Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles' heel?

Abstract

Mitochondria cooperate with their host cells by contributing to bioenergetics, metabolism, biosynthesis, and cell death or survival functions. Reactive oxygen species (ROS) generated by mitochondria participate in stress signalling in normal cells but also contribute to the initiation of nuclear or mitochondrial DNA mutations that promote neoplastic transformation. In cancer cells, mitochondrial ROS amplify the tumorigenic phenotype and accelerate the accumulation of additional mutations that lead to metastatic behaviour. As mitochondria carry out important functions in normal cells, disabling their function is not a feasible therapy for cancer. However, ROS signalling contributes to proliferation and survival in many cancers, so the targeted disruption of mitochondria-to-cell redox communication represents a promising avenue for future therapy.

Figure 1. Mitochondrial bioenergetic function

Pyruvate enters mitochondria via the mitochondrial pyruvate carrier (MPC), where it is decarboxylated and oxidized to acetyl-CoA by pyruvate dehydrogenase (PDH). Acetyl-CoA enters the tricarboxylic acid (TCA) cycle at citrate synthase. Subsequent steps in the cycle lead to the generation of reducing equivalents (NADH and NADPH) at dehydrogenase steps. Amino acids can also enter the TCA cycle by conversion to α-ketoglutarate, succinyl-CoA, fumarate, oxaloacetate or acetyl-CoA. For example, glutamine enters the TCA cycle after conversion to glutamate and subsequently to α-ketoglutarate. Reducing equivalents generated by the TCA cycle enter the electron transport chain and are eventually transferred to O₂. Reducing equivalents generated at complex I (NADH–ubiquinone oxidoreductase) and complex II (succinate dehydrogenase (SDH)) are transferred to complex III by ubiquinol (CoQH₂), a lipophilic quinone that carries a pair of electrons within the membrane. Electrons are transferred between complex III and complex IV by cytochrome c (cyt c), which carries a single electron coordinated to a haem group. Electrons that are transferred to complex IV (cyt c oxidase) are sequentially transferred to O₂, generating H₂O. Electron transfer steps at complexes I, III and IV are associated with proton translocation from the matrix to the intermembrane space, resulting in the generation of an electrochemical gradient across the inner membrane (ΔΨm). Complex V (ATP synthase) uses this gradient to catalyse the phosphorylation of ADP, yielding ATP. Exchange of ATP for ADP across the inner membrane is mediated by the adenine nucleotide transporter (ANT). Increases in ADP availability (as a result of cellular metabolic activity) tend to decrease ΔΨm slightly, which facilitates electron transfer at the steps involving proton extrusion. Hence, increases in cellular use of ATP cause an increase in mitochondrial respiration and ATP synthesis. By contrast, when ATP utilization falls, complex V activity decreases and the electron transport flux and oxygen consumption fall as a consequence of an increase in ΔΨm. Red highlighted proteins are known targets of reactive oxygen species (ROS). αKDH, α-ketoglutarate dehydrogenase; E_m, electrical field within the membrane; FH, fumarate hydratase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; PDHP, pyruvate dehydrogenase phosphatase; PDK, pyruvate dehydrogenase kinase; P_i, inorganic phosphate; SCoA-S, succinyl-CoA synthase; SH, cysteine thiol.

Figure 2. Mitochondrial reactive oxygen species (ROS) generation

Electrons derived from the oxidation of metabolic intermediates can lead to the generation of ROS at specific sites in mitochondria. Transfer of a single electron to O₂ yields superoxide (O₂^•−) which is converted to hydrogen peroxide (H₂O₂) by superoxide dismutase in the matrix (SOD2; also known as MnSOD), or in the intermembrane space (SOD1; also known as CuZn–SOD). The H₂O₂ is degraded in the matrix by glutathione peroxidase 1 (GPX1) or peroxiredoxins (PRDX3 or PRDX5) using reducing equivalents obtained from the oxidation of reduced glutathione (GSH)^–. Oxidized glutathione (GSSG) is reduced by glutathione reductase, which obtains its equivalents from NADPH oxidation. H₂O₂ generated in the matrix can oxidize proteins, lipids or mitochondrial DNA (mtDNA). Oxidized proteins are repaired by thioredoxin 2 (TRX2) or glutaredoxin (GRX). TRX2 and GRX are subsequently reduced by thioredoxin reductase 2 or by glutathione. Lipid hydroperoxides are reduced by GPX4. Ultimately, all ROS removal depends on the availability of GSH, which is maintained by the availability of NADPH in the respective compartments. H₂O₂ can potentially leak to the intermembrane space and the cytosol when excessive ROS generation occurs or antioxidant mechanisms fail. Complexes I, II and III can potentially generate ROS in the matrix compartment. Superoxide can be released to the intermembrane space from complex III, owing to generation from ubisemiquinone at the outer ubiquinone binding site (Qo) of complex III. The electrical gradient across the inner membrane (−180 mV) creates a strong electrical field within the membrane (257 kV per cm) that accelerates superoxide anions from the membrane into the intermembrane space. Paradoxically, cellular hypoxia augments the rate of ROS generation at that site, leading to the production of H₂O₂ in the intermembrane space^,,,,. Subsequent diffusion to the cytosol triggers redox-dependent inhibition of prolyl hydroxylases (PHDs), negative regulators of hypoxia-inducible factor-α (HIFα) stabilization. Thus, mitochondria-derived ROS can promote cancer initiation through oxidative stress, and cancer cell progression through the activation of transcription by HIF. CoQ, ubiquinone; cyt c, cytochrome c; GSHR, glutathione reductase; Qi, inner ubiquinone binding site on complex III; TH, mitochondrial trans-hydrogenase; TRX1R, TRX1 receptor; VDAC, voltage-dependent anion channel.

Figure 3. Hypoxia and pseudohypoxia activate mitochondrial reactive oxygen species (ROS) generation and oxidant signalling that drives the tumour cell phenotype

Hypoxia triggers ROS generation by the mitochondrial electron transport chain (ETC), leading to activation of hypoxia-inducible factor 1 (HIF1) and HIF2 through prolyl hydroxylase (PHD) inhibition. Hypoxia also activates other responses and transcription factors through ROS-dependent signalling. Pseudohypoxia mimetics trigger the effects of hypoxia by activating or inhibiting steps in the hypoxia response pathway. Oncogenes including KRAS and MYC can further drive ROS generation. Mutations in mitochondrial DNA (mtDNA)- or nuclear DNA-encoded proteins can augment mitochondrial ROS generation and mimic the effects of hypoxia^,,. Succinate dehydrogenase (SDH) mutations in subunits SDHB, SDHC or SDHD augment ROS generation from the ETC. Fumarate hydratase loss-of-function mutations lead to oxidative stress that activates redox signalling. Cobalt chloride (CoCl₂) increases oxidant generation by redox cycling, thereby augmenting ROS generation. Depending on the site of action, pseudohypoxic activators may only mimic some of the responses seen during authentic hypoxia. For example, chemical inhibitors of PHD (dimethyloxallyl glycine (DMOG) and desferrioxamine (DFO)) only trigger the activation of HIF1 and HIF2 without activating other pathways. AP1, activating protein 1; IMS, intermembrane space; NF-κB, nuclear factor-κB; NRF2, nuclear respiratory factor 2.

Figure 4. Mutations in tricarboxylic acid (TCA) cycle enzymes drive tumour cell progression through the generation of oxidant signalling

Inactivating mutations in fumarate hydratase (FH) block TCA cycle function, causing accumulation of fumarate and succinate. Fumarate reacts with reduced glutathione (GSH) to produce succinated glutathione (GSF), an oncometabolite that is degraded by glutathione reductase at the expense of NADPH. Released GSH is then available to recombine with fumarate in a futile cycle that consumes NADPH. This impairs the reactive oxygen species (ROS) detoxifying capacity of mitochondria, leading to an increased ROS release to the cytosol that inhibits prolyl hydroxylase and increases the stabilization of hypoxia inducible factor 1α (HIF1α). Mitochondria continue to metabolize α-ketoglutarate to produce citrate through reverse carboxylation. The supply of α-ketoglutarate is maintained by glutamate derived from glutamine deamination. Citrate that is produced by this reverse operation is then exported to the cytosol, where it is used to generate acetyl-CoA that is needed for lipid biosynthesis. Thus, TCA cycle inhibition can produce diverse effects in a tumour cell by driving ROS-dependent and ROS-independent signalling. αKGDH, α-ketoglutarate dehydrogenase; Acon, aconitase; KEAP1, kelch-like ECH-associated protein 1; MDH, malate dehydrogenase; MPC, mitochondrial pyruvate carrier; NRF2, nuclear respiratory factor 2; PDH, pyruvate dehydrogenase.