Anticancer targets in the glycolytic metabolism of tumors: a comprehensive review

Abstract

CANCER IS A METABOLIC DISEASE AND THE SOLUTION OF TWO METABOLIC EQUATIONS: to produce energy with limited resources and to fulfill the biosynthetic needs of proliferating cells. Both equations are solved when glycolysis is uncoupled from oxidative phosphorylation in the tricarboxylic acid cycle, a process known as the glycolytic switch. This review addresses in a comprehensive manner the main molecular events accounting for high-rate glycolysis in cancer. It starts from modulation of the Pasteur Effect allowing short-term adaptation to hypoxia, highlights the key role exerted by the hypoxia-inducible transcription factor HIF-1 in long-term adaptation to hypoxia, and summarizes the current knowledge concerning the necessary involvement of aerobic glycolysis (the Warburg effect) in cancer cell proliferation. Based on the many observations positioning glycolysis as a central player in malignancy, the most advanced anticancer treatments targeting tumor glycolysis are briefly reviewed.

Keywords: HIF-1; Warburg effect; biosynthesis; cancer; cataplerosis; glycolytic switch; hypoxia; metabolism.

Figure 1

Allosteric regulations of glycolysis confer metabolic plasticity with respect to local pO₂. Enzymes are represented in italicized blue font and their substrates in bold black. Because the glycolytic flux is nominally faster than OXPHOS, the Pasteur Effect has been evolutionary selected to couple both metabolic rates. The energy metabolites glucose-6-P, ATP, and citrate restrain the glycolytic flux through allosteric inhibition of key glycolytic enzymes, as represented by the red arrows. Inhibition is at its climax when oxygen is not a limiting substrate for OXPHOS, thus allowing the full oxidation of glucose. When oxygen levels are limited or when the pO₂ fluctuates, full glucose oxidation, and consequently the levels of ATP and citrate produced oxidatively are decreased. The Pasteur Effect is reset to less pronounced inhibition, thus allowing accelerated glycolysis to compensate for defective ATP production. An extreme situation characterized by full inhibition of the Pasteur Effect is met under severe hypoxia. The energetic crisis is associated with an increase in the cellular levels of fructose-1,6-bisP, ADP, AMP, and inorganic phosphate (Pi). These molecules exert a series of allosteric stimulations (represented by the green arrows) that accelerate the glycolytic flux. Glycolysis thus becomes the main source of cellular ATP production, a rescue situation allowing short-term cell survival until the pO₂ is restored. Other abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; P, phosphate; PPP, pentose phosphate pathway; TCA, tricarboxylic acid (cycle).

Figure 2

Simplified scheme depicting HIF-1 activation by hypoxia. Both HIF-1α and HIF-1β subunits are constitutively transcribed, but only the HIF-1β protein is stably expressed into the cell nucleus. HIF-1 activity primarily depends on the stability of the HIF-1α protein. Under normoxia, HIF-1α is hydroxylated at two proline residues by the prolylhydroxylase PHD2, and addressed to the von Hippel–Lindau (VHL) complex for ubiquitylation (Ub) and further proteasome-mediated degradation. Oxygen is a limiting substrate for the PHD2 reaction. Under hypoxia, the HIF-1α protein is expressed, migrates to the nucleus, and binds to HIF-1β, the adaptator p300, DNA polymerase II, and to the consensus DNA motif hypoxia-responsive element (HRE) in the promoting region of target genes. HIF-1-target gene products promote glycolysis, angiogenesis, and erythropoiesis, and regulate vasomotion. Adapted from Harris (2002).

Figure 3

HIF-1 promotes the expression of glycolytic enzymes and transporters. Enzymes are represented in italicized blue font and their substrates in bold black. Green arrows point at HIF-1-target gene products directly involved in the acceleration of the glycolytic flux. In the simplified scheme, only those that have been identified as potential therapeutic targets are highlighted. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT, glucose transporter; MCT4, monocarboxylate transporter 4; P, phosphate; PPP, pentose phosphate pathway; TCA, tricarboxylic acid (cycle).

Figure 4

Lactate dehydrogenases. In eukaryote cells, lactate dehydrogenases (LDHs) are tetrameric enzymes catalyzing the reversible reduction of pyruvate into lactate. The LDH-B gene is constitutively transcribed and encodes subunit LDH-H, whereas transcription of the LDH-A gene, which is inducible by hypoxia due to the presence of a consensus HIF-1-binding motif (hypoxia-responsive element, HRE), encodes the LDH-M subunit. Arrangement of the subunits to forms active tetramers may lead to the formation of five distinct enzymes, LDH1 to LDH5. Compared to LDH-H, LDH-M has a higher K_m and a higher V_max for pyruvate reduction. Consequently, LDH5/LDH-4M preferentially catalyzes the reduction of pyruvate into lactate, LDH1/LDH-4H preferentially catalyzes the oxidation of lactate into pyruvate, and LDH2, LDH3, and LDH4 have intermediate enzymatic activities.

Figure 5

Model according to which tumors behave as metabolic symbionts. Tumor heterogeneity includes metabolism. At a remote location from perfused blood vessels, hypoxic tumors cells rely on glycolysis for survival and proliferation. High ATP production mandatorily depends on high glucose availability and is associated with the release of lactate, a process facilitated by monocarboxylate transporter 4 (MCT4). In contrast, although they also express glucose transporters (GLUT), oxygenated tumor cells have a metabolic preference for lactate versus glucose. MCT1 is a transporter is adapted for lactate uptake (Sonveaux et al., 2008). In the presence of oxygen, lactate is oxidized to pyruvate by lactate dehydrogenase 1 (LDH1) and pyruvate fuels the tricarboxylic acid (TCA) cycle to produce ATP. The metabolic preference of oxidative tumor cells for lactate allows hypoxic tumor cells to get access to high levels of glucose. This metabolic cooperativity is key for tumor cell survival under hypoxia in vivo.

Figure 6

Major cellular pathways involved in the cellular export of protons. The maintenance of high-rate glycolysis requires the export of protons that would otherwise create intracellular acidification leading to cell death. Monocarboxylate transporter 4 (MCT4) is a passive lactate (Lac⁻)-proton symporter adapted for the export of protons. Carbonic anhydrase-9 (CA9) is a transmembrane enzyme promoting the reversible hydratation of CO₂. Acidity is exported under the form of cell-permeable CO₂, followed by CA9-facilitated hydratation of CO₂ to carbonic acid. Carbonic acid then readily dissociates to proton and bicarbonate extracellularly. Bicarbonate may be recaptured by the sodium bicarbonate cotransporter (NBC) to react with a proton intracellularly. Then, bicarbonate gets dehydrated to yield CO₂ for export. V-ATPase is expressed at the plasma membrane of several tumor cell types where it acts as a proton pump fueled by ATP. The sodium–proton exchanger 1 (NHE1) is a passive proton–sodium antiporter. The sodium–potassium (NaK) ATPase promotes the export of sodium that would otherwise accumulate as a consequence of NHE1, CA9, and NBC activities. MCT4 is thus the only truly passive system for proton export. Other abbreviation: Pi, inorganic phosphate.

Figure 7

Simplified scheme highlighting glycolysis as a biosynthetic hub. Enzymes are represented in italicized blue font and their substrates in bold black. Tumor cell proliferation relies on aerobic glycolysis for ATP production and also to redirect carbohydrates toward biosynthetic routes. When allosterically activated by fructose-1,6- bisphosphate (F1,6BP), pyruvate kinase M2 (PKM2) promotes pyruvate (and ATP) synthesis. Pyruvate fuels either the production of lactate (for NAD⁺ production further supporting glycolytic ATP production), alanine [through the reversible alanine aminotransferase (ALAT) reaction during which a nitrogen group is transferred from glutamate onto pyruvate to yield alanine], and/or it can be used to replenish the tricarboxylic acid (TCA) cycle. The fate of pyruvate is oriented by the activity of pyruvate dehydrogenase kinase 1 (PDK1) repressing pyruvate dehydrogenase (PDH) in the mitochondrion. When PKM2 is off (for example when accumulating alanine allosterically promotes dimer formation), glucose-6-P is directed toward the pentose phosphate pathway (PPP) to yield ribulose-5P (for DNA synthesis) and NADPH (2 molecules per molecule of G6P). In addition to glucose, glutamine is an important source of organic acids for proliferating cells. Glutamine uptake is mediated by glutamine transporters (GLT) and glutaminases present in the cytosol or in the mitochondrion generate glutamate. Glutamate fuels the TCA cycle and is also a nitrogen donor for alanine synthesis (ALAT reaction). Uncoupled mitochondria are major providers of biosynthetic precursors and produce reactive oxygen species (ROS). During cataplerosis, citrate, and isocitrate are exported to fuel lipogenesis, malate is exported and converted into pyruvate for NADPH production by the malic enzyme (ME), and glutamate may serve to regenerate glutamine as a precursor for amino-acid synthesis or to be exchanged against extracellular amino-acids. NADPH, produced either from the PPP or from malate, has two main roles: it is a necessary cofactor for lipogenesis (HMG-CoA reductase step) and it is used as a reductant for the regeneration of glutathione (GSH) from its oxidized disulfide form (GSSG). As an anti-oxidant, GSH detoxifies ROS. Other abbreviations: GLUT, glucose transporter; IDH, isocitrate dehydrogenase; MCT, monocarboxylate transporter.