American Journal of Clinical Oncology:
Targeting Glucose Metabolism: An Emerging Concept for Anticancer Therapy
Madhok, Brijesh M. MBBS, MS, MRCS; Yeluri, Sashidhar MBBS, MS, MRCS; Perry, Sarah L. BSc(Hons); Hughes, Thomas A. BA(Hons), DPhil; Jayne, David G. MD, FRCS
Leeds Institute of Molecular Medicine, University of Leeds, Leeds, United Kingdom
The authors declare no conflicts of interest.
Reprints: Brijesh M. Madhok, MBBS, MS, MRCS, Section of Translational Anaesthesia and Surgery, Level 7 Clinical Sciences Building, St. James's University Hospital, LS9 7TF Leeds, United Kingdom. e-mail: firstname.lastname@example.org.
Mortality from locally advanced and metastatic cancer remains high despite advances in our understanding of the molecular basis of the disease and improved adjuvant therapies. Recently, there has been an increased interest in cancer metabolomics, and in particular, the potential for targeting glucose metabolism, for therapeutic gain. This interest stems from the fact that cancer cells metabolize glucose very differently from normal cells. Cancer cells preferentially switch to aerobic glycolysis rather than oxidative phosphorylation as their means of glucose metabolism. This metabolic switch is believed to enhance cancer cell survival. Several therapeutic agents that target tumor metabolism have shown significant cancer cell cytotoxicity in preclinical studies, and some have progressed to clinical trials. In this review, we discuss the alteration of carbohydrate metabolism seen in cancer cells, the underlying mechanisms, and opportunities for targeting cancer metabolism for therapeutic purposes.
CARBOHYDRATE METABOLISM IN CANCER CELLS: A WEALTH OF POTENTIAL DRUG TARGETS
Normal cells rely on glycolysis for the production of adenosine 5′-triphosphate (ATP) in hypoxic conditions, but cancer cells are highly dependent on glycolysis even under normoxia. This phenomenon of “aerobic glycolysis” was first discovered by Otto Warburg in the 1920s, and hence it is known as the “Warburg effect.”1 Warburg suggested that aerobic glycolysis was a consequence of attenuated mitochondrial function in cancer cells, and hence they switch to glycolysis for energy production. He opined that mitochondrial dysfunction was responsible for the origin of cancer. There is now mounting evidence that cancer cells undergo increased glycolysis and reduced oxidative phosphorylation.2,3 Mechanisms underlying the Warburg effect seem to be multifacto-rial,4 with factors such as the hypoxic tumor microenvironment,5 hypoxia inducible factor 1 (HIF1),6,7 mitochondrial dysfunction,8 p53 mutations,9 and oncogenes10,11 playing important roles. Intriguingly, recent research indicates that pyruvate kinase, which regulates the final rate-limiting step of glycolysis plays a key role in the Warburg phenotype. In particular, increased expression of the M2 isoform of pyruvate kinase and its phosphorylation mediated by tyrosine kinase have been shown to promote aerobic glycolysis in tumors.12,13 This shift in basic glucose metabolism results from changes in expression or activity of a wide range of metabolic enzymes and other glucose-related molecules. Changes include increased expression of glucose transporters (GLUT) facilitating increased uptake of glucose from the extracellular environment, increased expression of most of the enzymes of glycolysis leading to increased glycolytic activity, and increased expression of pyruvate dehydrogenase kinase (PDK) resulting in reduced entry of pyruvate into oxidative phosphorylation via suppression of pyruvate dehydrogenase (PDH) activity.14,15 These changes give the cancer cells a selective growth advantage and favor cancer progression by downregulating apoptotic pathways (Fig. 1), and cause acidification of the extracellular environment, which is both toxic to the noncancer cells and enhances cancer-cell invasion and metastasis.5,16
The metabolic differences between cancer cells and normal cells present 2 potential therapeutic strategies. The first is to inhibit any of the glycolytic enzymes, the assumption being that cancer cells are more dependent on their activity for production of ATP than normal cells, and as a result the treatment would preferentially target the cancer cells. The second is to promote oxidative phosphorylation, with the intention of reactivating metabolic pathways that have been downregulated in cancer cells and reactivating apoptotic mechanisms.
A further possibility is to influence specific upstream regulators that are responsible for these metabolic changes, and thereby simultaneously inhibit glycolysis and promote oxidative phosphorylation. The metabolic changes are induced directly or indirectly by deregulated expression of various oncogenes (eg, c-myc and akt) or loss of tumor suppressor activity (eg, p53).9,10,17 However, each of these also influences many other aspects of cancer cell behavior and it is unclear whether the metabolic changes are a cause or an effect of their oncogenic influence. In contrast, 1 upstream factor, HIF1, directly regulates the expression of many of these key metabolic enzymes.6 Solid tumors are known to have increased expression of the HIFl α subunit,18,19 and it is thought that this has a substantial controlling influence on their metabolism. HIF1 itself represents a drug target which potentially has a diverse impact on metabolism.
Below, we describe strategies employed to manipulate the activity of individual proteins involved with glucose metabolism, under the headings of inhibition of glucose metabolism, promotion of oxidative phosphorylation, and inhibition of HIF1.
Inhibitin of Glycolysis
Inhibiting glycolysis may deprive cancer cells of ATP and curtail tumor growth. This effect has been demonstrated in vitro, and some glycolytic inhibitors are in phase II/III clinical trials. Importantly, these drugs appear to be more effective in terms of killing tumor cells in conditions of tumor hypoxia, which is usually associated with resistance to conventional chemotherapy or irradiation. Strategies to inhibit most of the individual enzymes of glycolysis have been attempted, and are described below. The role of each enzyme in the pathway is depicted in Figure 2.
Lonidamine (LND) was one of the first antiglycolytic drugs to be tested as a cancer therapeutic. It acts as a selective inhibitor of aerobic glycolysis in cancer cells, due to its inhibitory effects on mitochondrial bound hexokinase,20 although the mechanisms behind this remain poorly understood. Preclinical studies demonstrated that LND enhanced the cytotoxic effects of traditional chemotherapeutic agents,21,22 and it is in this context that LND entered clinical trials. Initial studies showed that LND could increase response rates to doxorubicin in liver metastases from breast cancer,23 however, 2 large phase III clinical trials failed to reproduce this.24,25 Similarly, LND gave no significant benefit in terms of disease-free or overall survival from early stage breast cancer when combined with Epirubicin and Cyclophosphamide.26 LND did, however, improve response rates and overall survival when combined with mitomycin-C or vindesine for treatment of non–small-cell lung cancer.27 Early phase trials have also been carried out for advanced ovarian cancer and have demonstrated that LND is safe and well tolerated when combined with conventional chemotherapy.28,29 Use of LND has now been approved in Europe in some cancer chemotherapy protocols,30 although this is not common practice.
2-Deoxyglucose (2-DG) is a glucose analogue that competitively inhibits hexokinase activity by acting as an alternative substrate. 2-DG is phosphorylated by hexokinase to form 2-DG-P, which cannot be metabolized further. 2-DG has been shown to deplete cellular ATP and cause cell death in hypoxic tumors31 while additionally, in normoxia, cancer cells apparently undergo cell death by an alternative mechanism involving changes to N-linked glycosylation patterns.32 Following promising results from many experimental studies in which 2-DG reduced cancer cell growth,33–36 2-DG has advanced into phase I/II clinical trials. Initial data demonstrate that 2-DG is well-tolerated at doses that were effective in animal models37,38 and further early phase trials have either been recently completed (ClinicalTrials.gov identifier: NCT00096707) or are underway (NCT00633087, NCT00247403). As yet, there are very few published data on the efficacy of 2-DG in clinical cancer therapy although initial results indicate that the combination of 2-DG with docetaxel is safe, and 2-DG has some antitumor effects in both breast cancer and head and neck tumors.39
3-Bromopyruvate (3-BrPA) is a lactate analogue that, unlike 2-DG, directly inhibits hexokinase by alkylation of its sulfhydryl groups.40 3-BrPA is reported to be a more potent hexokinase inhibitor, being effective in μM concentrations, as compared with 2-DG that is only effective at mM concentrations. In addition, 3-BrPA may exhibit an extra level of cancer specificity since it is taken up by lactate transporters that are frequently overexpressed in cancer cells.41,42 Accordingly, studies have demonstrated that it leads to ATP depletion and cell death in malignant cells.4 However, these promising results must be tempered with the knowledge that 3-BrPA is a strong alkylating agent that is likely to interact with molecules other than hexokinase, hence it is certainly not a highly-specific hexokinase inhibitor.43 Despite this concern, 3-BrPA has shown very encouraging anticancer activity as a single agent in a range of animal models bearing hepatocellular and breast cancer.44–46 In addition, in animal tumor models, 3-BrPA appears to synergize with some established anticancer agents, including Geldanamycin, an inhibitor of heat shock protein 90, and cisplatin, adeoxyribonucleic acid (DNA) damaging agent.47,48 Despite this wealth of encouraging data, 3-BrPA has not progressed to clinical trials, which may reflect concerns regarding its potential adverse effects.
Other molecules known to inhibit hexokinase, such as 5-thio-glucose, Mannoheptulose, and methyl jasmonate are currently being investigated for their anticancer effects.43,49
Glucose-6-phophate isomerase (GPI) is an especially interesting component of the glycolytic cascade as, in addition to its enzymatic role, it also functions as a secreted ligand, autocrine motility factor (AMF), that can regulate proliferation and invasion.50 Expression of GPI/AMF and its receptor AMFR is often increased in cancer, and is associated with poor prognosis.51,52 Downregulation of GPI/AMF expression using small interfering ribonucleic acid (siRNA) in human fibrosarcoma cells in culture sensitized the cells to oxidative stress leading to cellular senescence and reduced proliferation.53 Importantly, these results were reproduced by treatment of the cells with the GPI or AMF inhibitor, Erythrose 4-Phosphate. Several compounds, such as D-fructose-6-phosphate, 6-phosphogluconic acid and N-bromoacetyl-aminoethyl phosphate, have been suggested as potential GPI inhibitors.43
Control of 6-phosphofructo-l-kinase (PFK-1) activity is thought to have a key regulatory influence on glycolysis and therefore PFK-1 represents an attractive drug target. PFK-1 activity is believed to be indirectly upregulated in cancer cells as a result of overexpression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphotase (PFKFB). PFKFB is overexpressed in a variety of highly aggressive human carcinomas54 leading to high levels of fructose-2,6-biphosphate, which activate PFK-1 allosterically.55 The small molecule, 3PO [3-(3-pyridinyl)-l-(4-pyridinyl)-2-propen-l-one] was recently established as an inhibitor of PFKFB, leading to suppression of glycolysis and tumor growth in several cancer cells in vitro and in xenograft models of lung and breast cancer.56
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in various nonmetabolic functions in addition to its glycolytic role; these include DNA repair, regulation of transcription, and prevention of caspase-independent cell death. As with GPI, these extra functions are thought to contribute to cancer survival and growth making the molecule additionally attractive as a drug target.43,57 Lai et al demonstrated that iodoacetate, an inhibitor of GAPDH, reduced cell survival in several cancer cell lines.58 In addition, downregulation of GAPDH expression using ribonucleic acid interference sensitized resistant leukemic cells to imatinib and prednisolone.59,60 One concern with iodoacetate is that it is a strong alkylating agent, and hence may not be a selective inhibitor of GAPDH.49 Other compounds that have been shown to have anti-GAPDH activity are koningic acid, α-chlorohydrin, and ornidazole.4,61
Three major isozymes of enolase have been identified in mammals: α, β and γ. Downregulation of α-enolase expression employing siRNA has been shown to improve the cytotoxic effects of tamoxifen, even in tamoxifen-resistant breast cancer cells.62 Another study reported α-enolase to be a novel pancreatic ductal adenocarcinoma-associated antigen, and a-enolase specific T-cells reduced growth of pancreatic ductal adenocarcinoma cells in immunodeficient mice.63 Importantly, several compounds are currently being evaluated as potential enolase inhibitors. These include sodium fluoride, phosphonoacetohydroxamic acid, and 2-phospho-D-glyceric acid.43
Various compounds such as fluorophosphates, creatine phosphate, oxalate, L-phospholactate, and some phosphoenolpyruvate analogues with modified phosphate and carboxylate groups have been described as experimental inhibitors of pyruvate kinase (PK).43,64 The most widely tested PK inhibitor, TT-232, is a structural analogue of Somatostatin, which has been shown to reduce proliferation of various human malignancies both in vitro and in vivo.65,66 At least 2 phase II trials examining this PK inhibitor (TLN-232) are registered on the clinicaltrials.gov website: one in patients with recurring metastatic melanoma is currently suspended due to an ongoing dispute with the licensor (NCT00735332), however another phase II study in patients with refractory metastatic renal cell carcinoma has been completed (NCT00422786). Initial results indicate that TLN-232 is well tolerated without any clinically significant adverse effects, and is likely to have antitumor activity in patients with metastatic melanoma and advanced renal cell carcinoma.67
Inhibition of the Pentose Phosphate Pathway
Once glucose has been phosphorylated to glucoses-6-phosphate in the first step of glycolysis, it may undergo further metabolism through the pentose phosphate pathway. Thus, the pentose phosphate pathway is an alternative to glycolysis that generates NADPH and pentoses; NADPH plays an important role in preventing oxidative stress and pentoses are required for nucleic acids synthesis. Both NADPH and pentoses are needed in abundance by the rapidly proliferating cancer cells, and indeed tumor cells are known to upregulate their pentose phosphate pathway by increased expression of 2 key enzymes: transketolase, which controls the nonoxidative part of the pathway, and glucose-6-phosphate dehydrogenase (G6PD), which controls the oxidative part.68,69
Overexpression of one of the transketolase isoforms, Transketolase-like protein 1 (TKTL1), is associated with higher tumor grades and worse patient survival,70,71 whereas downregulation of TKTL1 by ribonucleic acid interference has been shown to inhibit tumor growth in vitro.71 Oxythiamine, a thiamine antagonist and inhibitor of transketolase, has been reported to have anticancer effects both in vitro and in vivo.72,73 Downregulation of G6PD with short hairpin ribonucleic acid has been shown to reduce cancer cell proliferation, enhance apoptosis, and render cancer cells more susceptible to oxidative stress.74 6-Aminonicotinamide (6-AN) inhibits G6PD, causes oxidative stress, and sensitizes human cancer cells to radiotherapy and Cisplatin.75,76 However 6-AN has significant neurotoxic effects, and hence is unlikely to be useful clinically.
Promotion of Oxidative Phosphorylation
The aim of promoting mitochondrial oxidative phosphorylation in cancer cells is to deviate the cells' metabolism away from dependence on glycolysis with the result of inducing apoptosis through the mitochondrial pathway. One of the ways this can be achieved is by inhibiting the enzyme lactate dehydrogenase (LDH), thus preventing conversion of pyruvate to lactate. This promotes conversion of pyruvate to acetyl CoA catalyzed by PDH, which can then enter oxidative phosphorylation. Downregulation of LDH-A by short hairpin ribonucleic acid has been shown to stimulate mitochondrial respiration, reduce proliferation of cancer cells in vitro under both normoxic and hypoxic conditions, and attenuate tumorigenicity.77 Another study recently confirmed that downregulation of LDH-A causes significant attenuation of tumor growth in a xenograft mouse model of renal cancer.78
An alternative strategy is to activate the enzyme PDH, thereby stimulating uptake of pyruvate into oxidative phosphorylation. PDH activity is regulated by a family of kinases, the PDKs, with hypophosphoryated PDH being highly active. In cancer cells, PDH is typically hyperphosphorylated and relatively inactive as a result of PDK over-expression.79 Downregulation of PDK activity with siRNA or with its pharmacological inhibitor dichloroacetate (DCA) has been shown to promote hypophosphorylation of PDH and consequently to increase mitochondrial oxidative phosphorylation, induce apoptosis, and inhibit cancer cell proliferation in vitro and in vivo.16,80,81 One study has reported that DCA inhibits proliferation in breast cancer cells, but does not to induce apoptosis,82 whereas in contrast, work in the authors' laboratory has shown that DCA reduces proliferation and induces apoptosis and growth arrest in colorectal cancer cells (Madhok et al unpublished data). Currently, there are 3 on-going phase I/II clinical trials evaluating the role of DCA in patients with recurrent and/or metastatic solid tumors, glioblastoma multiforme, and malignant gliomas (NCT00566410, NCT00703859, and NCT00540176).
Attenuation of HIF1 Activity
HTF1 is a critical upstream regulator of glucose metabolism, and its overexpression is thought to be a major event in the upregulation of glycolysis and downregulation of oxidative phosphorylation in many cancers. Therefore, many different approaches have been used in an attempt to downregulate HIF1 activity. Upregulation of HIF1 in cancer results from over activity of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways.83,84 These pathways have a wide range of cancer-related targets and although their inhibition may have an effect on HIF1 activity, it is likely that it would only be a contributory factor to any anticancer effects. For the purpose of this review, we have focused on therapeutic strategies that target HIF1 directly and modify glucose metabolism in the target cells, while referencing previously published reviews for a more complete description of HIF inhibitors.85–87
Topotecan was initially identified in a cell-based high-throughput screen for small molecule inhibitors of the HIF1 transcriptional activation pathway.88 Topotecan, a topoisomerase I inhibitor, inhibits HIFl α translation by a DNA damage-independent mechanism, and has been shown to have cytotoxic effects in glioblastoma xenograft mice.89,90 A recent study reported that the antitumor effects of topotecan are because of reduction in levels of HIF1 targeted genes, for example, GLUT, HK, PDK, and PFKFB, all of which are crucial in glucose metabolism, and the effects are augmented with concomitant use of bevacizumab, a humanized monoclonal antibody against VEGF.91 Topotecan is approved by FDA as second-line chemotherapy in patients with small-cell lung cancer or ovarian cancer. In addition, there are several on-going clinical trials registered on the clinical trials website to evaluate its role in various advanced cancers as a single agent, in combination therapy, or as a radiosensitizing agent.
Digoxin and other cardiac glycosides have been to found to inhibit HIFl α protein synthesis, causing reduced expression of HIFl α target genes such as HK and GLUT in cancer cells.92 Digoxin administration led to reduced tumor growth in mice bearing human tumor xenograft, indicating that digoxin, which has traditionally been used for treatment of cardiac failure for decades, might have a new role in cancer therapy. Currently, there is an ongoing phase II study evaluating the potential benefit of adding digoxin to erlotinib in patients with non-small-cell lung cancer (NCT00281021). Another inhibitor of HIFl α activity, PX-478, has been reported to have significant antitumour activity in xenograft mice bearing different types of human cancers by inhibiting expression of HIF1 target genes including GLUT.93 Currently, it is in a phase I clinical trial in patients with advanced solid tumors or lymphoma (NCT00522652).
Another compound, YC-1 that is a soluble guanylyl-cyclase stimulator, has been reported to have antitumour activity by inhibiting HIFlα stability and reducing expression of HEF1 inducible genes including glycolytic enzymes, aldolase, and enolase, in xenograft mice bearing various types of human cancers.94,95 In addition to attenuating glycolysis, inhibition of HIF has also been shown to promote mitochondrial oxidative phosphorylation in tumor cells. Treatment of xenograft mice bearing human cancer with HIF1 inhibitor, echinomycin, increased tumor oxygen consumption, which led to increased tumor hypoxia and improved response to hypoxia-specific cytotoxin tirapazamine (TPZ) (Table 1).96 However, earlier clinical trials with echinomycin failed to reveal significant cytotoxic effects in patients with various types of cancer.86
The manipulation of cellular glucose metabolism is an emerging and attractive area for the development of novel anticancer therapies. Recently many anticancer agents, which target specific molecules in the glycolytic pathway, have been developed or proposed. Some of these have progressed into clinical trials, but to date they have yet to be incorporated into routine clinical practice. Further research into their mechanisms of action and their influence on normal cells, particularly those cells that rely on glycolysis, such as neurons, erythrocytes, and skeletal muscle cells, is required. It is also possible that targeting a single pathway may not be sufficient and cancer cells may switch to alternative sources for energy production, such as fatty acid oxidation.97 Fatty Acid Synthase has been shown to be highly expressed in various cancers and its inhibition has cytotoxic effects.98 There is also evidence that cancer cells can switch metabolism depending on substrate availability.99 Hence, a combination of metabolic drugs targeting different pathways might be needed to avoid resistance to a single agent. Nevertheless, targeting tumor metabolic pathways for therapeutic exploitation is promising and may form an important component of anticancer treatment in the near future.
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