Mitochondria and cancer

Abstract

Contrary to conventional wisdom, functional mitochondria are essential for the cancer cell. Although mutations in mitochondrial genes are common in cancer cells, they do not inactivate mitochondrial energy metabolism but rather alter the mitochondrial bioenergetic and biosynthetic state. These states communicate with the nucleus through mitochondrial 'retrograde signalling' to modulate signal transduction pathways, transcriptional circuits and chromatin structure to meet the perceived mitochondrial and nuclear requirements of the cancer cell. Cancer cells then reprogramme adjacent stromal cells to optimize the cancer cell environment. These alterations activate out-of-context programmes that are important in development, stress response, wound healing and nutritional status.

Figure 1. Mitochondrial genome and mitochondrial biogenesis

The mitochondrial genome encompasses between one and two thousand nuclear-DNA-encoded mitochondrial genes and thousands of copies of the mitochondrial DNA (mtDNA). mtDNA has a high mutation rate, and de novo mtDNA mutations create a mixture of mutant and normal mtDNAs in cells, a state known as heteroplasmy. As the proportion of mutant mtDNAs increases, the energy output capacity of the cell declines until there is insufficient energy to sustain cellular function, termed the bioenergetic threshold. Mitochondria also constantly undergo fusion and fission, which permits complementation of mtDNAs in trans^,,,,. The mtDNA encodes 13 proteins, 22 tRNAs, and 12S and 16S rRNAs. The mtDNA is packaged in the nucleoid and is replicated by DNA polymerase-γ (pol-γ). It is transcribed by mitochondrial RNA polymerase (RNA pol) symmetrically from both stands as large polycistron transcripts in which the larger transcripts are punctuated by the tRNAs. Cleavage of the tRNAs out of the polycistron transcripts creates the mature rRNAs and mRNAs, which are then translated on mitochondrial-specific chloramphenicol- sensitive ribosomes, in which the polypeptides are initiated by N-formyl methionine. The mtDNA encodes seven (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6) of the 45 polypeptides of complex I; cytochrome b from the 11 polypeptides of complex III; three (cytochrome oxidase I (COI), COII and COIII) of the 13 polypeptides of complex IV; and two (ATP6 and ATP8) of the approximately 17 polypeptides of complex V. These proteins are central electron and proton carriers of the proton-transporting complexes and thus form the wiring diagram for oxidative phosphorylation (OXPHOS). All of the remaining mitochondrial proteins, including approximately 80 OXPHOS subunits and all four subunits of the non-proton-pumping complex II, are encoded by nuclear DNA (nDNA). The mRNAs from the nDNA-encoded subunits are translated on cytosolic ribosomes and the proteins are imported into the mitochondrion by transport through the outer (TOM) and inner (TIM) membrane complexes. TFAM, mitochondrial transcription factor A.

Figure 2. Mitochondrial bioenergetics and cancer cell mutations

Pyruvate from glycolysis is converted to acetyl-CoA, CO₂ and NADH by pyruvate dehydrogenase (PDH). PDH can be inactivated through phosphorylation by PDH kinase (PDHK) and reactivated through dephosphorylation by PDH phosphatase (PDHP). Acetyl-CoA enters the tricarboxylic acid (TCA) cycle by the citrate synthase (CS)-mediated reaction with oxaloacetate (OAA) to generate citrate. Citrate carbons then pass through the TCA cycle via the enzymes aconitase, isocitrate dehydrogenase (IDH) isoforms (IDH2 or IDH3), α-ketoglutarate dehydrogenase (αKDH), succinyl-CoA synthetase (SCS), succinate dehydrogenase (SDH; also known as complex II), fumarate hydratase (FH) and malate dehydrogenase (MDH). Cancer mutations have been identified in IDH2, SDH, and FH (shown as red ovals). FH defects are associated with induction of the haemoxygenase 1 (HMOX1), which degrades haem. Haem is synthesized by the condensation of succinyl-CoA and glycine by δ-aminolevulinic acid (ALA) synthetase (ALAS) to generate ALA. Succinyl-CoA conversion to succinate generates GTP, which can drive the condensation of OAA and CO₂ to phosphoenolpyruvate (PEP) by mitochondrial PEP carboxykinase (PEPCK-M). PEP can then be exported to the cytosol. NADH is generated by PDH, IDH3, αKDH and MDH, and can be oxidized by the electron transport chain (ETC). The ETC encompasses five multi-subunit complexes I–IV. NADH is oxidized by complex I (NADH dehydrogenase), and electrons from complexes I and II are transferred to coenzyme Q₁₀ (CoQ), then passed on to complex III (also known as the b-c1 complex), cytochrome c (cytc), complex IV (also known as cytochrome c oxidase (COX)), and finally to O₂ (half a molecule of O₂ per electron pair) to generate H₂O. As the electrons traverse complexes I, III and V, protons are pumped out across the mitochondrial inner membrane to generate the electrochemical gradient (ΔP = ΔΨ + Δμ^H+). ΔP is then used by complex V (H⁺-translocating ATP synthase) to condense ADP and inorganic phosphate (P_i) to ATP. The ADP and ATP are exchanged across the mitochondrial inner membrane by adenine nucleotide translocators (ANTs)^,,,. Cancer cell mitochondrial DNA (mtDNA) mutations have been reported in genes for complexes I, III, IV and V (shown as red squares). Hence, many of the mitochondrial gene mutations in cancer are intimately associated with oxidative phosphorylation (OXPHOS) and the redox regulation of reactive oxygen species (ROS).

Figure 3. Mitochondrial physiology

Mitochondria lie at the nexus of most biosynthetic pathways, produce much of the cellular energy through oxidative phosphorylation (OXPHOS), regulate mitochondrial and cellular redox status, generate most of the reactive oxygen species (ROS), regulate Ca²⁺ concentrations and can initiate apoptosis by the activation of the mitochondrial permeability transition pore (mtPTP). The mtPTP can be activated by a decreased membrane potential, high-energy phosphates (such as ADP), a more-oxidized redox status, and/or increased mitochondrial matrix Ca²⁺ and ROS concentrations. Reducing equivalents and acetyl-CoA enter the mitochondrion via pyruvate and fatty acids. Pyruvate is transported through the mitochondrial inner membrane by the pyruvate transporter (PT), binds to pyruvate dehydrogenase (PDH), which may be membrane-associated, and is oxidatively decarboxylated to produce acetyl-CoA. Inhibition of mitochondrial function results in pyruvate accumulation in the cytosol, where it can be reduced to lactate. Fatty acids are imported into the mitochondrion bound to carnitine. In the cytosol, fatty acids bound to CoA are transferred to carnitine, transported through the outer and inner mitochondrial membranes, and then transferred back to CoA for β-oxidation. The transfer of fatty acyl groups between CoA and carnitine is mediated by the carnitine palmitoyltransferases (not shown). As a by-product of OXPHOS — the substrates and products of which are transported through the outer membrane by the voltage-dependent anion channels (VDACs) — mitochondria generate ROS by the donation of excess electrons from complexes I and III directly to O₂ to generate superoxide anions (O₂^•−). Matrix O₂^•−, primarily from complex I, is dismutated to H₂O₂ by the mitochondrial matrix Mn superoxide dismutase (MnSOD; also known as SOD2), while intermembrane-space O₂^•−, which is primarily from complex III, is dismutated by Cu/Zn superoxide dismutase (Cu/ZnSOD; also known as SOD1). H₂O₂ can be reduced to water by glutathione peroxidase using reduced glutathione as an electron donor. Oxidized glutathione is reduced by glutathione reductase using NADPH as a reductant. In the presence of reduced transition metals, H₂O₂ can be reduced to hydroxyl radicals (^•OH), which are the most reactive ROS. The mtPTP is a protein complex that is thought to include the translocator protein (TSPO; also known as PBR), an unknown inner-membrane channel, adenine nucleotide translocators (ANTs) and the cyclosporine-A-sensitive cyclophilin D (CYPD; also known as PPID), which are regulatory, in association with the BCL-2 pro- and anti-apoptotic family members. When activated, the mtPTP forms a channel between the inner and outer membranes, which short-circuits ΔP. This is associated with the aggregation of BAX and BAD in the mitochondrial outer membrane to form a megachannel. The megachannel releases pro-apoptotic proteins from the intermembrane space into the cytosol to initiate the degradation of the cellular proteins and DNA^,,,. AIF, apoptosis-inducing factor; CoA-SH, coenzyme A with a free sulphydryl group; CoQ, coenzyme Q₁₀; ENDOG, mitochondrial endonuclease G; GPX, glutathione peroxidase; LDH, lactate dehydrogenase; OAA, oxaloacetate; SMAC, second mitochondria-derived activator of caspase; TCA, tricarboxylic acid. Modified, with permission, from REF. © (2005) Cold Spring Harbor Laboratory Press.

Figure 4. The mitochondrial NADPH shuttle system, and IDH 1 and IDH2 mutations

The mitochondrion can generate NADPH by the transfer of reducing equivalents from NADH to NADP⁺. This process is mediated by the mitochondrial inner membrane nicotinamide nucleoside transhydrogenase (NNT), which exploits ΔP to provide the additional reducing potential energy. NADH can either be generated within the mitochondrion or can be imported from the cytosol by the aspartate–glutamate and malate–α-ketoglutarate (αKG) shuttle system. Within the mitochondrion, NADPH can be used to reduce glutathione and thus to control mitochondrial reactive oxygen species (ROS) signalling. Alternatively, NADPH can be used to reduce mitochondrial thioredoxin 2 (TRX2), which regulates the thio-disulphide redox state of mitochondrial proteins. However, NADPH can energize the mitochondrial NADP⁺-dependent isocitrate dehydrogenase 2 (IDH2) to reductively carboxylate αKG to isocitrate. Isocitrate can then be converted to cis-aconitate and then citrate by mitochondrial aconitase. Citrate can be exported across the mitochondrial inner membrane into the cytosol by the citrate carrier where it can be converted to cis-aconitate and isocitrate by cytosolic aconitase. Cytosolic isocitrate can then be oxidatively decarboxylated by cytosolic NADP⁺-linked IDH1, producing cytosolic NADPH. The resulting αKG or its aminated derivative glutamate can then be recycled back into the mitochondrion. In the cytosol, the NADPH can be used to reduce glutathione for antioxidant defences, or the reducing equivalents can be funnelled through the nuclear–cytosol TRX1 (for which the reduced (SH₂) and oxidized (SS) forms are shown) and then through the bifunctional apurinic/apyrimidinic endonuclease 1 (APE1; also known as redox factor 1(REF1)) protein to reduce thiols in cytosolic and nuclear proteins, including the FOS and JUN transcription factors. Oncogenic mutations in IDH1 or IDH2 (shown as the red ovals with the asterisk) can result in a neomorphic function such that the αKG and NADPH generated by the wild-type IDH1 and IDH2 enzymes (shown as blue ovals) is converted to R(-)-2-hydroxyglutarate ((R)-2HG) and NADP⁺. This would deplete NADPH, thus increasing ROS production and altering the regulation of nuclear transcription factors^,. AGA, aspartate–glutamate antiporter; GSH, reduced glutathione monomer; GSSG, oxidized glutathione dimer; MαA, malate–αKG antiporter; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PKM, pyruvate kinase isoform M.