancer and its inhibition or silencing resulted in a profound
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decrease in tumor growth (Cheng et al., 2011; Lobo et al., 2000; Wang et al., 2010). The
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mechanism underlying this oncogenic expression of GLS was demonstrated to be dependent on the oncogenic transcription factor c-Myc, mediated by a suppression of miR-23a/b (Gao et al.,
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2009). c-Myc was further found to induce the upregulation of the glutamine importer SLC1A5, thereby stimulating glutamine catabolism and cell proliferation. Recently, another, c-Myc-
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independent mechanism was revealed: ErbB2 signaling leads to elevated GLS expression by activation of NF‐kB in ErbB2-positive breast cancer cells (Qie et al., 2013). In contrast, GLS2 is
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speculated to be a tumor suppressor in cells due to it being a p53 target, linking the common loss of p53 with the metabolic shift of tumors from mitochondrial OXPHOS towards glycolysis (Hu et al., 2010; Suzuki et al., 2010). Further strengthening this view are findings documenting the loss of the GAB GLS2 isoform in highly malignant glioblastomas and anaplastic astrocytomas in the brain, whereas artificial reintroduction of GLS2 led to decreased tumor growth and migration (Szeliga et al., 2009; Szeliga et al., 2005). Glutamate, the product of glutaminase enzyme activity, serves multiple roles in the cell. It might act as a precursor of the most important cellular antioxidant, glutathione, as well as a donor for amino groups for non-essential amino acids, such as serine, glycine, aspartate, and alanine. During oxidative glutaminolysis, glutamate is transformed to the TCA cycle intermediate α-ketoglutarate (αKG) by removal of the amine group. This reaction is either
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ACCEPTED MANUSCRIPT catalyzed by transaminases (such as glutamate pyruvate transaminase or glutamate oxaloacetate transaminase) for production of non-essential amino acids, or via oxidative deamination by
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glutamate dehydrogenase (GDH). When supply of glucose is abundant, the transamination
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pathway prevails, whereas under conditions of limited glucose, the GDH-mediated conversion is necessary to fuel carbon to the TCA cycle (Yang et al., 2009). In the latter case, glutamine, via
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αKG and the TCA cycle, serves both as an energy supply providing reducing equivalents and as
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a major precursor for anabolic processes.
Metabolized αKG can either be mediated by oxidative or reductive pathways (Holleran et
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al., 1995). αKG can be oxidized by α-ketoglutarate dehydrogenase (αKGDH) with formation of succinate, thereby fueling the TCA cycle for energy production. However, the reductive pathway
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is catalyzed by isocitrate dehydrogenase (IDH), carboxylating αKG to isocitrate, and finally citrate, which is both a TCA cycle substrate and an important precursor for cytosolic fatty acid
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synthesis. The reductive pathway is favored by cells under hypoxic conditions, enabling them to compensate for the drop in glucose-derived citrate production by using glutamine carboxylation to maintain citrate levels, and thereby sustaining lipid metabolism and cell proliferation (Metallo et al., 2012; Mullen et al., 2012; Scott et al., 2011; Wise et al., 2011). Recently, a mechanism for the hypoxic switch to reductive glutamine metabolism was proposed, showing a HIF1αdependent proteasomal degradation of the αKGDH subunit OGDH2, thereby causing a severe drop of αKGDH activity and a redirection of αKG to metabolization by IDH (Sun and Denko, 2014). The authors also demonstrated that this metabolic switch supports maintenance of lipid synthesis and is essential for hypoxic growth. Three different IDH isoforms exist in humans: the NAD+-dependent IDH3 as part of the TCA cycle, and the NADP+-dependent isoforms IDH1 and IDH2. IDH1 and IDH2 were shown
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ACCEPTED MANUSCRIPT to play an intriguing role in cancer: heterozygous mutations were identified for both isozymes and can be found in various malignancies, such as glioma, acute myeloid leukemia (AML),
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chondrosarcoma, cholangiocarcinoma, and angioimmunoblastic T-cell lymphoma (Duncan et al.,
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2012; Mardis et al., 2009; Parsons et al., 2008; Ward et al., 2010; Yan et al., 2009). These missense mutations in specific arginine residues of the enzyme active site have severe
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consequences, leading to formation of the oncometabolite D-2-hydroxyglutarate (D2HG) instead
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of αKG (Dang et al., 2009). This so far poorly studied metabolite can accumulate up to millimolar levels in the cell and is associated with epigenetic changes, impairment of defense
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mechanisms against ROS, and changes in redox homeostasis. The underlying mechanism was shown to be mainly mediated by D2HG acting as a competitive inhibitor of αKG-dependent
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dioxygenases, such as the ten-eleven translocation (TET) family of dioxygenases, JmjC-domaincontaining histone demethylases, prolyl hydroxylases (PHD) and lysyl hydroxylases (LHD)
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(Rose et al., 2011; Xu et al., 2011). How exactly these modulations of enzyme activities contribute to tumorigenesis is not fully understood, but are proposed to be based on epigenetic modifications, resulting in differential expression of genes involved in cell proliferation (TET proteins, JmjC demethylases), and changes in HIF1α levels (PHD), as well as aberrant extracellular matrix structures induced by changes in collagen synthesis (LHD) (Duncan et al., 2012; Lee et al., 2007; Lu et al., 2012; Turcan et al., 2012). Especially for glioblastoma, these findings have led to clinical relevance, since 60–80% of all astrocytomas, oligodendrogliomas, and oligoastrocytomas of WHO grade II and III harbor IDH1 mutations and are therefore regularly checked for D2HG as a marker for diagnostic and prognostic purposes (Hartmann et al., 2009). Furthermore, D2HG levels are measured in serum/plasma of AML (Pollyea et al., 2013) and in glioma patients using non-invasive proton magnetic resonance spectroscopy (MRS)
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ACCEPTED MANUSCRIPT (Andronesi et al., 2012; Choi et al., 2012; Elkhaled et al., 2012; Pope et al., 2012). Tumorspecific IDH1/2 mutations were further utilized as targets for chemotherapy and led to the
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development of specific small-molecule inhibitors with the aim of reducing D2HG levels and
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inhibiting tumorigenesis. Inhibitors were developed against mutant IDH1 R132H in glioma, as well as targeted to IDH2 R140Q in AML, resulting in decreased D2HG levels and inhibition of
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cell growth, and were proven to be successful in a glioma xenograft model for IDH1 R132H
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(Kim and DeBerardinis, 2013; Rohle et al., 2013; Wang et al., 2013). Nevertheless, it should be mentioned that their effect was mainly cytostatic and not cytotoxic, making them promising
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agents for a co-treatment setting with other chemotherapeutic drugs. Glutamine metabolism in tumors is clinically utilized via several therapeutic approaches,
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using either amino acid analogs or inhibitors directed against different steps of glutamine metabolism, such as glutamine import and glutaminase enzymatic activity. Therapeutic success
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of these approaches has shown large variation because in addition to targeting the glutamine pathway only in a non-selective manner, amino acid analogs, such as acivicin and azaserine, induced severe side effects such as gastrointestinal toxicity and neurotoxicity (Ahluwalia et al., 1990). Therefore, a more direct approach was developed by inhibiting the cellular glutamine transporter, sodium-dependent neutral amino acid transporter type 2 (ASCT2), by L-γ-glutamylp-nitroanilide (GPNA), which was shown to reduce cell proliferation in lung cancer (Hassanein et al., 2013). GPNA treatment had a striking side effect as concurrent uptake of the glycolytic inhibitor 3BP was significantly increased due to GPNA-mediated stabilization of monocarboxylate transporter (MCT1), making GPNA a promising adjuvant for 3-BP therapy (Cardaci et al., 2012). Other strategies focus on suppression of glutaminases by employing two different specific inhibitors, compound 968 and Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-
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ACCEPTED MANUSCRIPT yl)ethyl sulfide (BPTES), which both exhibit an anti-proliferative effect in cellular and xenograft models (Le et al., 2012; Wang et al., 2010).
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Radiolabeled amino acids, which have been used for imaging applications in humans for decades, are becoming more and more attractive for diagnostic purposes in cancer. Since not all
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tumors depend entirely on glucose as an energy source, 18F-FDG-based diagnostic PET scans are
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not applicable in some cancer types. In particular, tumors with active Myc/MycN, such as
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MycN-amplified neuroblastoma (Qing et al., 2012), have been shown to rely on glutamine and are thereby potentially suitable for glutamine-based PET, which is currently under development
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(Krasikova et al., 2011; Qu et al., 2012; Qu et al., 2011). Thus, glutamine-based PET could be of
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great value in complementing existing metabolic imaging techniques.
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Citrate
Citrate plays a central role in cellular metabolism as a substrate of the mitochondrial TCA cycle
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and also as a crucial switch between metabolic pathways, including OXPHOS, glycolysis, fatty acid synthesis, and gluconeogenesis. Citrate is formed in the mitochondria either by the canonical reaction (from acetyl-CoA and oxaloacetate catalyzed by citrate synthase), or from αKG via a reversed TCA cycle mediated by IDH. Within the mitochondria, the primary function of citrate is to fuel the TCA cycle, and thus contribute to cellular ATP production via OXPHOS. In addition, citrate may act as an allosteric regulator of several metabolic enzymes. Citrate was shown to repress the TCA cycle by inhibition of PDH and SDH activities (Taylor and Halperin, 1973)(Hillar et al., 1975), whereas after its cytosolic translocation via the specific citrate carrier (CIC) (Bisaccia et al., 1989; Kramer and Palmieri, 1989), it acts as a glycolytic inhibitor. Thus, in normal cells, citrate, together with ATP, plays a pivotal role in the mediation of the Pasteur effect (Salas et al., 1965) via attenuation of the enzymatic activity of phosphofructokinase 1 16
ACCEPTED MANUSCRIPT (PFK1). A similar effect was demonstrated for 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase (PFK2) (Chesney, 2006). In addition to its role in regulating catabolic processes,
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cytosolic citrate stimulates anabolic reactions such as lipid synthesis by enhancing acetyl-CoA
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carboxylase activity and by serving as a source for acetyl-CoA itself via cleavage by ATP-citrate lyase (ACLY), ultimately inducing fatty acid synthesis. ACLY was further shown to be
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important for metabolic modulation mediated by oncogenic PI3K/AKT signaling (Bauer et al.,
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2005), and its knockdown caused tumor suppression and cell differentiation (Hanai et al., 2013; Hatzivassiliou et al., 2005). The level of citrate in the cytosol is determined by the mitochondrial
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export rate and can be modulated by differential expression of the CIC gene. Several regulatory transcription factors, such as Sp1 (Iacobazzi et al., 2008) or NF-κB, can induce or inhibit CIC
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expression, thus linking CIC and cytosolic citrate levels with pro-inflammatory pathways (Infantino et al., 2011). In tumor cells, CIC was found to be transcriptionally upregulated and its
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inhibition has anti-tumor activity, potentially opening a therapeutic window for specific CIC inhibitors such as 1,2,3-benzene-tricarboxylate (BTA) or siRNA (Catalina-Rodriguez et al., 2012). Furthermore, citrate-mediated changes of fructose-2,6-bisphosphate (F2,6P) levels significantly influence tumor cell proliferation. Inhibition of PFK2 suppressed cell proliferation, whereas overexpression of PFK3 stimulated proliferation (Yalcin et al., 2009a; Yalcin et al., 2009b). Hence, changing cytosolic citrate levels is a powerful tool for modulation of cellular energy metabolism and important anabolic processes. Recently, another cancer-related role of citrate was proposed, suggesting the potential usage of citrate for therapeutic approaches in cancer. Citrate was shown to either induce cell death directly in various tumor cell lines (Lu et al., 2011; Yousefi et al., 2004) or facilitate toxic effects of conventional anticancer drugs such as cisplatin in malignant pleural mesothelioma
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ACCEPTED MANUSCRIPT cells (Zhang et al., 2009). These toxic effects were explained by the ability of citrate to chelate calcium and suppress glycolysis, and thereby reduce cellular ATP levels, cellular growth, and
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cellular capacity to repair DNA damage induced by cisplatin. A reduction of induced myeloid
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leukemia cell differentiation protein (Mcl-1) level has been reported to be a consequence of treating cells with citrate, causing loss of viability and ultimately cell death (Lincet et al., 2013).
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Even in a clinical setting, the anticancer effect of citrate has been demonstrated. Two
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cancer patients suffering from medullary thyroid cancer or primary peritoneal mesothelioma were reported to have improved in their health condition after oral administration of citrate as a
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sole anticancer treatment (Bucay, 2011; Halabe Bucay, 2009). An alternative mechanism of citrate-induced toxicity was proposed showing activation of initiator caspases by citrate in a set
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of neuroblastoma cell lines. Sensitivity of cells to citrate was dependent on the expression of caspase-8 (Kruspig et al., 2012b). This member of the initiator family of caspases can be
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activated by the so-called "induced proximity model" via adaptor-mediated clustering of zymogens (Salvesen and Dixit, 1999). Strikingly, in a study using a cell-free system, citrate was able to induce both dimerization and activation of caspase-8. The underlying mechanism was proposed to be the kosmotropic feature of citrate (Boatright et al., 2003; Pop et al., 2007). Kosmotropes are salts that promote and stabilize water–water interactions, and thereby are able to stabilize intermolecular interactions in macromolecules such as proteins. This kosmotropic property of citrate might also be involved in the activation of apical caspases in vivo. Similar to caspase-8, caspase-2 can also be stabilized and activated by citrate (Kruspig et al., 2012b). After conversion to acetyl-CoA, citrate was shown to affect caspase activation via another mechanism, i.e., modulation of protein N--acetylation. Acetyl-CoA is a key cofactor for N--acetylation and changes in its cytosolic abundance affect the activity of N-
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ACCEPTED MANUSCRIPT acetyltransferase protein complexes (NatA, NatB, NatC, NatD, and NatE), and consequently the post-transcriptional modification and activation of target proteins, including pro-apoptotic
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proteins such as caspase-2, -3, and -9 (Yi et al., 2007). This exemplifies the tight interplay
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between metabolism and apoptosis that has been shown for several metabolic pathways (Nutt et al., 2009; Nutt et al., 2005; Schafer et al., 2009; Vaughn and Deshmukh, 2008). Strikingly, this
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interconnection between apoptotic pathways and cellular metabolism is not unidirectional, since
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apoptotic protein Bcl-xL was shown to influence regulation of the mitochondrial membrane potential (Gottlieb et al., 2000; Vander Heiden et al., 1999). Overexpression of this anti-
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apoptotic Bcl-2 family member, a common feature in tumors in order to avoid apoptosis, was further shown to significantly decrease the level of cytosolic citrate, leading to an inhibition of
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N--acetylation of caspases and thereby contributing to the anti-apoptotic function of Bcl-xL (Yi et al., 2011). Citrate demonstrated a synergistic effect with ABT-737, a small-molecule inhibitor
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of anti-apoptotic members of the Bcl-2 family that is currently being tested in clinical trials. In combination, citrate and ABT-737 strongly inhibited the expression of Mcl-1 and thereby caused prominent apoptotic cell death (Lincet et al., 2013). The underlying mechanism of citratemediated downregulation of this anti-apoptotic protein is unclear as yet, but the authors speculated that citrate could cause a drop in the cellular ATP level by inhibition of glycolysis, leading to AMPK pathway activation, which in turn could lead to a GSK-3-mediated phosphorylation and consequent proteasomal degradation of Mcl-1. Summarizing the current knowledge of citrate’s role in cancer, it is an important metabolic switch involved in tumor-related adaptations and has the potential to be used as an anticancer agent, and thus citrate is a very attractive metabolite and it will be of great importance
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ACCEPTED MANUSCRIPT to further elucidate how the different molecular features of citrate orchestrate its overall effect on
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cancer cells.
Succinate
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Succinate provides electrons to the mitochondrial respiratory chain via Complex II, also known
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as succinate dehydrogenase (SDH) or succinate:ubiquinone oxidoreductase (SQR). This complex
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represents a unique structure, as it is the only enzyme that participates in both the TCA cycle and the electron transport chain (ETC). Reflecting its dual role, the four subunits of Complex II are
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divided according to their functional contribution, into the SDH part, consisting of SDHA and SDHB, and the SQR part, comprising SDHC and SDHD (Sun et al., 2005). Unlike all other
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mitochondrial respiratory complexes, Complex II is entirely nuclear encoded, and therefore lacks
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any contribution from the mitochondrial genome for its expression. Localized in the inner
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mitochondrial membrane, facing the matrix, the SDH part of Complex II catalyzes the oxidation of succinate to fumarate and the concurrent production of FADH2. The subunits SDHC and SDHD form the intramembranous SQR part of Complex II and mediate the transfer of electrons from the first catalytic step to reduce ubiquinone to ubiquinol, and subsequently to the respiratory Complex III. Other structural abnormalities further highlight the special role of Complex II within the respiratory chain. In contrast to Complexes I, III, and IV, which form a so-called respirasome, Complex II resides relatively separated from this supramolecular complex (Lenaz and Genova, 2009; Schagger and Pfeiffer, 2000). It was established that the complexes responsible for the leakage of electrons are Complex I and III. For many years, Complex II was not considered as a source of ROS. However, various recent publications focused attention on Complex II as an important site of ROS production in the form of superoxide (Gleason et al., 2011; Ishii et al., 2005; Moreno-Sanchez et al., 2013; Quinlan et al., 2012), and as a direct 20
ACCEPTED MANUSCRIPT consequence was also linked to ROS-mediated execution of apoptotic cell death (Ricci et al., 2003). First, overexpression of SDHC induced cell death and cells lacking this subunit of
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Complex II were much less sensitive towards several chemotherapeutic agents, such as cisplatin,
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doxorubicin, and etoposide (Albayrak et al., 2003). Later on, this effect was proposed to be mediated by a specific disintegration of Complex II, triggered by pH changes after treatment
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with apoptotic stimuli, causing a dissociation of SDHA/SDHB subunits, which functionally
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represent the SDH fraction of the complex. This results in an impairment of the SQR activity of the membrane-bound SDHC/SDHD subunits, while leaving the SDH activity of the dissociated
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SDHA/SDHB part intact, which in turn was shown to excessively produce superoxide, ultimately causing cell death (Lemarie et al., 2011). Based on these findings, Complex II was suggested to
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act as a cell death sensor, responding to acidification upon toxic stimuli by its disassembly and thereby further facilitating ROS-mediated apoptosis (Grimm, 2013). Reflecting this crucial
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function in cell death, it is of no surprise that Complex II was also found to be a tumor suppressor in various tumors. The first direct connection to cancer was established in familial paraganglioma and pheochromocytoma cases, which were found to carry germline mutations in SDHB, SDHC, and SDHD (Astuti et al., 2001a; Astuti et al., 2001b; Baysal et al., 2000; Niemann and Muller, 2000). Later, mutations were also reported in renal cell carcinoma and gastrointestinal stromal tumors (GIST) (Miettinen et al., 2011; Ricketts et al., 2008). Besides its role in cell death, another mechanism was discovered explaining how Complex II mutations can facilitate tumorigenesis. Early reports linking SDH mutations with familial cancer cases revealed a concomitant activation of hypoxia-inducible genes (Baysal, 2003), which was shown to be due to cytosolic accumulation of succinate, a result of impaired SDH function, ultimately leading to a stabilization and activation of HIF1α (Selak et al., 2005). Under normoxic conditions, this master
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ACCEPTED MANUSCRIPT regulator of hypoxic adaptation is targeted for proteasomal degradation by a process involving the von Hippel-Lindau (VHL) ubiquitin-ligase and HIF-PHD, which serve as intracellular
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oxygen-sensors since oxygen is a crucial cofactor for their enzymatic activity. Besides oxygen,
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this enzyme requires several other cofactors, including αKG, which is metabolized to succinate during this reaction. Elevated cytosolic succinate levels cause impairment of the 2-oxoglutarate
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(2-OG)-dependent HIF1-PHD activity by product inhibition, creating a so-called pseudo-hypoxic
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state due to HIF1α stabilization, independent of cellular oxygen levels. Mutations in SDH subunits can lead to activation of an oncogenic signaling pathway, leading to HIF1α stabilization
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and concomitant expression of genes involved in glycolysis, angiogenesis, and metastasis, and thereby facilitating tumorigenesis. Interestingly, cytosolic succinate levels were also shown to be
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elevated by inflammatory processes independently of SDH mutations. Under these conditions,
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succinate is derived from glutamine-dependent anaplerosis, as well as the γ-aminobutyric acid
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(GABA) shunt, and was shown to serve as an inflammatory signal, as well as leading to HIF1α activation (Tannahill et al., 2013). Hence, the tumorigenic effect of inflammatory processes could be in part due to an increase in cytosolic succinate levels. Succinate and Complex II are not only associated with tumorigenesis, but also utilized as targets for anticancer therapy. Therapeutic approaches vary depending on the type of tumor and their specific characteristics. Despite only representing a small fraction of tumors, as mentioned earlier, certain familial cancers carry SDH mutations that were shown to be important for tumorigenesis. As a promising new approach for treatment of these cancers, cell-permeable αKG derivatives were successfully tested in both cellular and xenograft models. The underlying rationale is that αKG, a cofactor for PHD enzymes, restores enzymatic activity of these
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ACCEPTED MANUSCRIPT hydroxylases, causing HIF1α destabilization and thereby reverses the pseudo-hypoxic condition and pro-tumorigenic effect (MacKenzie et al., 2007; Tennant et al., 2009).
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For the vast majority of tumors, which lack mutations in the Complex II subunits, different chemotherapeutic drugs targeting Complex II have been tested. The redox-silent
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vitamin E analog -tocopheryl succinate (α-TOS) was shown as early as 1982 to have antiproliferative effects in mouse melanoma cells (Prasad and Edwards-Prasad, 1982), and
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furthermore, proved to be effective in a large variety of experimental cancers such as colon, breast, and neuroblastoma, in both in vitro and in vivo settings (Neuzil et al., 2001b; Stapelberg
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et al., 2004; Swettenham et al., 2005). The underlying mechanism for apoptosis induction by αTOS was investigated in several studies, providing different models for its mode of action, but
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without finding the specific molecular target of α-TOS. In a study by Dong and colleagues, this target was identified to be the ubiquinone (UbQ) binding site of Complex II, and the authors
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provided evidence for the involvement of superoxide production in this process (Dong et al., 2008). Later on, this model was extended by findings that α-TOS additionally triggers an increase in cellular calcium levels, which in combination with ROS production leads to mitochondrial destabilization and cell death (Gogvadze et al., 2010). This compound is particularly attractive as a potential anticancer drug, as it was shown to be selective for cancers, while being non-toxic to normal cells (Neuzil et al., 2001a), and to kill cancer cells irrespective of their p53 or MycN status (Kruspig et al., 2012a). The cancer-specificity of α-TOS is thought to be based on a higher antioxidant capacity and elevated activity of intracellular esterases in non-malignant cells, which can cleave and thereby detoxify α-TOS into its two non-toxic components, i.e., antioxidant α-tocopherol and succinate (Carini et al., 1990; Fariss et al., 2001; Neuzil and Massa, 2005). A large effort has been made to improve the molecular properties of α-
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ACCEPTED MANUSCRIPT TOS by structural modifications of this compound, leading to a more specific accumulation in the mitochondria and a higher efficacy (Kovarova et al., 2014; Prochazka et al., 2013). It should
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be taken into account that despite its well-documented toxic effect, when used at low
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concentrations in a co-treatment setting with conventional anticancer drugs, α-TOS was shown to either sensitize cells to cell death, in the case of combination with etoposide, or to protect cells,
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when treated together with cisplatin (Kruspig et al., 2013). Thus, α-TOS should be used in
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combined treatment with conventionally used anticancer drugs cautiously. Recent observations demonstrate that targeting Complex II can modulate the cellular
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response to anticancer treatment. Stimulation of Complex II activity by supplying exogenous succinate protected cells from cisplatin-induced apoptosis as assessed by caspase-3-like activity,
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release of cytochrome c, or Poly (ADP-ribose) polymerase (PARP) cleavage (Kruspig et al., 2013). Succinate usually shows poor passage through the plasma membrane, but when applied at
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high concentrations it can slowly penetrate into the cell. This effect of succinate is apparently based on the ability of this substrate to monopolize the respiratory chain of mitochondria (Krebs et al., 1961). One of the consequences of this monopolization is an even greater reduction of mitochondrial pyridine nucleotides than that achieved by Complex I substrates (Chance and Hollunger, 1961). The redox state of mitochondrial pyridine nucleotides is involved in the regulation of various processes, such as the detoxification of hydrogen peroxide and organic hydroperoxides via the glutathione-dependent defense system (Sies and Summer, 1975), or the induction of mitochondrial permeability transition (MPT) (Costantini et al., 1996). Indeed, mitochondria oxidizing succinate can accumulate 3–4-times more Ca2+ before the onset of MPT as compared to mitochondria oxidizing NAD-dependent substrates (Kondrashova et al., 1982). Moreover, oxidation of succinate makes mitochondria resistant to oxidative stress; in the
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ACCEPTED MANUSCRIPT absence, as well as in the presence, of the oxidant t-butylhydroperoxide, mitochondria retained more Ca2+ with succinate than with -hydroxybutyrate as the respiratory substrate (Gogvadze et
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al., 1996). In contrast, inhibition of Complex II by thenoyltrifluoroacetone (TTFA) markedly
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stimulated cell death induced by low doses of cisplatin (unpublished data).
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Lately, similar to IDH mutations, mutations in SDH and in fumarate hydratase (FH) were reported, leading to accumulation of succinate or fumarate, respectively, and concomitant
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inhibition of several α-KG-dependent dioxygenases by product inhibition (Xiao et al., 2012). As discussed earlier, this class of enzymes, which includes histone demethylases and DNA
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hydroxylases, plays an important role in hydroxylation reactions of several different substrates in the cell, such as proteins and DNA. As a result of SDH mutations, the accumulation of succinate
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was linked to a hypermethylated phenotype in GIST, as well as paraganglioma (Killian et al., 2013; Letouze et al., 2013). The severity of these epigenetic modifications, leading to gene
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silencing, correlates with the aggressiveness of certain tumor subtypes, because the highly malignant SDHB-mutated tumors were also shown to have the most pronounced hypermethylated state (Letouze et al., 2013). These findings highlight a new and interesting interplay between the TCA cycle and tumor-specific epigenetic changes, and further represent a potential therapeutic target in these malignancies. Clinically relevant are the findings that patients harboring mutations in SDHB and SDHD also had a higher probability of increased succinate levels in plasma (Hobert et al., 2012), which suggests that this could be explored for diagnostic purposes, thereby providing a more cost-effective and easy method of screening for cancers harboring SDH mutations.
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ACCEPTED MANUSCRIPT Concluding remarks Changes in the abundance, localization, and metabolism of mitochondrial substrates are a crucial
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part of the specific metabolic aberrations found in the majority of cancers. Moreover, it has
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become clear that some metabolites by themselves can directly drive tumorigenesis, exemplified by succinate and its ability to induce pseudo-hypoxic conditions and alter gene expression by
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DNA hypermethylation in cancer cells harboring SDH mutations. In addition, mitochondrial
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substrates are directly or indirectly being utilized for clinical approaches, such as diagnosis or anticancer therapy. Although several chemotherapeutical interventions targeting different aspects
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of mitochondrial metabolism are already being tested either in laboratories or in clinical trials, future research should focus on tackling the shortcomings of conventional anticancer therapy and
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providing a better understanding of the role of mitochondrial metabolism in cancer biology.
Acknowledgements
The work in the author’s laboratories was supported by grants from the Swedish and Stockholm Cancer Societies, the Swedish Childhood Cancer Foundation, the Swedish Research Council, the Russian Science Foundation, and the Russian Foundation for Basic Research.
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ACCEPTED MANUSCRIPT Figure 1: Cellular glucose and glutamine metabolism. Overview of cellular glucose and glutamine metabolism, as well as the mitochondrial
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tricarboxylic acid cycle. Metabolic enzymes that are discussed within this review are indicated.
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Figure adapted from BioCarta.
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Figure 2: Role of succinate in cancer metabolism and tumorigenesis, and potential
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chemotherapeutic interventions.
Under physiological conditions (black), succinate, as a substrate of the TCA cycle, is oxidized to
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fumarate by the SDHA subunit of Complex II/SDH, and electrons are transferred further via
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subunit SDHB to the ubiquinone binding site formed by subunits SDHC and SDHD. Ubiquinone is reduced to ubiquinol, and subsequently relays the electrons to Complex III of the respiratory
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chain. Specific drugs (blue) may be used to inhibit this process at different subunits of the SDH complex. 3-NPA and malonate bind and block the SDHA subunit and thereby prevent oxidation of succinate. α-TOS and thenoyltrifluoroacetone (TTFA) bind and inhibit the ubiquinone binding site, which may lead to leakage of electrons and the formation of ROS in the form of superoxide radicals. Mutations (red) in different subunits of SDH are found in several cancers and can cause ROS formation and accumulation of succinate. Increased cytosolic succinate levels may induce HIF1α stabilization by product inhibition of HIF-1-PHD, resulting in concomitant pseudohypoxic conditions, and thus induction of angiogenesis, glycolysis, and metastasis. Further, accumulation of succinate in the cytosol may lead to DNA hypermethylation by inhibition of αKG-dependent dioxygenases. Exogenous (green) supply of succinate can induce a reversed electron flow, monopolization of the respiratory chain, and an increase in the pool of pyridine
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Figure adapted from BioCarta and David Goodsell & RCSB Protein Data Bank.
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Figure 3: Effects of citrate treatment on cellular metabolism and mechanisms of citrateinduced toxicity.
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Citrate treatment may cause suppression of the TCA cycle by inhibition of PDH and SDH
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activity, as well as glycolysis, by attenuating PFK1. Increased cytosolic citrate can enforce fatty acid synthesis after conversion to acetyl-CoA by ACLY. Dimerization and activation of apical
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caspase-2/-8, induced by the kosmotropic feature of citrate, as well post-transcriptional
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adapted from BioCarta.
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modification by N--acetylation of caspase-2/-3/-9 can facilitate apoptotic cell death. Figure
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References
AC CE P
TE
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MA
NU
SC
RI
Ahluwalia, G.S., Grem, J.L., Hao, Z., Cooney, D.A., 1990. Metabolism and action of amino acid analog anti-cancer agents. Pharmacology & therapeutics 46, 243-271. Albayrak, T., Scherhammer, V., Schoenfeld, N., Braziulis, E., Mund, T., Bauer, M.K.A., Scheffler, I.E., Grimm, S., 2003. The tumor suppressor cybL, a component of the respiratory chain, mediates apoptosis induction. Mol Biol Cell 14, 3082-3096. Aledo, J.C., Gomez-Fabre, P.M., Olalla, L., Marquez, J., 2000. Identification of two human glutaminase loci and tissue-specific expression of the two related genes. Mammalian genome : official journal of the International Mammalian Genome Society 11, 1107-1110. Andronesi, O.C., Kim, G.S., Gerstner, E., Batchelor, T., Tzika, A.A., Fantin, V.R., Vander Heiden, M.G., Sorensen, A.G., 2012. Detection of 2-hydroxyglutarate in IDH-mutated glioma patients by in vivo spectral-editing and 2D correlation magnetic resonance spectroscopy. Science translational medicine 4, 116ra114. Astuti, D., Douglas, F., Lennard, T.W., Aligianis, I.A., Woodward, E.R., Evans, D.G., Eng, C., Latif, F., Maher, E.R., 2001a. Germline SDHD mutation in familial phaeochromocytoma. Lancet 357, 1181-1182. Astuti, D., Latif, F., Dallol, A., Dahia, P.L., Douglas, F., George, E., Skoldberg, F., Husebye, E.S., Eng, C., Maher, E.R., 2001b. Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. American journal of human genetics 69, 49-54. Bauer, D.E., Hatzivassiliou, G., Zhao, F., Andreadis, C., Thompson, C.B., 2005. ATP citrate lyase is an important component of cell growth and transformation. Oncogene 24, 6314-6322. Baysal, B.E., 2003. On the association of succinate dehydrogenase mutations with hereditary paraganglioma. Trends in endocrinology and metabolism: TEM 14, 453-459. Baysal, B.E., Ferrell, R.E., Willett-Brozick, J.E., Lawrence, E.C., Myssiorek, D., Bosch, A., van der Mey, A., Taschner, P.E., Rubinstein, W.S., Myers, E.N., Richard, C.W., 3rd, Cornelisse, C.J., Devilee, P., Devlin, B., 2000. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 287, 848-851. Bisaccia, F., De Palma, A., Palmieri, F., 1989. Identification and purification of the tricarboxylate carrier from rat liver mitochondria. Biochimica et biophysica acta 977, 171-176. Bluemlein, K., Gruning, N.M., Feichtinger, R.G., Lehrach, H., Kofler, B., Ralser, M., 2011. No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis. Oncotarget 2, 393-400. Boatright, K.M., Renatus, M., Scott, F.L., Sperandio, S., Shin, H., Pedersen, I.M., Ricci, J.E., Edris, W.A., Sutherlin, D.P., Green, D.R., Salvesen, G.S., 2003. A unified model for apical caspase activation. Molecular cell 11, 529-541. Bucay, A.H., 2011. Clinical report: a patient with primary peritoneal mesothelioma that has improved after taking citric acid orally. Clinics and research in hepatology and gastroenterology 35, 241. Cardaci, S., Rizza, S., Filomeni, G., Bernardini, R., Bertocchi, F., Mattei, M., Paci, M., Rotilio, G., Ciriolo, M.R., 2012. Glutamine deprivation enhances antitumor activity of 3-bromopyruvate through the stabilization of monocarboxylate transporter-1. Cancer research 72, 4526-4536. Carini, R., Poli, G., Dianzani, M.U., Maddix, S.P., Slater, T.F., Cheeseman, K.H., 1990. Comparative evaluation of the antioxidant activity of alpha-tocopherol, alpha-tocopherol polyethylene glycol 1000 succinate and alpha-tocopherol succinate in isolated hepatocytes and liver microsomal suspensions. Biochemical pharmacology 39, 1597-1601. 29
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Catalina-Rodriguez, O., Kolukula, V.K., Tomita, Y., Preet, A., Palmieri, F., Wellstein, A., Byers, S., Giaccia, A.J., Glasgow, E., Albanese, C., Avantaggiati, M.L., 2012. The mitochondrial citrate transporter, CIC, is essential for mitochondrial homeostasis. Oncotarget 3, 1220-1235. Chance, B., Hollunger, G., 1961. The interaction of energy and electron transfer reactions in mitochondria. I. General properties and nature of the products of succinate-linked reduction of pyridine nucleotide. The Journal of biological chemistry 236, 1534-1543. Chen, M., Zhang, J., Manley, J.L., 2010. Turning on a fuel switch of cancer: hnRNP proteins regulate alternative splicing of pyruvate kinase mRNA. Cancer research 70, 8977-8980. Cheng, T., Sudderth, J., Yang, C., Mullen, A.R., Jin, E.S., Mates, J.M., DeBerardinis, R.J., 2011. Pyruvate carboxylase is required for glutamine-independent growth of tumor cells. Proceedings of the National Academy of Sciences of the United States of America 108, 8674-8679. Chesney, J., 2006. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and tumor cell glycolysis. Current opinion in clinical nutrition and metabolic care 9, 535-539. Choi, C., Ganji, S.K., DeBerardinis, R.J., Hatanpaa, K.J., Rakheja, D., Kovacs, Z., Yang, X.L., Mashimo, T., Raisanen, J.M., Marin-Valencia, I., Pascual, J.M., Madden, C.J., Mickey, B.E., Malloy, C.R., Bachoo, R.M., Maher, E.A., 2012. 2-hydroxyglutarate detection by magnetic resonance spectroscopy in IDH-mutated patients with gliomas. Nature medicine 18, 624-629. Christofk, H.R., Vander Heiden, M.G., Harris, M.H., Ramanathan, A., Gerszten, R.E., Wei, R., Fleming, M.D., Schreiber, S.L., Cantley, L.C., 2008. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452, 230-233. Costantini, P., Chernyak, B.V., Petronilli, V., Bernardi, P., 1996. Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. The Journal of biological chemistry 271, 6746-6751. Dang, L., White, D.W., Gross, S., Bennett, B.D., Bittinger, M.A., Driggers, E.M., Fantin, V.R., Jang, H.G., Jin, S., Keenan, M.C., Marks, K.M., Prins, R.M., Ward, P.S., Yen, K.E., Liau, L.M., Rabinowitz, J.D., Cantley, L.C., Thompson, C.B., Vander Heiden, M.G., Su, S.M., 2009. Cancer-associated IDH1 mutations produce 2hydroxyglutarate. Nature 462, 739-744. de la Rosa, V., Campos-Sandoval, J.A., Martin-Rufian, M., Cardona, C., Mates, J.M., Segura, J.A., Alonso, F.J., Marquez, J., 2009. A novel glutaminase isoform in mammalian tissues. Neurochemistry international 55, 76-84. De Milito, A., Fais, S., 2005. Tumor acidity, chemoresistance and proton pump inhibitors. Future oncology 1, 779-786. Denko, N.C., 2008. Hypoxia, HIF1 and glucose metabolism in the solid tumour. Nature reviews. Cancer 8, 705-713. Dong, L.F., Low, P., Dyason, J.C., Wang, X.F., Prochazka, L., Witting, P.K., Freeman, R., Swettenham, E., Valis, K., Liu, J., Zobalova, R., Turanek, J., Spitz, D.R., Domann, F.E., Scheffler, I.E., Ralph, S.J., Neuzil, J., 2008. Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II. Oncogene 27, 4324-4335. Duncan, C.G., Barwick, B.G., Jin, G., Rago, C., Kapoor-Vazirani, P., Powell, D.R., Chi, J.T., Bigner, D.D., Vertino, P.M., Yan, H., 2012. A heterozygous IDH1R132H/WT mutation induces genome-wide alterations in DNA methylation. Genome research 22, 2339-2355. Eagle, H., 1955. Nutrition needs of mammalian cells in tissue culture. Science 122, 501-514. Elgadi, K.M., Meguid, R.A., Qian, M., Souba, W.W., Abcouwer, S.F., 1999. Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alternative splicing. Physiological genomics 1, 51-62. Elkhaled, A., Jalbert, L.E., Phillips, J.J., Yoshihara, H.A., Parvataneni, R., Srinivasan, R., Bourne, G., Berger, M.S., Chang, S.M., Cha, S., Nelson, S.J., 2012. Magnetic resonance of 2-hydroxyglutarate in IDH1mutated low-grade gliomas. Science translational medicine 4, 116ra115. 30
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Estrella, V., Chen, T., Lloyd, M., Wojtkowiak, J., Cornnell, H.H., Ibrahim-Hashim, A., Bailey, K., Balagurunathan, Y., Rothberg, J.M., Sloane, B.F., Johnson, J., Gatenby, R.A., Gillies, R.J., 2013. Acidity generated by the tumor microenvironment drives local invasion. Cancer research 73, 1524-1535. Fariss, M.W., Nicholls-Grzemski, F.A., Tirmenstein, M.A., Zhang, J.G., 2001. Enhanced antioxidant and cytoprotective abilities of vitamin E succinate is associated with a rapid uptake advantage in rat hepatocytes and mitochondria. Free radical biology & medicine 31, 530-541. Ganapathy-Kanniappan, S., Geschwind, J.F., Kunjithapatham, R., Buijs, M., Vossen, J.A., Tchernyshyov, I., Cole, R.N., Syed, L.H., Rao, P.P., Ota, S., Vali, M., 2009. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is pyruvylated during 3-bromopyruvate mediated cancer cell death. Anticancer research 29, 4909-4918. Gao, P., Tchernyshyov, I., Chang, T.C., Lee, Y.S., Kita, K., Ochi, T., Zeller, K.I., De Marzo, A.M., Van Eyk, J.E., Mendell, J.T., Dang, C.V., 2009. c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Nature 458, 762-765. Gao, X., Wang, H., Yang, J.J., Liu, X., Liu, Z.R., 2012. Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Molecular cell 45, 598-609. Geschwind, J.F., Ko, Y.H., Torbenson, M.S., Magee, C., Pedersen, P.L., 2002. Novel therapy for liver cancer: direct intraarterial injection of a potent inhibitor of ATP production. Cancer research 62, 39093913. Gleason, C., Huang, S., Thatcher, L.F., Foley, R.C., Anderson, C.R., Carroll, A.J., Millar, A.H., Singh, K.B., 2011. Mitochondrial complex II has a key role in mitochondrial-derived reactive oxygen species influence on plant stress gene regulation and defense. Proceedings of the National Academy of Sciences of the United States of America 108, 10768-10773. Gogvadze, V., Norberg, E., Orrenius, S., Zhivotovsky, B., 2010. Involvement of Ca2+ and ROS in alphatocopheryl succinate-induced mitochondrial permeabilization. International journal of cancer. Journal international du cancer 127, 1823-1832. Gogvadze, V., Schweizer, M., Richter, C., 1996. Control of the pyridine nucleotide-linked Ca2+ release from mitochondria by respiratory substrates. Cell calcium 19, 521-526. Gomez-Fabre, P.M., Aledo, J.C., Del Castillo-Olivares, A., Alonso, F.J., Nunez De Castro, I., Campos, J.A., Marquez, J., 2000. Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase. The Biochemical journal 345 Pt 2, 365-375. Gottlieb, E., Vander Heiden, M.G., Thompson, C.B., 2000. Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis. Molecular and cellular biology 20, 5680-5689. Grimm, S., 2013. Respiratory chain complex II as general sensor for apoptosis. Bba-Bioenergetics 1827, 565-572. Halabe Bucay, A., 2009. Hypothesis proved...citric acid (citrate) does improve cancer: a case of a patient suffering from medullary thyroid cancer. Medical hypotheses 73, 271. Halestrap, A.P., 1975. The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. The Biochemical journal 148, 85-96. Hanahan, D., Weinberg, R.A., 2011. Hallmarks of cancer: the next generation. Cell 144, 646-674. Hanai, J.I., Doro, N., Seth, P., Sukhatme, V.P., 2013. ATP citrate lyase knockdown impacts cancer stem cells in vitro. Cell death & disease 4, e696. Hartmann, C., Meyer, J., Balss, J., Capper, D., Mueller, W., Christians, A., Felsberg, J., Wolter, M., Mawrin, C., Wick, W., Weller, M., Herold-Mende, C., Unterberg, A., Jeuken, J.W., Wesseling, P., Reifenberger, G., von Deimling, A., 2009. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta neuropathologica 118, 469-474.
31
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Hassanein, M., Hoeksema, M.D., Shiota, M., Qian, J., Harris, B.K., Chen, H., Clark, J.E., Alborn, W.E., Eisenberg, R., Massion, P.P., 2013. SLC1A5 mediates glutamine transport required for lung cancer cell growth and survival. Clinical cancer research : an official journal of the American Association for Cancer Research 19, 560-570. Hatzivassiliou, G., Zhao, F., Bauer, D.E., Andreadis, C., Shaw, A.N., Dhanak, D., Hingorani, S.R., Tuveson, D.A., Thompson, C.B., 2005. ATP citrate lyase inhibition can suppress tumor cell growth. Cancer cell 8, 311-321. Hillar, M., Lott, V., Lennox, B., 1975. Correlation of the effects of citric acid cycle metabolites on succinate oxidation by rat liver mitochondria and submitochondrial particles. Journal of bioenergetics 7, 1-16. Hobert, J.A., Mester, J.L., Moline, J., Eng, C., 2012. Elevated plasma succinate in PTEN, SDHB, and SDHD mutation-positive individuals. Genetics in medicine : official journal of the American College of Medical Genetics 14, 616-619. Holleran, A.L., Briscoe, D.A., Fiskum, G., Kelleher, J.K., 1995. Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation. Molecular and cellular biochemistry 152, 95-101. Hoshino, A., Hirst, J.A., Fujii, H., 2007. Regulation of cell proliferation by interleukin-3-induced nuclear translocation of pyruvate kinase. The Journal of biological chemistry 282, 17706-17711. Hu, W., Zhang, C., Wu, R., Sun, Y., Levine, A., Feng, Z., 2010. Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function. Proceedings of the National Academy of Sciences of the United States of America 107, 7455-7460. Iacobazzi, V., Infantino, V., Palmieri, F., 2008. Epigenetic mechanisms and Sp1 regulate mitochondrial citrate carrier gene expression. Biochemical and biophysical research communications 376, 15-20. Icard, P., Poulain, L., Lincet, H., 2012. Understanding the central role of citrate in the metabolism of cancer cells. Biochimica et biophysica acta 1825, 111-116. Ihrlund, L.S., Hernlund, E., Khan, O., Shoshan, M.C., 2008. 3-Bromopyruvate as inhibitor of tumour cell energy metabolism and chemopotentiator of platinum drugs. Molecular oncology 2, 94-101. Infantino, V., Convertini, P., Cucci, L., Panaro, M.A., Di Noia, M.A., Calvello, R., Palmieri, F., Iacobazzi, V., 2011. The mitochondrial citrate carrier: a new player in inflammation. The Biochemical journal 438, 433436. Ishii, T., Yasuda, K., Akatsuka, A., Hino, O., Hartman, P.S., Ishii, N., 2005. A mutation in the SDHC gene of complex II increases oxidative stress, resulting in apoptosis and tumorigenesis. Cancer research 65, 203209. Jiang, B.H., Jiang, G., Zheng, J.Z., Lu, Z., Hunter, T., Vogt, P.K., 2001. Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research 12, 363-369. Jurica, M.S., Mesecar, A., Heath, P.J., Shi, W., Nowak, T., Stoddard, B.L., 1998. The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate. Structure 6, 195-210. Kato, M., Wynn, R.M., Chuang, J.L., Tso, S.C., Machius, M., Li, J., Chuang, D.T., 2008. Structural basis for inactivation of the human pyruvate dehydrogenase complex by phosphorylation: role of disordered phosphorylation loops. Structure 16, 1849-1859. Killian, J.K., Kim, S.Y., Miettinen, M., Smith, C., Merino, M., Tsokos, M., Quezado, M., Smith, W.I., Jr., Jahromi, M.S., Xekouki, P., Szarek, E., Walker, R.L., Lasota, J., Raffeld, M., Klotzle, B., Wang, Z., Jones, L., Zhu, Y., Wang, Y., Waterfall, J.J., O'Sullivan, M.J., Bibikova, M., Pacak, K., Stratakis, C., Janeway, K.A., Schiffman, J.D., Fan, J.B., Helman, L., Meltzer, P.S., 2013. Succinate dehydrogenase mutation underlies global epigenomic divergence in gastrointestinal stromal tumor. Cancer discovery 3, 648-657. Kim, J., DeBerardinis, R.J., 2013. Cancer. Silencing a metabolic oncogene. Science 340, 558-559.
32
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Ko, Y.H., Pedersen, P.L., Geschwind, J.F., 2001. Glucose catabolism in the rabbit VX2 tumor model for liver cancer: characterization and targeting hexokinase. Cancer letters 173, 83-91. Kondrashova, M.N., Gogvadze, V.G., Medvedev, B.I., Babsky, A.M., 1982. Succinic acid oxidation as the only energy support of intensive Ca2+ uptake by mitochondria. Biochemical and biophysical research communications 109, 376-381. Kovarova, J., Bajzikova, M., Vondrusova, M., Stursa, J., Goodwin, J., Nguyen, M., Zobalova, R., Pesdar, E.A., Truksa, J., Tomasetti, M., Dong, L.F., Neuzil, J., 2014. Mitochondrial targeting of alpha-tocopheryl succinate enhances its anti-mesothelioma efficacy. Redox report : communications in free radical research 19, 16-25. Kramer, R., Palmieri, F., 1989. Molecular aspects of isolated and reconstituted carrier proteins from animal mitochondria. Biochimica et biophysica acta 974, 1-23. Krasikova, R.N., Kuznetsova, O.F., Fedorova, O.S., Belokon, Y.N., Maleev, V.I., Mu, L., Ametamey, S., Schubiger, P.A., Friebe, M., Berndt, M., Koglin, N., Mueller, A., Graham, K., Lehmann, L., Dinkelborg, L.M., 2011. 4-[18F]fluoroglutamic acid (BAY 85-8050), a new amino acid radiotracer for PET imaging of tumors: synthesis and in vitro characterization. Journal of medicinal chemistry 54, 406-410. Krebs, H.A., Eggleston, L.V., D'Alessandro, A., 1961. The effect of succinate and amytal on the reduction of acetoacetate in animal tissues. The Biochemical journal 79, 537-549. Kruspig, B., Nilchian, A., Bejarano, I., Orrenius, S., Zhivotovsky, B., Gogvadze, V., 2012a. Targeting mitochondria by alpha-tocopheryl succinate kills neuroblastoma cells irrespective of MycN oncogene expression. Cellular and Molecular Life Sciences 69, 2091-2099. Kruspig, B., Nilchian, A., Orrenius, S., Zhivotovsky, B., Gogvadze, V., 2012b. Citrate kills tumor cells through activation of apical caspases. Cellular and Molecular Life Sciences 69, 4229-4237. Kruspig, B., Zhivotovsky, B., Gogvadze, V., 2013. Contrasting effects of alpha-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis. Mitochondrion 13, 533-538. Kung, H.N., Marks, J.R., Chi, J.T., 2011. Glutamine synthetase is a genetic determinant of cell typespecific glutamine independence in breast epithelia. PLoS genetics 7, e1002229. Le, A., Lane, A.N., Hamaker, M., Bose, S., Gouw, A., Barbi, J., Tsukamoto, T., Rojas, C.J., Slusher, B.S., Zhang, H., Zimmerman, L.J., Liebler, D.C., Slebos, R.J., Lorkiewicz, P.K., Higashi, R.M., Fan, T.W., Dang, C.V., 2012. Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells. Cell metabolism 15, 110-121. Lee, B.H., Tothova, Z., Levine, R.L., Anderson, K., Buza-Vidas, N., Cullen, D.E., McDowell, E.P., Adelsperger, J., Frohling, S., Huntly, B.J., Beran, M., Jacobsen, S.E., Gilliland, D.G., 2007. FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia. Cancer cell 12, 367-380. Lee, J., Kim, H.K., Han, Y.M., Kim, J., 2008. Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. The international journal of biochemistry & cell biology 40, 1043-1054. Lemarie, A., Huc, L., Pazarentzos, E., Mahul-Mellier, A.L., Grimm, S., 2011. Specific disintegration of complex II succinate: ubiquinone oxidoreductase links pH changes to oxidative stress for apoptosis induction. Cell death and differentiation 18, 338-349. Lenaz, G., Genova, M.L., 2009. Mobility and function of coenzyme Q (ubiquinone) in the mitochondrial respiratory chain. Biochimica et biophysica acta 1787, 563-573. Letouze, E., Martinelli, C., Loriot, C., Burnichon, N., Abermil, N., Ottolenghi, C., Janin, M., Menara, M., Nguyen, A.T., Benit, P., Buffet, A., Marcaillou, C., Bertherat, J., Amar, L., Rustin, P., De Reynies, A., Gimenez-Roqueplo, A.P., Favier, J., 2013. SDH mutations establish a hypermethylator phenotype in paraganglioma. Cancer cell 23, 739-752.
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RI
PT
Lincet, H., Kafara, P., Giffard, F., Abeilard-Lemoisson, E., Duval, M., Louis, M.H., Poulain, L., Icard, P., 2013. Inhibition of Mcl-1 expression by citrate enhances the effect of Bcl-xL inhibitors on human ovarian carcinoma cells. Journal of ovarian research 6, 72. Lobo, C., Ruiz-Bellido, M.A., Aledo, J.C., Marquez, J., Nunez De Castro, I., Alonso, F.J., 2000. Inhibition of glutaminase expression by antisense mRNA decreases growth and tumourigenicity of tumour cells. The Biochemical journal 348 Pt 2, 257-261. Lu, C., Ward, P.S., Kapoor, G.S., Rohle, D., Turcan, S., Abdel-Wahab, O., Edwards, C.R., Khanin, R., Figueroa, M.E., Melnick, A., Wellen, K.E., O'Rourke, D.M., Berger, S.L., Chan, T.A., Levine, R.L., Mellinghoff, I.K., Thompson, C.B., 2012. IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature 483, 474-478. Lu, Y., Zhang, X., Zhang, H., Lan, J., Huang, G., Varin, E., Lincet, H., Poulain, L., Icard, P., 2011. Citrate induces apoptotic cell death: a promising way to treat gastric carcinoma? Anticancer research 31, 797805. Luo, W., Hu, H., Chang, R., Zhong, J., Knabel, M., O'Meally, R., Cole, R.N., Pandey, A., Semenza, G.L., 2011. Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell 145, 732744. MacKenzie, E.D., Selak, M.A., Tennant, D.A., Payne, L.J., Crosby, S., Frederiksen, C.M., Watson, D.G., Gottlieb, E., 2007. Cell-permeating alpha-ketoglutarate derivatives alleviate pseudohypoxia in succinate dehydrogenase-deficient cells. Molecular and cellular biology 27, 3282-3289. Mardis, E.R., Ding, L., Dooling, D.J., Larson, D.E., McLellan, M.D., Chen, K., Koboldt, D.C., Fulton, R.S., Delehaunty, K.D., McGrath, S.D., Fulton, L.A., Locke, D.P., Magrini, V.J., Abbott, R.M., Vickery, T.L., Reed, J.S., Robinson, J.S., Wylie, T., Smith, S.M., Carmichael, L., Eldred, J.M., Harris, C.C., Walker, J., Peck, J.B., Du, F., Dukes, A.F., Sanderson, G.E., Brummett, A.M., Clark, E., McMichael, J.F., Meyer, R.J., Schindler, J.K., Pohl, C.S., Wallis, J.W., Shi, X., Lin, L., Schmidt, H., Tang, Y., Haipek, C., Wiechert, M.E., Ivy, J.V., Kalicki, J., Elliott, G., Ries, R.E., Payton, J.E., Westervelt, P., Tomasson, M.H., Watson, M.A., Baty, J., Heath, S., Shannon, W.D., Nagarajan, R., Link, D.C., Walter, M.J., Graubert, T.A., DiPersio, J.F., Wilson, R.K., Ley, T.J., 2009. Recurring mutations found by sequencing an acute myeloid leukemia genome. The New England journal of medicine 361, 1058-1066. Matsumoto, S., Saito, K., Yasui, H., Morris, H.D., Munasinghe, J.P., Lizak, M., Merkle, H., ArdenkjaerLarsen, J.H., Choudhuri, R., Devasahayam, N., Subramanian, S., Koretsky, A.P., Mitchell, J.B., Krishna, M.C., 2013. EPR oxygen imaging and hyperpolarized 13C MRI of pyruvate metabolism as noninvasive biomarkers of tumor treatment response to a glycolysis inhibitor 3-bromopyruvate. Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine 69, 1443-1450. Metallo, C.M., Gameiro, P.A., Bell, E.L., Mattaini, K.R., Yang, J., Hiller, K., Jewell, C.M., Johnson, Z.R., Irvine, D.J., Guarente, L., Kelleher, J.K., Vander Heiden, M.G., Iliopoulos, O., Stephanopoulos, G., 2012. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 481, 380-384. Miettinen, M., Wang, Z.F., Sarlomo-Rikala, M., Osuch, C., Rutkowski, P., Lasota, J., 2011. Succinate dehydrogenase-deficient GISTs: a clinicopathologic, immunohistochemical, and molecular genetic study of 66 gastric GISTs with predilection to young age. The American journal of surgical pathology 35, 17121721. Moreno-Sanchez, R., Hernandez-Esquivel, L., Rivero-Segura, N.A., Marin-Hernandez, A., Neuzil, J., Ralph, S.J., Rodriguez-Enriquez, S., 2013. Reactive oxygen species are generated by the respiratory complex II-evidence for lack of contribution of the reverse electron flow in complex I. The FEBS journal 280, 927938. Mullen, A.R., Wheaton, W.W., Jin, E.S., Chen, P.H., Sullivan, L.B., Cheng, T., Yang, Y., Linehan, W.M., Chandel, N.S., DeBerardinis, R.J., 2012. Reductive carboxylation supports growth in tumour cells with defective mitochondria. Nature 481, 385-388. 34
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Neuzil, J., Massa, H., 2005. Hepatic processing determines dual activity of alpha-tocopheryl succinate: a novel paradigm for a shift in biological activity due to pro-vitamin-to-vitamin conversion. Biochemical and biophysical research communications 327, 1024-1027. Neuzil, J., Weber, T., Gellert, N., Weber, C., 2001a. Selective cancer cell killing by alpha-tocopheryl succinate. British journal of cancer 84, 87-89. Neuzil, J., Weber, T., Schroder, A., Lu, M., Ostermann, G., Gellert, N., Mayne, G.C., Olejnicka, B., NegreSalvayre, A., Sticha, M., Coffey, R.J., Weber, C., 2001b. Induction of cancer cell apoptosis by alphatocopheryl succinate: molecular pathways and structural requirements. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 15, 403-415. Niemann, S., Muller, U., 2000. Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nature genetics 26, 268-270. Noguchi, T., Inoue, H., Tanaka, T., 1986. The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. The Journal of biological chemistry 261, 13807-13812. Noguchi, T., Yamada, K., Inoue, H., Matsuda, T., Tanaka, T., 1987. The L- and R-type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters. The Journal of biological chemistry 262, 14366-14371. Nutt, L.K., Buchakjian, M.R., Gan, E., Darbandi, R., Yoon, S.Y., Wu, J.Q., Miyamoto, Y.J., Gibbons, J.A., Andersen, J.L., Freel, C.D., Tang, W., He, C., Kurokawa, M., Wang, Y., Margolis, S.S., Fissore, R.A., Kornbluth, S., 2009. Metabolic control of oocyte apoptosis mediated by 14-3-3zeta-regulated dephosphorylation of caspase-2. Developmental cell 16, 856-866. Nutt, L.K., Margolis, S.S., Jensen, M., Herman, C.E., Dunphy, W.G., Rathmell, J.C., Kornbluth, S., 2005. Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2. Cell 123, 89-103. Parsons, D.W., Jones, S., Zhang, X., Lin, J.C., Leary, R.J., Angenendt, P., Mankoo, P., Carter, H., Siu, I.M., Gallia, G.L., Olivi, A., McLendon, R., Rasheed, B.A., Keir, S., Nikolskaya, T., Nikolsky, Y., Busam, D.A., Tekleab, H., Diaz, L.A., Jr., Hartigan, J., Smith, D.R., Strausberg, R.L., Marie, S.K., Shinjo, S.M., Yan, H., Riggins, G.J., Bigner, D.D., Karchin, R., Papadopoulos, N., Parmigiani, G., Vogelstein, B., Velculescu, V.E., Kinzler, K.W., 2008. An integrated genomic analysis of human glioblastoma multiforme. Science 321, 1807-1812. Pollyea, D.A., Kohrt, H.E., Zhang, B., Zehnder, J., Schenkein, D., Fantin, V., Straley, K., Vasanthakumar, A., Abdel-Wahab, O., Levine, R., Godley, L.A., Medeiros, B.C., 2013. 2-Hydroxyglutarate in IDH mutant acute myeloid leukemia: predicting patient responses, minimal residual disease and correlations with methylcytosine and hydroxymethylcytosine levels. Leukemia & lymphoma 54, 408-410. Pop, C., Fitzgerald, P., Green, D.R., Salvesen, G.S., 2007. Role of proteolysis in caspase-8 activation and stabilization. Biochemistry 46, 4398-4407. Pope, W.B., Prins, R.M., Albert Thomas, M., Nagarajan, R., Yen, K.E., Bittinger, M.A., Salamon, N., Chou, A.P., Yong, W.H., Soto, H., Wilson, N., Driggers, E., Jang, H.G., Su, S.M., Schenkein, D.P., Lai, A., Cloughesy, T.F., Kornblum, H.I., Wu, H., Fantin, V.R., Liau, L.M., 2012. Non-invasive detection of 2hydroxyglutarate and other metabolites in IDH1 mutant glioma patients using magnetic resonance spectroscopy. Journal of neuro-oncology 107, 197-205. Prasad, K.N., Edwards-Prasad, J., 1982. Effects of tocopherol (vitamin E) acid succinate on morphological alterations and growth inhibition in melanoma cells in culture. Cancer research 42, 550-555. Prochazka, L., Koudelka, S., Dong, L.F., Stursa, J., Goodwin, J., Neca, J., Slavik, J., Ciganek, M., Masek, J., Kluckova, K., Nguyen, M., Turanek, J., Neuzil, J., 2013. Mitochondrial targeting overcomes ABCA1dependent resistance of lung carcinoma to alpha-tocopheryl succinate. Apoptosis : an international journal on programmed cell death 18, 286-299.
35
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Qie, S., Chu, C., Li, W., Wang, C., Sang, N., 2013. ErbB2 activation upregulates glutaminase 1 expression which promotes breast cancer cell proliferation. Journal of cellular biochemistry. Qing, G., Li, B., Vu, A., Skuli, N., Walton, Z.E., Liu, X., Mayes, P.A., Wise, D.R., Thompson, C.B., Maris, J.M., Hogarty, M.D., Simon, M.C., 2012. ATF4 regulates MYC-mediated neuroblastoma cell death upon glutamine deprivation. Cancer cell 22, 631-644. Qu, W., Oya, S., Lieberman, B.P., Ploessl, K., Wang, L., Wise, D.R., Divgi, C.R., Chodosh, L.A., Thompson, C.B., Kung, H.F., 2012. Preparation and characterization of L-[5-11C]-glutamine for metabolic imaging of tumors. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 53, 98-105. Qu, W., Zha, Z., Ploessl, K., Lieberman, B.P., Zhu, L., Wise, D.R., Thompson, C.B., Kung, H.F., 2011. Synthesis of optically pure 4-fluoro-glutamines as potential metabolic imaging agents for tumors. Journal of the American Chemical Society 133, 1122-1133. Quinlan, C.L., Orr, A.L., Perevoshchikova, I.V., Treberg, J.R., Ackrell, B.A., Brand, M.D., 2012. Mitochondrial complex II can generate reactive oxygen species at high rates in both the forward and reverse reactions. The Journal of biological chemistry 287, 27255-27264. Ricci, J.E., Gottlieb, R.A., Green, D.R., 2003. Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis. The Journal of cell biology 160, 65-75. Ricketts, C., Woodward, E.R., Killick, P., Morris, M.R., Astuti, D., Latif, F., Maher, E.R., 2008. Germline SDHB mutations and familial renal cell carcinoma. Journal of the National Cancer Institute 100, 12601262. Rohle, D., Popovici-Muller, J., Palaskas, N., Turcan, S., Grommes, C., Campos, C., Tsoi, J., Clark, O., Oldrini, B., Komisopoulou, E., Kunii, K., Pedraza, A., Schalm, S., Silverman, L., Miller, A., Wang, F., Yang, H., Chen, Y., Kernytsky, A., Rosenblum, M.K., Liu, W., Biller, S.A., Su, S.M., Brennan, C.W., Chan, T.A., Graeber, T.G., Yen, K.E., Mellinghoff, I.K., 2013. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science 340, 626-630. Rose, N.R., McDonough, M.A., King, O.N., Kawamura, A., Schofield, C.J., 2011. Inhibition of 2oxoglutarate dependent oxygenases. Chemical Society reviews 40, 4364-4397. Salas, M.L., Vinuela, E., Salas, M., Sols, A., 1965. Citrate Inhibition of Phosphofructokinase and the Pasteur Effect. Biochemical and biophysical research communications 19, 371-376. Salvesen, G.S., Dixit, V.M., 1999. Caspase activation: the induced-proximity model. Proceedings of the National Academy of Sciences of the United States of America 96, 10964-10967. Schafer, Z.T., Grassian, A.R., Song, L., Jiang, Z., Gerhart-Hines, Z., Irie, H.Y., Gao, S., Puigserver, P., Brugge, J.S., 2009. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 461, 109-113. Schagger, H., Pfeiffer, K., 2000. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. The EMBO journal 19, 1777-1783. Schodel, J., Oikonomopoulos, S., Ragoussis, J., Pugh, C.W., Ratcliffe, P.J., Mole, D.R., 2011. Highresolution genome-wide mapping of HIF-binding sites by ChIP-seq. Blood 117, e207-217. Scott, D.A., Richardson, A.D., Filipp, F.V., Knutzen, C.A., Chiang, G.G., Ronai, Z.A., Osterman, A.L., Smith, J.W., 2011. Comparative metabolic flux profiling of melanoma cell lines: beyond the Warburg effect. The Journal of biological chemistry 286, 42626-42634. Selak, M.A., Armour, S.M., MacKenzie, E.D., Boulahbel, H., Watson, D.G., Mansfield, K.D., Pan, Y., Simon, M.C., Thompson, C.B., Gottlieb, E., 2005. Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer cell 7, 77-85. Semenza, G.L., 2010. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 29, 625-634. Shapiro, R.A., Farrell, L., Srinivasan, M., Curthoys, N.P., 1991. Isolation, characterization, and in vitro expression of a cDNA that encodes the kidney isoenzyme of the mitochondrial glutaminase. The Journal of biological chemistry 266, 18792-18796. 36
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
Sies, H., Summer, K.H., 1975. Hydroperoxide-metabolizing systems in rat liver. European journal of biochemistry / FEBS 57, 503-512. Smith, E.M., Watford, M., 1990. Molecular cloning of a cDNA for rat hepatic glutaminase. Sequence similarity to kidney-type glutaminase. The Journal of biological chemistry 265, 10631-10636. Stapelberg, M., Tomasetti, M., Alleva, R., Gellert, N., Procopio, A., Neuzil, J., 2004. alpha-Tocopheryl succinate inhibits proliferation of mesothelioma cells by selective down-regulation of fibroblast growth factor receptors. Biochemical and biophysical research communications 318, 636-641. Stetak, A., Veress, R., Ovadi, J., Csermely, P., Keri, G., Ullrich, A., 2007. Nuclear translocation of the tumor marker pyruvate kinase M2 induces programmed cell death. Cancer research 67, 1602-1608. Sun, F., Huo, X., Zhai, Y., Wang, A., Xu, J., Su, D., Bartlam, M., Rao, Z., 2005. Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121, 1043-1057. Sun, Q., Chen, X., Ma, J., Peng, H., Wang, F., Zha, X., Wang, Y., Jing, Y., Yang, H., Chen, R., Chang, L., Zhang, Y., Goto, J., Onda, H., Chen, T., Wang, M.R., Lu, Y., You, H., Kwiatkowski, D., Zhang, H., 2011. Mammalian target of rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth. Proceedings of the National Academy of Sciences of the United States of America 108, 4129-4134. Sun, R.C., Denko, N.C., 2014. Hypoxic Regulation of Glutamine Metabolism through HIF1 and SIAH2 Supports Lipid Synthesis that Is Necessary for Tumor Growth. Cell metabolism 19, 285-292. Suzuki, S., Tanaka, T., Poyurovsky, M.V., Nagano, H., Mayama, T., Ohkubo, S., Lokshin, M., Hosokawa, H., Nakayama, T., Suzuki, Y., Sugano, S., Sato, E., Nagao, T., Yokote, K., Tatsuno, I., Prives, C., 2010. Phosphate-activated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive oxygen species. Proceedings of the National Academy of Sciences of the United States of America 107, 7461-7466. Swettenham, E., Witting, P.K., Salvatore, B.A., Neuzil, J., 2005. Alpha-tocopheryl succinate selectively induces apoptosis in neuroblastoma cells: potential therapy of malignancies of the nervous system? Journal of neurochemistry 94, 1448-1456. Szeliga, M., Obara-Michlewska, M., Matyja, E., Lazarczyk, M., Lobo, C., Hilgier, W., Alonso, F.J., Marquez, J., Albrecht, J., 2009. Transfection with liver-type glutaminase cDNA alters gene expression and reduces survival, migration and proliferation of T98G glioma cells. Glia 57, 1014-1023. Szeliga, M., Sidoryk, M., Matyja, E., Kowalczyk, P., Albrecht, J., 2005. Lack of expression of the liver-type glutaminase (LGA) mRNA in human malignant gliomas. Neuroscience letters 374, 171-173. Tannahill, G.M., Curtis, A.M., Adamik, J., Palsson-McDermott, E.M., McGettrick, A.F., Goel, G., Frezza, C., Bernard, N.J., Kelly, B., Foley, N.H., Zheng, L., Gardet, A., Tong, Z., Jany, S.S., Corr, S.C., Haneklaus, M., Caffrey, B.E., Pierce, K., Walmsley, S., Beasley, F.C., Cummins, E., Nizet, V., Whyte, M., Taylor, C.T., Lin, H., Masters, S.L., Gottlieb, E., Kelly, V.P., Clish, C., Auron, P.E., Xavier, R.J., O'Neill, L.A., 2013. Succinate is an inflammatory signal that induces IL-1beta through HIF-1alpha. Nature 496, 238-242. Taylor, W.M., Halperin, M.L., 1973. Regulation of pyruvate dehydrogenase in muscle. Inhibition by citrate. The Journal of biological chemistry 248, 6080-6083. Tennant, D.A., Frezza, C., MacKenzie, E.D., Nguyen, Q.D., Zheng, L., Selak, M.A., Roberts, D.L., Dive, C., Watson, D.G., Aboagye, E.O., Gottlieb, E., 2009. Reactivating HIF prolyl hydroxylases under hypoxia results in metabolic catastrophe and cell death. Oncogene 28, 4009-4021. Thangaraju, M., Karunakaran, S.K., Itagaki, S., Gopal, E., Elangovan, S., Prasad, P.D., Ganapathy, V., 2009. Transport by SLC5A8 with subsequent inhibition of histone deacetylase 1 (HDAC1) and HDAC3 underlies the antitumor activity of 3-bromopyruvate. Cancer 115, 4655-4666. Turcan, S., Rohle, D., Goenka, A., Walsh, L.A., Fang, F., Yilmaz, E., Campos, C., Fabius, A.W., Lu, C., Ward, P.S., Thompson, C.B., Kaufman, A., Guryanova, O., Levine, R., Heguy, A., Viale, A., Morris, L.G., Huse, J.T., Mellinghoff, I.K., Chan, T.A., 2012. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 483, 479-483. 37
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
NU
SC
RI
PT
van den Heuvel, A.P., Jing, J., Wooster, R.F., Bachman, K.E., 2012. Analysis of glutamine dependency in non-small cell lung cancer: GLS1 splice variant GAC is essential for cancer cell growth. Cancer biology & therapy 13, 1185-1194. Vander Heiden, M.G., Chandel, N.S., Schumacker, P.T., Thompson, C.B., 1999. Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange. Molecular cell 3, 159-167. Vaughn, A.E., Deshmukh, M., 2008. Glucose metabolism inhibits apoptosis in neurons and cancer cells by redox inactivation of cytochrome c. Nature cell biology 10, 1477-1483. Wang, F., Travins, J., DeLaBarre, B., Penard-Lacronique, V., Schalm, S., Hansen, E., Straley, K., Kernytsky, A., Liu, W., Gliser, C., Yang, H., Gross, S., Artin, E., Saada, V., Mylonas, E., Quivoron, C., Popovici-Muller, J., Saunders, J.O., Salituro, F.G., Yan, S., Murray, S., Wei, W., Gao, Y., Dang, L., Dorsch, M., Agresta, S., Schenkein, D.P., Biller, S.A., Su, S.M., de Botton, S., Yen, K.E., 2013. Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation. Science 340, 622-626. Wang, H.J., Hsieh, Y.J., Cheng, W.C., Lin, C.P., Lin, Y.S., Yang, S.F., Chen, C.C., Izumiya, Y., Yu, J.S., Kung, H.J., Wang, W.C., 2014. JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1alphamediated glucose metabolism. Proceedings of the National Academy of Sciences of the United States of America 111, 279-284. Wang, J.B., Erickson, J.W., Fuji, R., Ramachandran, S., Gao, P., Dinavahi, R., Wilson, K.F., Ambrosio, A.L., Dias, S.M., Dang, C.V., Cerione, R.A., 2010. Targeting mitochondrial glutaminase activity inhibits oncogenic transformation. Cancer cell 18, 207-219. Warburg, O., Wind, F., Negelein, E., 1927. The Metabolism of Tumors in the Body. The Journal of general physiology 8, 519-530. Ward, P.S., Patel, J., Wise, D.R., Abdel-Wahab, O., Bennett, B.D., Coller, H.A., Cross, J.R., Fantin, V.R., Hedvat, C.V., Perl, A.E., Rabinowitz, J.D., Carroll, M., Su, S.M., Sharp, K.A., Levine, R.L., Thompson, C.B., 2010. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer cell 17, 225-234. Waygood, E.B., Sanwal, B.D., 1972. Regulation of pyruvate kinases by succinyl coenzyme A. Biochemical and biophysical research communications 48, 402-407. Waygood, E.B., Sanwal, B.D., 1974. The control of pyruvate kinases of Escherichia coli. I. Physicochemical and regulatory properties of the enzyme activated by fructose 1,6-diphosphate. The Journal of biological chemistry 249, 265-274. Wise, D.R., DeBerardinis, R.J., Mancuso, A., Sayed, N., Zhang, X.Y., Pfeiffer, H.K., Nissim, I., Daikhin, E., Yudkoff, M., McMahon, S.B., Thompson, C.B., 2008. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proceedings of the National Academy of Sciences of the United States of America 105, 18782-18787. Wise, D.R., Thompson, C.B., 2010. Glutamine addiction: a new therapeutic target in cancer. Trends in biochemical sciences 35, 427-433. Wise, D.R., Ward, P.S., Shay, J.E., Cross, J.R., Gruber, J.J., Sachdeva, U.M., Platt, J.M., DeMatteo, R.G., Simon, M.C., Thompson, C.B., 2011. Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of alpha-ketoglutarate to citrate to support cell growth and viability. Proceedings of the National Academy of Sciences of the United States of America 108, 19611-19616. Wong, N., Ojo, D., Yan, J., Tang, D., 2014. PKM2 contributes to cancer metabolism. Cancer letters. Wouters, B.G., Koritzinsky, M., 2008. Hypoxia signalling through mTOR and the unfolded protein response in cancer. Nature reviews. Cancer 8, 851-864. Xiao, M., Yang, H., Xu, W., Ma, S., Lin, H., Zhu, H., Liu, L., Liu, Y., Yang, C., Xu, Y., Zhao, S., Ye, D., Xiong, Y., Guan, K.L., 2012. Inhibition of alpha-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors. Genes & development 26, 1326-1338. 38
ACCEPTED MANUSCRIPT
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RI
PT
Xu, W., Yang, H., Liu, Y., Yang, Y., Wang, P., Kim, S.H., Ito, S., Yang, C., Wang, P., Xiao, M.T., Liu, L.X., Jiang, W.Q., Liu, J., Zhang, J.Y., Wang, B., Frye, S., Zhang, Y., Xu, Y.H., Lei, Q.Y., Guan, K.L., Zhao, S.M., Xiong, Y., 2011. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutaratedependent dioxygenases. Cancer cell 19, 17-30. Yalcin, A., Clem, B.F., Simmons, A., Lane, A., Nelson, K., Clem, A.L., Brock, E., Siow, D., Wattenberg, B., Telang, S., Chesney, J., 2009a. Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases. The Journal of biological chemistry 284, 24223-24232. Yalcin, A., Telang, S., Clem, B., Chesney, J., 2009b. Regulation of glucose metabolism by 6phosphofructo-2-kinase/fructose-2,6-bisphosphatases in cancer. Experimental and molecular pathology 86, 174-179. Yan, H., Parsons, D.W., Jin, G., McLendon, R., Rasheed, B.A., Yuan, W., Kos, I., Batinic-Haberle, I., Jones, S., Riggins, G.J., Friedman, H., Friedman, A., Reardon, D., Herndon, J., Kinzler, K.W., Velculescu, V.E., Vogelstein, B., Bigner, D.D., 2009. IDH1 and IDH2 mutations in gliomas. The New England journal of medicine 360, 765-773. Yang, C., Sudderth, J., Dang, T., Bachoo, R.M., McDonald, J.G., DeBerardinis, R.J., 2009. Glioblastoma cells require glutamate dehydrogenase to survive impairments of glucose metabolism or Akt signaling. Cancer research 69, 7986-7993. Yang, W., Xia, Y., Cao, Y., Zheng, Y., Bu, W., Zhang, L., You, M.J., Koh, M.Y., Cote, G., Aldape, K., Li, Y., Verma, I.M., Chiao, P.J., Lu, Z., 2012. EGFR-induced and PKCepsilon monoubiquitylation-dependent NFkappaB activation upregulates PKM2 expression and promotes tumorigenesis. Molecular cell 48, 771784. Yang, W., Xia, Y., Ji, H., Zheng, Y., Liang, J., Huang, W., Gao, X., Aldape, K., Lu, Z., 2011. Nuclear PKM2 regulates beta-catenin transactivation upon EGFR activation. Nature 480, 118-122. Yi, C.H., Pan, H., Seebacher, J., Jang, I.H., Hyberts, S.G., Heffron, G.J., Vander Heiden, M.G., Yang, R., Li, F., Locasale, J.W., Sharfi, H., Zhai, B., Rodriguez-Mias, R., Luithardt, H., Cantley, L.C., Daley, G.Q., Asara, J.M., Gygi, S.P., Wagner, G., Liu, C.F., Yuan, J., 2011. Metabolic regulation of protein N-alpha-acetylation by Bcl-xL promotes cell survival. Cell 146, 607-620. Yi, C.H., Sogah, D.K., Boyce, M., Degterev, A., Christofferson, D.E., Yuan, J., 2007. A genome-wide RNAi screen reveals multiple regulators of caspase activation. The Journal of cell biology 179, 619-626. Yousefi, S., Owens, J.W., Cesario, T.C., 2004. Citrate shows specific, dose-dependent lympholytic activity in neoplastic cell lines. Leukemia & lymphoma 45, 1657-1665. Yuneva, M., Zamboni, N., Oefner, P., Sachidanandam, R., Lazebnik, Y., 2007. Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells. The Journal of cell biology 178, 93-105. Zhang, X., Varin, E., Allouche, S., Lu, Y., Poulain, L., Icard, P., 2009. Effect of citrate on malignant pleural mesothelioma cells: a synergistic effect with cisplatin. Anticancer research 29, 1249-1254.
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Highlights Mitochondria play crucial role in various cell death modalities Metabolic aberrations are hallmarks of cancer Metabolic switch in tumor cells is associated with alteration in the abundance, utilization, and localization of various mitochondrial substrates Targeting mitochondria is a promising strategy to combat cancer
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