Biochemical Pharmacology 92 (2014) 3–11

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Review - Part of the Special Issue: Metabolism 2014 – Alterations of metabolic pathways as therapeutic targets

Glycogen metabolism in cancer Christos E. Zois b,1,2, Elena Favaro a,1,3, Adrian L. Harris b,* a

Cell Death and Metabolism, Danish Cancer Society Research Center, Strandboulevarden 49, 2100 Copenhagen, Denmark Molecular Oncology Laboratories, Oxford University, Department of Oncology, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 June 2014 Accepted 4 September 2014 Available online 16 September 2014

Since its identification more than 150 years ago, there has been an extensive characterisation of glycogen metabolism and its regulatory pathways in the two main glycogen storage organs of the body, i.e. liver and muscle. In recent years, glycogen metabolism has also been demonstrated to be upregulated in many tumour types, suggesting it is an important aspect of cancer cell pathophysiology. Here, we provide an overview of glycogen metabolism and its regulation, with a focus on its role in metabolic reprogramming of cancer cells. The various methods to detect glycogen in tumours in vivo are also reviewed. Finally, we discuss the targeting of glycogen metabolism as a strategy for cancer treatment. ß 2014 Elsevier Inc. All rights reserved.

Keywords: Glycogen metabolism Hypoxia Pentose shunt Cancer

Contents 1. 2. 3. 4. 5.

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Glycogen metabolism overview . . . . . . . . . . . . . . . . . Regulation of glycogen metabolism . . . . . . . . . . . . . . Role of glycogen in cancer. . . . . . . . . . . . . . . . . . . . . . Methods for assessing glycogen stores and turnover. Pharmacological inhibition of glycogen metabolism . 5.1. Active-site inhibitors . . . . . . . . . . . . . . . . . . . . AMP-site inhibitors . . . . . . . . . . . . . . . . . . . . . . 5.2. Indole-carboxamide-site inhibitors . . . . . . . . . 5.3. Purine-nucleoside-site inhibitors . . . . . . . . . . . 5.4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Glycogen metabolism overview Glycogen is a branched polymer of glucose that acts as an intracellular glucose store. Most tissues are capable of storing

* Corresponding author. Tel.: +44 1865 222 457; fax: +44 1865 222 431. E-mail addresses: [email protected] (C.E. Zois), [email protected] (E. Favaro), [email protected] (A.L. Harris). 1 Equal first authors. 2 Tel.: +44 1865 222 457; fax: +44 1865 222 431. 3 Tel.: +45 3525 7733. http://dx.doi.org/10.1016/j.bcp.2014.09.001 0006-2952/ß 2014 Elsevier Inc. All rights reserved.

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glycogen. However, liver and skeletal muscle contain the most abundant depots in the body. Glycogen metabolism in the liver is important for the regulation of blood glucose homeostasis, whereas in the muscle, glycogen utilisation is the main fuel to provide glucose and ATP needed for muscle contraction in prolonged high-intensity exercise. Other tissues capable of metabolising glycogen include the brain and the adipose tissue. In the brain, glycogen is found both in astrocytes and neurons and its degradation in the former is thought to spare blood glucose for neurons during high brain activation [1], whereas in the latter, it promotes neuronal tolerance to hypoxic stress [2]. In the adipose tissue, glycogen is tightly coupled with lipid metabolism and potentially functions as a metabolic sensor to

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coordinate lipogenic programmes [3]. Presence of glycogen has been described also in various cancer cells and tumours. The level of glycogen accumulation varies greatly across tumours. The levels of glycogen were demonstrated to be particularly high in breast, kidney, uterus, bladder, ovary, skin and brain cancer cell lines. Glycogen content in these cells and in human colorectal cancer tissues was inversely correlated with proliferation rate, suggesting that glycogen is consumed to sustain cancer cell growth [4,5]. In particular, ‘clear cell carcinomas’ represent a subset of tumours, which are characterised by a prominent cellular enrichment in glycogen. Their name derives from the clear, vacuolated appearance of cellular cytoplasm caused by extraction of glycogen during histology processing. Both glycogen synthesis and degradation involve the activity of several enzymes and regulatory proteins. Synthesis is performed in the cytosol from extracellular glucose transported into the cells through glucose transporters (direct pathway) or from gluconeogenic substrates, such as lactate and amino acids (indirect pathway). The indirect pathway occurs mainly in the liver, following either the intrahepatic or extrahepatic conversion of gluconeogenic precursors into glucose [6]. The first step of glycogen synthesis consists on the autoglucosylation of the core protein, glycogenin (GYG). This provides an oligosaccharide primer for glycogen synthase enzyme (GYS), which elongates the glucose chain by attaching (activated) uridine diphospho-glucose (UDP-glucose) units through a-1,4 glycosidic bonds. Once the elongating chain consists of approximately 11 units, the glycogen branching enzyme (GBE) transfers a chain of 7 units to an adjacent chain through a a-1,6 glycosidic bond [7] (Fig. 1). The coordinated function of these two enzymes proceeds to form spherical granules named b-particles, which are 20–50 nm in diameter and contain up to 55,000 glucose units. The b-particles can aggregate to form larger a-rosettes, of 200 nm in diameter.

The highly branched structure of glycogen exerts two functions: first, it increases the solubility of the molecule in the cytosol and secondly, it provides a multitude of docking sites for glycogen binding proteins. These include glycogen metabolising enzymes and regulatory proteins, which bind to glycogen through carbohydrate binding modules. Glycogen degradation can occur through two different pathways: a well-characterised cytosolic pathway and a less-defined lysosomal pathway. In the cytosol, glycogen breakdown is catalysed by two enzymes, namely glycogen phosphorylase and glycogen debranching enzyme (DBE). Glycogen phosphorylase cleaves a-1,4 glycosidic linkages by the addition of orthophosphate to yield glucose-1phosphate (glucose-1P), the free glucose form after glycogen phosphorylase action. When glycogen phosphorylase reaches the fourth unit from a branching point, DBE transfers three units to the end of another glycogen chain and catalyses the hydrolysis of the a1,6 glycosidic linkage, yielding free glucose [7,8]. Glucose-1P, by phosphoglucomutase (PGM)-dependent conversion into glucose-6P, has three possible fates: (1) it can be metabolised through the glycolytic pathway and generate ATP, (2) it can enter the pentose phosphate pathway and be used to fuel anabolic reactions and to sustain the antioxidant defences of the cell, or (3) in the liver and other tissues expressing the enzyme glucose 6-phosphatase, it can be dephosphorylated into free glucose and exit the cell. Alternatively, glycogen can be transferred to the autophagosomes and delivered to the lysosomal compartment, where it is hydrolysed by alpha-acid glycosidase (GAA) in a process called glycogen autophagy or glycophagy [9]. The role of glycophagy is well characterised in the liver of newborn animals, where it provides energy substrates during a period of starvation prior to the beginning of feeding. In adult animals, little is know on the role of glycophagy, however, the importance of this pathway is suggested by the severe consequences of GAA defects in people

Fig. 1. Schematic representation of glycogen synthesis and breakdown in the cytosol. The precursor used for glycogen synthesis comprises UTP-activated glucose units (UDPglucose). Glycogen synthesis in the cytosol begins with the formation of glycogenin (GYG)-bound oligosaccharide primers and proceeds with the elongation of the primers by glycogen synthase (GYS), which binds activated glucose units to the non-reducing end of the forming glycogen chain. Glycogen degradation consists on the phosphorolytic cleavage of glucose-1-P, catalysed by glycogen phosphorylase (PYGL/B/M), and the removal of branches catalysed by the debranching enzyme (DBE). DBE transfers three glucose blocks to another glycogen chain, and then hydrolytically cleaves the remaining glucose of the branch, generating free glucose. Glycogen-derived glucose-1-P and free glucose can enter the glycolytic or the pentose phosphate pathway (PPP). HK: hexokinase; glucose-6/1-p: glucose-6/1-phosphate; PGM: phosphoglucomutase; UGP: UDPglucose pyrophosphatase; UDP-glucose: uridine diphospho-glucose; GYG: glycogenin; GYS: glycogen synthase; GBE: glycogen branching enzyme; PYGL/B/M: glycogen phosphorylase, liver/brain/muscle; DBE: debranching enzyme; PPP: pentose phosphate pathway.

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Table 1 Glycogen storage diseases. List of glycogen storage disorders with corresponding enzymatic defect, organ involvement and prominent clinical features. Disease type (name)

Enzyme defect

Organs involved

Clinical features

Type 0

Glycogen synthase (muscle (GYS1) or liver (GYS2))

Type I (von Gierke) Type II (Pompe)

Glucose-6-phosphatase or glucose-6-phosphate translocase Acid alpha-glucosidase (GAA)

Skeletal muscle and myocardium (GYS1); liver (GYS2) Liver, kidney

Type III (Cori)

Amylo-alpha-1, 6-glucosidase (AGL)

Muscle pain and weakness, syncope, cardiac arrest (GYS1); fasting hypoglycaemia, ketosis, usually mild (GYS2). Hepatomegaly, renomegaly, growth retardation. Many live into adulthood. Lethal cardiorespiratory failure (infantile-onset); muscle weakness and respiratory insufficiency (late onset). Hepatomegaly, hyperlipidaemia, cardiomyopathy.

Type IV (Andersen)

Glycogen branching enzyme I (GBEI)

Type V (McArdle)

Glycogen phosphorylase, muscle form (PYGM) Glycogen phosphorylase, liver form (PYGL) Phosphofructokinase Phosphorylase kinase

Skeletal muscle

Epilepsy, progressive myoclonus type 2A (EPM2A), epilepsy, progressive myoclonus type 2B (EPM2B)

Brain (neurons), liver, kidney, muscle

Type VI (Hers) Type VII (Tarui) Type IX (phosphorylase b kinase deficiency) Progressive myoclonus epilepsy, Lafora type (Lafora disease)

affected by Pompe disease (glycogen storage disease II) (see Table 1). The exact mechanism by which glycogen is engulfed in the autophagosomes is not yet defined, although a number of observations point to the selective nature of the process. These include: (i) the spatial distribution of glycogen-enriched vesicles in close proximity of cytosolic glycogen, with rare occurrence of other organelles or cell components in the same vesicles and (ii) the specific inhibition of GAA activity (and unchanged activity of another lysosomal enzyme, acid phosphatase) and accumulation of autophagosomal glycogen (without evidence of accumulation in other organelles), following either glucose or insulin administration [10]. Recent evidence in fibroblast-like and hepatic cells suggests that glycogen engulfment into lysosomes could be mediated by anchoring through starch binding domain-containing protein 1 (Stbd1)/genethonin 1 and interaction of this with the Atg8 autophagy protein g-aminobutyric acid type A receptorassociated protein-like 1 (GABARAPL1) [11,12]. Analysis of the glycogen-associated proteome could be instrumental for the identification of other important players and regulators of this alternative glycogen catabolic pathway. Notably, two recent studies have provided a comprehensive analysis of the composition of the glycogen-associated proteome in liver and adipose tissue, respectively [13,14]. Although both studies listed GAA in their data sets, Stbd1 was only identified in the liver glycogen proteome, possibly indicating a tissue-specific dependence on this process or the activity of different glycophagy receptors in different tissues. Moreover, these studies confirmed the indication that glycogen metabolism is ‘spatially regulated’. Electron microscopy imaging of cellular glycogen had in fact already shown association with cellular components, including mitochondria, Golgi apparatus, polyribosomes and endoplasmic reticulum (ER) [15]. The glycogen proteome analyses identified several associated proteins residents in subcellular compartments, such as the ER, the mitochondria and the cytoskeleton, with some differences between the two studies, suggesting a tissue-specialisation for glycogen metabolic functions. In these studies, the identification of glycogen-interacting proteins relied on either direct binding to glycogen or indirect interaction and co-purification as part of complexes or cellular structures and membranes bound to glycogen [13,14]. Inherited mutations of several proteins involved in glycogen turnover are responsible for glycogen storage disorders, which are

Skeletal muscle, myocardium

Liver, skeletal muscle, myocardium Liver, spleen

Liver Skeletal muscle Liver, skeletal muscle

Severity varies greatly, from early childhood death due to liver failure, to mild symptomatology and survival to adulthood. Exercise-induced muscle cramps and pain, otherwise normal. Similar to type I, but milder. Similar to type V. Mild hepatomegaly, hypoglycaemia and growth retardation. Usually improves with age. Myoclonus, seizures, hallucinations, progressive neurologic degeneration. Most patients die within 10 years of onset.

characterised by a different degree of liver or muscle symptomatology, as summarised in Table 1 [7]. In particular, a new emerging glycogen storage disease is represented by Lafora disease. This fatal disorder is characterised by the accumulation of poorly branched, insoluble glycogen aggregates (‘Lafora bodies’) in most tissues of affected patients, who prevalently present with neurological symptoms, including myoclonus, seizures and progressive neurodegeneration. In the brain, glycogen is usually found in astrocytes, whereas in neurons, glycogen synthesis is kept in an inactive form and glycogen levels are not detectable [16]. The disorder is caused by loss-of-function mutations in two genes, namely epilepsy, progressive myoclonus type 2A (EPM2A) and epilepsy, progressive myoclonus type 2B (EPM2B), encoding the proteins Laforin and Malin, respectively [8]. Laforin is a phosphatase that can directly bind glycogen through a carbohydratebinding domain and other glycogen metabolising proteins. Malin is an E3-ubiquitin ligase that is recruited to its substrates through the interaction with Laforin [8]. The pathogenesis of Lafora disease has not been fully elucidated, however the main hypotheses under study focus on functions of the Laforin–Malin complex that have been linked to (i) imbalance between glycogen synthesis and branching activities and (ii) accumulation of abnormally phosphorylated glycogen [8,17]. Recent experimental evidences in a mouse model of Lafora disease proposes glycogen accumulation as a direct cause of neuronal degeneration and indicates glycogen synthesis as a putative target for the treatment of the disease [18]. 2. Regulation of glycogen metabolism Glycogen metabolism is under allosteric regulation to meet the needs of the cell, and under hormonal regulation to meet the needs of the organism. Regulation occurs on the enzymes glycogen phosphorylase and glycogen synthase, and involves allosteric effects and covalent modifications of enzymes. Three enzymes control glycogen breakdown: glycogen phosphorylase, glycogen debranching enzyme and phosphoglucomutase. Gycogen phosphorylase (GP) is the rate-limiting enzyme and in mammals has three isoforms, liver (PYGL, 97 kDa), muscle (PYGM, 97 kDa) and the brain (PYGB, 96.6 kDa). For simplicity, hereafter glycogen phosphorylase will be referred to as GP for all isoforms. The liver enzyme serves the glycaemic demands of the body in general, whereas the brain and muscle isozymes supply just those tissues. GP has an essential cofactor, pryridoxal

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phosphate (PLP) and is regulated by allosteric effectors and by phosphorylation of a single serine residue at the N-terminus [19–22]. Phosphorylation of glycogen phosphorylase b (GPb, less active) to glycogen phosphorylase a (GPa, active) is catalysed by phosphorylase kinase, which is activated by protein kinase A and increased levels of calcium [19–22] (Fig. 2A and B). GPs are dimers of two identical monomers and allosteric effectors bind to five regulatory sites in each monomer: catalytic site (C-site, binds glucose, glucose 1-phosphate and inorganic phosphate), the purine nucleotide inhibitor or caffeinebinding site located near the active site, an AMP allosteric site that also bind ATP and G6P, glycogen site (G-site), and the indole site that binds indole carboxamide ligands [19–22]. Protein phosphatase 1 (PP1) dephosphorylates the GPa to the inactive form, GPb. Both forms of GP can be found in the T (tense state) and R-states (relaxed state), where T is the inactive state because it has a low affinity for substrate and R is the active state, given its greater affinity for substrate [19–22]. Phosphorylation and allosteric ligands (AMP, inorganic phosphate, glucose 1-phosphate) stabilise the R-state, while glucose, ATP, purine nucleosides and G6P stabilise the less active T-state. Moreover, glucose and other ligands that stabilise the T-state promote the dephosphorylation of the GPa to GPb by PP1 (Fig. 2B). The phosphorylation control of GP is a response to messages from the extracellular environment, signalled by hormones, whilst allosteric control is a response to intracellular sensors of the cell metabolic status [19–22]. During physical activity, hormonal stimulation (glucagon, epinephrine or adrenaline) activates the enzyme phosphorylase kinase, which in turn phosphorylates and converts the less active form GPb into the more active form, GPa (Fig. 2A) [19–22]. Glycogen synthase is regulated by multiple phosphorylation/ dephosphorylation events and by allosteric effectors. The liver isoform (GYS2, 81 kDa) is about 70% identical to the muscle isoform (GYS1, 84 kDa) and has several phosphorylation sites near the N- and C-terminus [23]. These sites are phosphorylated in vitro by kinases such as protein kinase A, phosphorylase kinase, protein kinase C, protein kinases CK1 and CK2, glycogen synthase kinase 3 and AMP-activate protein kinase [20,21]. Like glycogen phosphorylase, glycogen synthase can exist in a phosphorylated (glycogen synthase b) and a dephosphorylated form (glycogen synthase a). Phosphorylation causes inactivation of the enzyme by decreasing the affinity for UDP-glucose [20,21], while glucose 6-phosphate (G6P) is an allosteric activator of the phosphorylated form [24,25] (Fig. 2A). G6P regulates the activity of glycogen synthase not only by allosteric activation but also by making the protein a more suitable substrate for dephosphorylation by protein phosphatase-1 (PP1) [24]. Dephosphorylation of glycogen synthase is catalysed by PP1, which removes a phosphate group by hydrolysis [26,27]. Insulin activates the insulin receptor tyrosine kinase, which further activates the PI3K and Akt pathway and stimulates glycogen synthesis via inhibition of glycogen synthase 3 (GSK3) also activation of the PP1. [28,29] (Fig. 2A). Moreover, insulin stimulates glucose uptake via translocation of glucose transporters to the plasma membrane [28,29]. Hypoxia regulates the activity of glycogen synthase via the induction of the protein phosphatase 1, regulatory subunit 3 (PPP1R3C) and stimulates glycogen accumulation [30]. 3. Role of glycogen in cancer The presence of glycogen has been demonstrated in a variety of cancer cells and tumours, where glycogen content was inversely correlated with proliferation rate [4]. However, only recently, investigators have turned their attention to glycogen metabolism and recognised it as an important pathway that is metabolically

reprogrammed in cancer cells. There are still several questions that need to be addressed, concerning both the regulation and the role of glycogen metabolism in cancer. A recent study has identified the oncogene Rab25 as a positive regulator of glycogen synthesis in cancer cells [31]. RAB25 is amplified/overexpressed in many human cancers and its regulation of glycogen metabolism is mediated, in part, via AKT-dependent inhibition of GSK3, which in turns catalyses the inhibitory phosphorylation of GYS. The resulting glycogen stores represent an energy source and a survival mechanism for cancer cells affected by nutrient stress. It would be of great interest to investigate whether other oncogenic pathways are associated with glycogen metabolic reprogramming in cancer cells. For example, the oncogene MYC is a master regulator of tumour cell metabolism, including glucose metabolism [32]. It would be worth studying MYC to see if it can also regulate glycogen turnover and if this contributes to drive tumorigenesis. GSK3 functions in diverse cellular processes, including proliferation, motility and survival, and participates in tumour development [33]. It has yet to be demonstrated whether its role in tumorigenesis and cancer progression has any relationship to its ability to regulate glycogen synthesis. Similarly, it should be investigated whether altered glycogen metabolism plays any role on the development of clear cell carcinomas driven by VHL mutations. A number of studies have demonstrated that glycogen turnover can be induced in hypoxia in cancer cell lines in vitro and in tumours in vivo, similarly to non-cancer cells [30,34–38]. In particular, it has been documented that glycogen levels are increased in perinecrotic (hypoxic) areas of tumours and can be induced by anti-angiogenic treatment with bevacizumab [38]. Consistent with these observations, both glycogen synthetic and breakdown enzymes are induced in hypoxia. In particular, the synthetic enzymes and regulatory proteins UTP:Glucose-1-P urydylyltransferase (UGP2), GYS1, GBE, PGM and PPP1R3C, are induced in hypoxia in a hypoxia inducible factor (HIF)-dependent manner [30,35,36,39]. HIF-dependent regulation of glycogen synthesis has also been confirmed in the human renal clear cell carcinoma cell lines RCC4 and 786-O, which are characterised by constitutive HIF-1a and/or HIF-2a activation, due to a defect in the tumour suppressor protein and HIF-negative regulator von Hippel–Lindau (VHL) [40]. These findings suggest that constitutive activation of the synthetic pathway could contribute to glycogen enrichment and to the clear aspect of VHL-defective clear cell renal carcinomas. It remains to be determined whether increased glycogen accumulation contributes to tumourigenesis in von Hippel–Lindau patients and other clear cell carcinoma patients. Glycogen breakdown enzymes, both in the cytosolic (PYGL) and lysosomal (GAA) degradation pathways, are induced in cancer cells after prolonged hypoxic exposure as compared to the acute induction of synthetic enzymes [38], therefore preventing the occurrence of a futile cycle of glycogen synthesis and degradation. The regulation of PYGL and GAA in hypoxic conditions has not been characterised, although a recent study excluded HIF as a potential player in PYGL induction [38]. The PhKG1 (the g-subunit) of the holoenzyme phosphorylase kinase, involved in activation of glycogen phosphorylase, has been shown upregulated in several human tumour samples, also involved in both aspects of tumour progression, angiogenesis and tumour metabolism [41]. This study indicates that PhKG1 could be a potential target candidate therapy. Why do cancer cells store glycogen? The most intuitive explanation is that glycogen accumulation improves survival of cancer cells in response to impoverishment of nutrients and oxygen of the microenvironment (Fig. 3) [35,36]. Importantly, glycogen represents a source of ATP that can be rapidly metabolised without oxygen requirements, as opposed to the other main source of energy for the cells, i.e. fatty acids.

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Fig. 2. Regulation of glycogen metabolism by glycogen synthase and glycogen phosphorylase. (A) Glucagon, epinephrine and physical activity induce cAMP via cognate receptors. cAMP activates protein kinase A (PKA). PKA converts the inactive phosphorylase kinase (PK) to an active form. Also, PKA converts the active form of glycogen synthase a (GSa) to an inactive form glycogen synthase b (GSb). The active PK phosphorylates and activates glycogen phosphorylase a (GPa). The protein phosphatase 1 (PP1) dephosphorylates GPa and active PK and forms the less active GPb and inactive PK. GPa catalyses the degradation of glycogen to G1P and also inhibits the glycogen PP1 which converts the inactive glycogen synthase (GSb) to active glycogen synthase a (GSa). PP1 is regulated by another inhibitor called phosphoprotein phosphatase inhibitor (PI-1). PI-1 can be phosphorylated (activated) by PKA. PKA phosphorylation turns on GP and turns off GS. It also activates PI-1, which turns off the phosphatase (PP1) that would normally activate GS by dephosphorylating it. Insulin activates the insulin receptor tyrosine kinase, which further activates the PI3K and Akt and stimulates glycogen synthesis via inhibition of glycogen synthase 3 (GSK3) also activation of the PP1. Insulin stimulates the glucose uptake via translocation of glucose transporters to the plasma membrane. (B) Glycogen phosphorylase is converted to GPa by phosphorylation via phosphorylase kinase (PK), while dephosphorylation via protein phosphatase 1 (PP1) turns it back to GPb. GPa and GPb exist in equilibrium between a more active R-state (R-relaxed) and less active T-state (T-tense). Under allosteric control AMP converts the inactive T-states (GPa and GPb) to active R-states. Allosteric inhibitors such as ATP, glucose, glucose 6-phosphate and caffeine can alter the equilibrium back to the inactive Tstates of GP [19–22]. (C). Glycogen synthase kinase 3 (GSK3), protein kinase A (PKA), phosphorylase kinase (PK), protein kinase C (PKC), protein kinases CK1 and CK2, glycogen and AMP-activate protein kinase phosphorylate and inactivate glycogen synthase. Protein phosphatase 1 dephosphorylates the GSb and converts it to active GSa form. ATP, glucose, glucose 6-phosphate and caffeine activate the GS while calcium and cAMP deactivates the GS. (D) Four major classes of glycogen phosphorylase inhibitors.

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However, other studies suggested an additional pathophysiological function of glycogen that goes beyond its role as an energy source in situations of increased glucose demand [38,42,43]. First, disruption of glycogen breakdown in cancer cells was associated with profound phenotypic consequences, despite abundant availability of extracellular glucose. Second, inhibition of glycogen degradation has been linked to reduced activity of biosynthetic pathways and antioxidant defences of the cell. In particular, in one study, PYGL depletion was associated with a reduction in cell proliferation, decreased functioning of the pentose phosphate pathway (PPP) and increased levels of reactive oxygen species (ROS) (Fig. 3) [38]. Another study demonstrated that inhibition of glycogen phosphorylase using a small molecule inhibitor (CP-320626) promoted apoptosis by limiting glucose oxidation, nucleic acid and lipid synthesis in a human pancreatic cancer cell line [42]. A more recent proteomic analysis performed on these cells revealed the activation of various stress-induced pathways, as well as increased levels of the cell cycle regulators p21, p27 and p53 [43]. It remains to be demonstrated whether glycogen itself acts as a source of precursors for biosynthetic pathways and if the preferential channelling of glycogen-derived glucose through these pathways is dictated by subcellular compartmentalisation of glycogen. Alternatively, glycogen could act as a metabolic sensor for the cells, fine-tuning the activity of biosynthetic and catabolic pathways. The putative signalling functions of glycogen may arise due to its ability to directly regulate the activity of the master

regulator of intracellular energy homeostasis, AMP-activated protein kinase (AMPK) [44]. Cells normally regulate their glycogen levels very carefully and glycogen metabolising enzymes may simply act as intracellular ‘glycogen sculptors’ in this respect. Future studies will be required to address these open questions. 4. Methods for assessing glycogen stores and turnover Several techniques have been described for detecting and assessing the level of glycogen in cells, tissues and entire organs, such as electron microscopy, immunochistochemical, biochemical, live fluorescence imaging and radioisotope methods. Electron microscopy is the only technique able to describe in detail the fine structure of the cell, and its various compartments. The fine structure of glycogen was first described by Revel and colleagues in 1960, as roughly circular granules from 150 to 400 A in diameter [45]. Today, electron microscopy is a powerful tool to analyse glycogen within the cell, and its localisation. However, due to the lack of expertise, cost and time effectiveness, this technique cannot be used routinely. Routinely, the presence of glycogen can be examined histochemically in both cryosections and formalinfixed, paraffin-embedded sections by the periodic acid-Schiff (PAS) reaction [46]. Glycogen sub-cellular distribution can be studied by immunohistochemistry and immunofluorescent techniques by using a specific antibody against glycogen [47–49], which was developed by Dr Baba [50]. Notably, it is very important to maintain optimal preservation of glycogen in biopsies. Up to 70% of glycogen can be lost using formalin fixation, and this is due to the

Fig. 3. Roles of glycogen metabolism in cancer cells. Under normal fed conditions, cancer cells present a low rate of glycogen turnover (top-left panel). Glycogen synthesis in cancer cells is promoted by oncogene activation (Rab25) or tumour suppressor inhibition (VHL). Glycogen-derived glucose contributes to ATP generation, ROS scavenging and biosynthesis of macromolecules, particularly nucleotides, and also proteins and lipids, required for cell proliferation. After acute exposure to hypoxia, glycogen synthesis is induced through HIF-mediated induction of GYS (top-right panel). Prolonged hypoxia and/or starvation are characterised by reduced glycogen synthesis and augmented glycogen degradation via increased GP expression (bottom-right panel). Glycogen-derived glucose sustains the energetic requirements of the cell and provides protection from ROS. In cancer cells lacking GP, glycogen accumulation, decreased synthesis of macromolecules and increased levels of ROS, can additively contribute to senescence (bottom-left panel). VHL: von Hippel–Lindau protein; GYS: glycogen synthase; GP: glycogen phosphorylase; ROS: reactive oxygen species; PPP: pentose phosphate pathway.

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soluble nature of the predominant form of glycogen in the cytoplasm [51]. Three biochemical methods have been used for analysis of glycogen content in tissue homogenates: (i) acid hydrolysis of the tissue followed by enzymatic analysis of glucose, (ii) enzymatic hydrolysis with amylo-a-1,4-a-1,6-glucosidase followed by analysis of glucose and (iii) analysis of glucose-1-P produced by degradation of glycogen with phosphorylase and debranching complex [52]. 2-NBDG (2-{N-[7-nitrobenz-2-oxa-1,3-diazol 4-yl] amino}-2deoxyglucose) is a fluorescent glucosamine derivative that enters the cells through glucose transporters, is phosphorylated by the enzyme hexokinase and cannot be metabolised through glycolysis but is incorporated into glycogen granules. Therefore, this probe is suitable for glycogen quantification and imaging [53]. However, this technique is ideal only for in vitro measurements and cannot be used for glycogen in vivo determinations, due to the limited tissue penetration of the emitted green fluorescence. More recently, a new technique has been developed to detect de novo glycogenesis in tumour cells in culture and in tumour xenografts in vivo, by using a novel radiotracer, (18)F-N-(methyl-(2-fluoroethyl)-1H-[1,2,3]triazole-4-yl)glucosamine (18F-NFTG) [54]. This technique shows great promise for future clinical applications, allowing tumours stratification according to glycogen content and correlation with treatment response. 5. Pharmacological inhibition of glycogen metabolism The evidence that glycogen metabolism is reprogrammed in cancer cells indicates that targeting of glycogen metabolism could represent a new strategy for cancer treatment. GS and GP are the key enzymes involved in glycogen metabolism. There are no specific inhibitors or activators developed for GS, and the regulation of GS activity is complex. Two major kinases, GSK3beta (GSK3beta) and AMPK, involved also in GS phosphorylation and thus inactivation, have been studied extensively as therapeutic targets for several diseases, including diabetes, cancer and neurological disorders. Glycogen synthase kinase 3beta is regulated by serine (inhibitory) and tyrosine (stimulatory) phosphorylation and had been initially identified as a key regulator of insulin-dependent glycogen synthesis. It is now known that GSK3beta functions in diverse cellular processes including proliferation, differentiation, motility and survival. The role of GSK3beta in tumorigenesis and cancer progression remains controversial [33]. Extensive reviews on a range of the GSK3beta inhibitors and activators have been reported [55–58]. Also, 50 -adenosine monophosphate-activated protein kinase (AMPK) has emerged as an important regulator of cellular energy homeostasis both at cellular level and whole-body levels by inhibiting anabolic and activating catabolic processes, and therefore considered as a promising target for treatment of metabolic diseases/disorders particularly type 2 diabetes, obesity and cancer [59]. Activation of AMPK by either 5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside (AICAR) or hydrogen peroxide was associated with a decrease in the activity ratio of GS [60]. GSK3 forms a stable complex with AMPK through interactions with the AMPK b regulatory subunit and phosphorylates the AMPK a catalytic subunit and inhibits its activity [61]. Thus GSK inhibitors might also increase the AMPK activity. Also, a range of AMPK activators have been reported [62]. Preclinical and clinical studies will be required to identify the best candidates to target, and the clinical features associated with glycogen storage disorders (Table 1) can assist in predicting the toxicity associated with depletion of the different glycogen metabolism enzymes. As there is so much literature dealing with GSK3beta and AMPK inhibitors and activators, and because these

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kinases have multifaced and complex functions, this review is mainly focused on the GP inhibitors as a potential new anticancer treatment for combination therapy. In particular, GP defects in Hers’ disease patients are associated with mild symptomatology. Interestingly, GP inhibition has been studied as a potential therapeutic target for type 2 diabetes [22]. GP is the key enzyme that drives glycogenolysis, and therefore comprises an important target in the attempt to reduce postprandial glucose levels by lowering hepatic glucose production. Glycogenolysis and GP could also potentially represent targets to tackle cancer cachexia, a tumour associated syndrome characterised by loss of adipose tissue and skeletal muscle due to hypermetabolism. Interestingly, an overall increased glucose flux through gluconeogenesis and glycogenolysis has been shown to be increased in weight-losing (cachectic) cancer patients and such flux has been estimated to contribute up to 40% of the increase in energy expenditure in metastatic cancer [63]. Also, a potential new treatment strategy for cancer cachexia has been proposed recently via inhibition of parathyroid-hormonerelated protein (PTHRP) which signals through the PTH receptor, a G protein-coupled receptor (GPCR) that activates the cyclic-AMPdependent protein kinase A (PKA) pathway [64,65], a major pathway for GS inactivation and GP activation. Extensive reviews on a range of GP inhibitors and proposed sites of actions have been reported [19–21,66–70]. Binding sites for physiological and pharmacological ligands have been identified by crystallography. Four major regulation sites have been used to develop glycogen phosphorylase inhibitors: the catalytic-active site (C-site), nucleotide binding site (adenosine monophosphate (AMP)-site), phosphorylation site (P-site) and the indole-site [19] (Fig. 2D). The presence and absence of an allosteric ligand such as glucose and AMP can influence the inhibitory activity, thus researchers need to be careful when using the glycogen phosphorylase inhibitors. 5.1. Active-site inhibitors Glucose analogues such as N-acetyl-b-D-glucosamine and the bicyclic compound glucopyranose spirohydantoin have been synthesised, however these two analogues have lower affinity for liver than muscle isoforms of GP. The most potent active site inhibitor of liver glycogen phosphorylase a is DAB (azasugar 1, 4-dideoxy-1,4-amino-D-arabinitol) [71,72]. DAB is a potent inhibitor of GPa in hepatocytes in both in the absence and presence of glucagon, with half-maximal effect at 1–2 mM and greater than 80% inhibition of glycogenolysis at 5–20 mM [15]. DAB is also an indirect inhibitor of glycogen synthesis [73]. 5.2. AMP-site inhibitors Ligands of the AMP activator site cause both inactivation and allosteric inhibition of glycogen phosphorylase a. A pro-drug, the dihydropyridine derivative (BAY3401), is metabolised to 1, 4-dihydropyridine-2,3-dicarboxylate (BAY1807) and binds to the AMP-site and cause the conversion of GPa to GPb in liver and skeletal muscle [74]. Researchers at Novo-Nordisk [75], Merck [76] and Sanofi Aventis [77,78] have identified several other compounds that bound to the AMP site, with EC50 values in the nanomolar range. Phthalic acid derivatives that bind at the AMPactive site and are potent inhibitors of liver and muscle GPa [75]. Diacid analogues that bind at the AMP site not only are very potent but have approximately 10-fold selectivity against liver versus muscle glycogen phosphorylase (GP) in in vitro assays [76]. Acyl ureas derivatives, which bind at the AMP-active site, were discovered as a novel class of inhibitors for glycogen phosphorylase [78].

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5.3. Indole-carboxamide-site inhibitors There is one indole site to each subunit and in the native T-state of the GPb the site is occupied by 30 water molecules [21]. This site is referred to as the indole site since almost all inhibitors that bind to the site have been based on an indole2-carboxamide scaffold. Researchers from Pfizer [79–83], Merck [84] and AstraZeneca [85–87] have identified several indole-carboxamide site inhibitors. One of these inhibitors is ingliforib (CP-368296) which inhibits the GP isoforms with IC50 values of 52 nM (liver), 150 nM (brain) and 352 nM (muscle). 5.4. Purine-nucleoside-site inhibitors The purine nucleoside-site inhibitors include purines (e.g. caffeine), flavopiridol, nucleosides (e.g. adenosine) and nucleotides (e.g. AMP, IMP, ATP, NADH and FAD) [19–22]. Flavopiridol, an anti tumour drug and inhibitor of cyclin-dependent kinases, was identified as a high-affinity ligand of the purine nucleoside site [88,89]. Further, olefin derivatives of flavopiridol have been synthesised with similar potency on glycogen phosphorylase but and high potency in hepatocytes [90]. The olefin derivatives inhibit glycogenolysis with similar potency in the absence and presence of glucagon, while the indole carboxamide CP-91149 has high potency in the absence of glucagon [73]. 6. Concluding remarks Glycogen is a key energy store in cancer cells. Glycogen turnover allows cells to adapt and survive under adverse oxygen and nutrient conditions within the tumour microenvironment. Therefore, targeting glycogen metabolism in cancer cells could provide a novel strategy for combinational anticancer treatment. Cancer cells can be classified based on those with high or low glycogen content as well as with high or low flux of glycogen metabolism, both in normal cell culture conditions and after hypoxia or other stress. This classification could be instrumental for the identification and the characterisation of cancer cells with increased glycogen requirements. The observation that glycogen accumulates under hypoxic conditions as a survival mechanism, suggests the idea that combination treatment of glycogen breakdown inhibition with anti-angiogenic drugs (e.g. bevacizumab) could provide a potent strategy and should be investigated in future. Moreover, autophagy potential contribution the glycogen breakdown suggests that blocking both degradation pathways could provide synergism as anti-cancer treatment. Glycogen breakdown supports the pentose phosphate pathway, which generates nucleotides required for proliferation and DNA repair, as well as NADPH, which is an important reducing agent for ROS scavenging and nucleotide, amino acid and lipid synthesis [91]. Considering this major role of glycogen metabolism in the pentose phosphate pathway, combination treatment of glycogen inhibition with DNA damaging agents, ROS generating agents, mitochondria inhibition agents, and ionising radiation might be a potent strategy for anticancer treatment. Acknowledgments C.E.Z. is a Marie Curie Fellow (IEF), FP7-People-2012-IEF-330071. A.L.H is funded by Cancer Research UK (C602/A11359), the Breast Cancer Research Foundation Oxford NHS Biomedical Research Centre (A93022), CRUK Imaging and Cancer Centre (C5255/A16466). E.F. is the recipient of a Federation of European Biochemical Societies (FEBS) Fellowship.

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