The role of mitochondrial electron transport in tumorigenesis and metastasis

Background

Tumor formation and spread via the circulatory and lymphatic drainage systems is associated with metabolic reprogramming that often includes increased glycolytic metabolism relative to mitochondrial energy production. However, cells within a tumor are not identical due to genetic change, clonal evolution and layers of epigenetic reprogramming. In addition, cell hierarchy impinges on metabolic status while tumor cell phenotype and metabolic status will be influenced by the local microenvironment including stromal cells, developing blood and lymphatic vessels and innate and adaptive immune cells. Mitochondrial mutations and changes in mitochondrial electron transport contribute to metabolic remodeling in cancer in ways that are poorly understood.

Scope of Review

This review concerns the role of mitochondria, mitochondrial mutations and mitochondrial electron transport function in tumorigenesis and metastasis.

Major Conclusions

It is concluded that mitochondrial electron transport is required for tumor initiation, growth and metastasis. Nevertheless, defects in mitochondrial electron transport that compromise mitochondrial energy metabolism can contribute to tumor formation and spread. These apparently contradictory phenomena can be reconciled by cells in individual tumors in a particular environment adapting dynamically to optimally balance mitochondrial genome changes and bioenergetic status.

General Significance

Tumors are complex evolving biological systems characterized by genetic and adaptive epigenetic changes. Understanding the complexity of these changes in terms of bioenergetics and metabolic changes will permit the development of better combination anticancer therapies. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Cardiolipin Regulates the Activity of the Reconstituted Mitochondrial Calcium Uniporter by Modifying the Structure of the Liposome Bilayer

Reconstitution of mitochondrial calcium transport activity requires the incorporation of membrane proteins into a lipidic ambient. Calcium uptake has been measured previously using Cytochrome oxidase vesicles. The enrichment of these vesicles with cardiolipin, an acidic phospholipid that is found only in the inner mitochondrial membrane of eukaryotic cells, strongly inhibits calcium transport, in remarkable contrast with the activation effect that cardiolipin exerts upon other mitochondrial transporters and enzymes. The relation of the inactivation of calcium transport to the physical state of the bilayer was studied by following the polarization changes of 1,6-diphenyl-1,3,5-hexatriene (DPH) and by flow cytometry in the cardiolipin-enriched liposomes with incorporated mitochondrial solubilized proteins. Non-bilayer molecular arrangements in the cardiolipin-supplemented liposomes, detected by flow cytometry, may produce the fluidity changes observed by fluorescence polarization of DPH. Fluidity changes correlate with the abolition of calcium uptake, but have no effect on the establishment of a membrane potential in the vesicles required for calcium transport activity. Changes in the membrane structure and uniporter function are observed in the combined presence of cardiolipin and calcium leading to a modified lipid configuration.     

A new method for quantifying mitochondrial axonal transport

Axonal transport of mitochondria is critical for neuronal survival and function. Automatically quantifying and analyzing mitochondrial movement in a large quantity remain challenging. Here, we report an efficient method for imaging and quantifying axonal mitochondrial transport using microfluidic-chamber-cultured neurons together with a newly developed analysis package named “MitoQuant”. This tool-kit consists of an automated program for tracking mitochondrial movement inside live neuronal axons and a transient-velocity analysis program for analyzing dynamic movement patterns of mitochondria. Using this method, we examined axonal mitochondrial movement both in cultured mammalian neurons and in motor neuron axons of Drosophila in vivo. In 3 different paradigms (temperature changes, drug treatment and genetic manipulation) that affect mitochondria, we have shown that this new method is highly efficient and sensitive for detecting changes in mitochondrial movement. The method significantly enhanced our ability to quantitatively analyze axonal mitochondrial movement and allowed us to detect dynamic changes in axonal mitochondrial transport that were not detected by traditional kymographic analyses.     

The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability

Mitochondria are well known as sites of electron transport and generators of cellular ATP. Mitochondria also appear to be sites of cell survival regulation. In the process of programmed cell death, mediators of apoptosis can be released from mitochondria through disruptions in the outer mitochondrial membrane; these mediators then participate in the activation of caspases and of DNA degradation. Thus the regulation of outer mitochondrial membrane integrity is an important control point for apoptosis. The Bcl-2 family is made up of outer mitochondrial membrane proteins that can regulate cell survival, but the mechanisms by which Bcl-2 family proteins act remain controversial. Most metabolites are permeant to the outer membrane through the voltage dependent anion channel (VDAC), and Bcl-2 family proteins appear to be able to regulate VDAC function. In addition, many Bcl-2 family proteins can form channels in vitro, and some pro-apoptotic members may form multimeric channels large enough to release apoptosis promoting proteins from the intermembrane space. Alternatively, Bcl-2 family proteins have been hypothesized to coordinate the permeability of both the outer and inner mitochondrial membranes through the permeability transition (PT) pore. Increasing evidence suggests that alterations in cellular metabolism can lead to pro-apoptotic changes, including changes in intracellular pH, redox potential and ion transport. By regulating mitochondrial membrane physiology, Bcl-2 proteins also affect mitochondrial energy generation, and thus influence cellular bioenergetics. Cell Death and Differentiation (2000) 7, 1182 - 1191     

Changes in mitochondrial activity evoked by cholecystokinin in isolated mouse pancreatic acinar cells

In the present study, we have employed confocal laser scanning microscopy to investigate the effect that stimulation of mouse pancreatic acinar cells with the secretagogue cholecystokinin (CCK) has on mitochondrial activity. We have monitored changes in cytosolic as well as mitochondrial Ca2+ concentrations, mitochondrial membrane potential and FAD autofluorescence by loading the cells with fluo-3, rhod-2 or JC-1, respectively. Our results show that stimulation of cells with cholecystokinin led to release of Ca2+ from intracellular stores that then accumulated into mitochondria. In the presence of the hormone a depolarization of mitochondrial membrane potential was observed, which partially recovered; in addition a transient increase in FAD autofluorescence could be observed. Similarly, treatment of cells with thapsigargin induced increases in mitochondrial Ca2+ and FAD autofluorescence, and depolarized mitochondria. Pretreament of cells with thapsigargin blocked cholecystokinin-evoked changes. Similar results were obtained when the cells were incubated in the presence of rotenone, which blocks the mitochondrial electron transport chain. Our findings are consistent with changes in mitochondrial activity in response to stimulation of pancreatic acinar cells with cholecystokinin. Following stimulation, mitochondria take up Ca2+ that could in turn activate the mitochondrial machinery that may match the energy supply necessary for the cell function during secretion, suggesting that Ca2+ can act as a regulator of mitochondrial activity.     

Mitochondrial movers and shapers: Recent insights into regulators of fission,fusion and transport

Mitochondria are highly dynamic organelles that undergo rapid morphological adaptations influencing their number, transport, cellular distribution, and function, which in turn facilitate the integration of mitochondrial function with physiological changes in the cell. These mitochondrial dynamics are dependent on tightly regulated processes such as fission, fusion, and attachment to the cytoskeleton, and their defects are observed in various pathophysiological conditions including cancer, cardiovascular disease, and neurodegeneration. Various studies over the years have identified key molecular players and uncovered the mechanisms that mediate and regulate these processes and have highlighted their complexity and context-specificity. This review focuses on the recent studies that have contributed to the understanding of processes that influence mitochondrial morphology including fission, fusion, and transport in the cell.     

Differential effect of three-repeat and four-repeat tau on mitochondrial axonal transport

Tau protein is present in six different splice forms in the human brain and interacts with microtubules via either 3 or 4 microtubule binding repeats. An increased ratio of 3 repeat to 4 repeat isoforms is associated with neurodegeneration in inherited forms of frontotemporal dementia. Tau over-expression diminishes axonal transport in several systems, but differential effects of 3 repeat and 4 repeat isoforms have not been studied. We examined the effects of tau on mitochondrial transport and found that both 3 repeat and 4 repeat tau change normal mitochondrial distribution within the cell body and reduce mitochondrial localization to axons; 4 repeat tau has a greater effect than 3 repeat tau. Further, we observed that the 3 repeat and 4 repeat tau cause different alterations in retrograde and anterograde transport dynamics with 3 repeat tau having a slightly stronger effect on axon transport dynamics. Our results indicate that tau-induced changes in axonal transport may be an underlying theme in neurodegenerative diseases associated with isoform specific changes in tau's interaction with microtubules.     

Disruption of Mitochondrial DNA Replication in Drosophila Increases Mitochondrial Fast Axonal Transport In Vivo

Mutations in mitochondrial DNA polymerase (pol γ) cause several progressive human diseases including Parkinson''s disease, Alper''s syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol γ leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol γ deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol γ−α mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases.     

Reactive oxygen species act remotely to cause synapse loss in a Drosophila model of developmental mitochondrial encephalopathy

Mitochondrial dysfunction is a hallmark of many neurodegenerative diseases, yet its precise role in disease pathology remains unclear. To examine this link directly, we subtly perturbed electron transport chain function in the Drosophila retina, creating a model of Leigh Syndrome, an early-onset neurodegenerative disorder. Using mutations that affect mitochondrial complex II, we demonstrate that mild disruptions of mitochondrial function have no effect on the initial stages of photoreceptor development, but cause degeneration of their synapses and cell bodies in late pupal and adult animals. In this model, synapse loss is caused by reactive oxygen species (ROS) production, not energy depletion, as ATP levels are normal in mutant photoreceptors, and both pharmacological and targeted genetic manipulations that reduce ROS levels prevent synapse degeneration. Intriguingly, these manipulations of ROS uncouple synaptic effects from degenerative changes in the cell body, suggesting that mitochondrial dysfunction activates two genetically separable processes, one that induces morphological changes in the cell body, and another that causes synapse loss. Finally, by blocking mitochondrial trafficking into the axon using a mutation affecting a mitochondrial transport complex, we find that ROS action restricted to the cell body is sufficient to cause synaptic degeneration, demonstrating that ROS need not act locally at the synapse. Thus, alterations in electron transport chain function explain many of the neurodegenerative changes seen in both early- and late-onset disorders.     

Pathophysiology of mitochondrial volume homeostasis: Potassium transport and permeability transition

Regulation of mitochondrial volume is a key issue in cellular pathophysiology. Mitochondrial volume and shape changes can occur following regulated fission-fusion events, which are modulated by a complex network of cytosolic and mitochondrial proteins; and through regulation of ion transport across the inner membrane. In this review we will cover mitochondrial volume homeostasis that depends on (i) monovalent cation transport across the inner membrane, a regulated process that couples electrophoretic K⁺ influx on K⁺ channels to K⁺ extrusion through the K⁺-H⁺ exchanger; (ii) the permeability transition, a loss of inner membrane permeability that may be instrumental in triggering cell death. Specific emphasis will be placed on molecular advances on the nature of the transport protein(s) involved, and/or on diseases that depend on mitochondrial volume dysregulation.     

Mitochondrial cholesterol import

All animal subcellular membranes require cholesterol, which influences membrane fluidity and permeability, fission and fusion processes, and membrane protein function. The distribution of cholesterol among subcellular membranes is highly heterogeneous and the cholesterol content of each membrane must be carefully regulated. Compared to other subcellular membranes, mitochondrial membranes are cholesterol-poor, particularly the inner mitochondrial membrane (IMM). As a result, steroidogenesis can be controlled through the delivery of cholesterol to the IMM, where it is converted to pregnenolone. The low basal levels of cholesterol also make mitochondria sensitive to changes in cholesterol content, which can have a relatively large impact on the biophysical and functional characteristics of mitochondrial membranes. Increased mitochondrial cholesterol levels have been observed in diverse pathological conditions including cancer, steatohepatitis, Alzheimer disease and Niemann-Pick Type C1-deficiency, and are associated with increased oxidative stress, impaired oxidative phosphorylation, and changes in the susceptibility to apoptosis, among other alterations in mitochondrial function. Mitochondria are not included in the vesicular trafficking network; therefore, cholesterol transport to mitochondria is mostly achieved through the activity of lipid transfer proteins at membrane contact sites or by cytosolic, diffusible lipid transfer proteins. Here we will give an overview of the main mechanisms involved in mitochondrial cholesterol import, focusing on the steroidogenic acute regulatory protein StAR/STARD1 and other members of the StAR-related lipid transfer (START) domain protein family, and we will discuss how changes in mitochondrial cholesterol levels can arise and affect mitochondrial function. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

The mitochondrial oxoglutarate carrier: from identification to mechanism

The 2-oxoglutarate carrier (OGC) belongs to the mitochondrial carrier protein family whose members are responsible for the exchange of metabolites, cofactors and nucleotides between the cytoplasm and mitochondrial matrix. Initially, OGC was characterized by determining substrate specificity, kinetic parameters of transport, inhibitors and molecular probes that form covalent bonds with specific residues. It was shown that OGC specifically transports oxoglutarate and certain carboxylic acids. The substrate specificity combination of OGC is unique, although many of its substrates are also transported by other mitochondrial carriers. The abundant recombinant expression of bovine OGC in Escherichia coli and its ability to functionally reconstitute into proteoliposomes made it possible to deduce the individual contribution of each and every residue of OGC to the transport activity by a complete set of cys-scanning mutants. These studies give experimental support for a substrate binding site constituted by three major contact points on the even-numbered α-helices and identifies other residues as important for transport function through their crucial positions in the structure for conserved interactions and the conformational changes of the carrier during the transport cycle. The results of these investigations have led to utilize OGC as a model protein for understanding the transport mechanism of mitochondrial carriers.     

A role for mitochondrial aquaporins in cellular life-and-death decisions?

Mitochondria dominate the process of life-and-death decisions of the cell. Continuous generation of ATP is essential for cell sustenance, but, on the other hand, mitochondria play a central role in the orchestra of events that lead to apoptotic cell death. Changes of mitochondrial volume contribute to the modulation of physiological mitochondrial function, and several ion permeability pathways located in the inner mitochondrial membrane have been implicated in the mediation of physiological swelling-contraction reactions, such as the K+ cycle. However, the channels and transporters involved in these processes have not yet been identified. Osmotic swelling is also one of the fundamental characteristics exhibited by mitochondria in pathological situations, which activates downstream cascades, culminating in apoptosis. The permeability transition pore has long been postulated to be the primary mediator for water movement in mitochondrial swelling during cell death, but its molecular identity remains obscure. Inevitably, accumulating evidence shows that mitochondrial swelling induced by apoptotic stimuli can also occur independently of permeability transition pore activation. Recently, a novel mechanism for osmotic swelling of mitochondria has been described. Aquaporin-8 and -9 channels have been identified in the inner mitochondrial membrane of various tissues, including the kidney, liver, and brain, where they may mediate water transport associated with physiological volume changes, contribute to the transport of metabolic substrates, and/or participate in osmotic swelling induced by apoptotic stimuli. Hence, the recent discovery that aquaporins are expressed in mitochondria opens up new areas of investigation in health and disease.     

Lipid peroxidation associated cardiolipin loss and membrane depolarization in rat brain mitochondria

Oxidative stress induced by Fe2+ (50 microM) and ascorbate (2 mM) in isolated rat brain mitochondria incubated in vitro leads to an enhanced lipid peroxidation, cardiolipin loss and an increased formation of protein carbonyls. These changes are associated with a loss of mitochondrial membrane potential (depolarization) and an impaired activity of electron transport chain (ETC) as measured by MTT reduction assay. Butylated hydroxytoluene (0.2 mM), an inhibitor of lipid peroxidation, can prevent significantly the loss of cardiolipin, the increased protein carbonyl formation and the decrease in mitochondrial membrane potential induced by Fe2+ and ascorbate, implying that the changes are secondary to membrane lipid peroxidation. However, iron-ascorbate induced impairment of mitochondrial ETC activity is apparently independent of lipid peroxidation process. The structural and functional derangement of mitochondria induced by oxidative stress as reported here may have implications in neuronal damage associated with brain aging and neurodegenerative disorders.     

α-Ketoglutarate regulation of glutamine transport and deamidation by renal mitochondria

α-ketoglutarate was found to be a potent inhibitor of glutamine transport and deamidation in mitochondria isolated from rat kidney; physiological concentrations of the ketoacid (～0.3mM) reduced transport and deamidation 45–60 percent. The observed concentration-inhibition relationship between α-ketoglutarate and mitochondrial glutamine transport and deamidation indicated that changes in renal concentration of the ketoacid occurring during conditions associated with an increase in glutamine deamidation (e.g. metabolic acidosis) would have significant effects on glutamine transport and deamidation by renal mitochondria in vivo. The inhibitory effect of α-ketoglutarate was specific; several of the other major organic acids found in renal cells stimulated rather than inhibited mitochondrial glutamine transport.     

Optimal Dynamics for Quality Control in Spatially Distributed Mitochondrial Networks

Recent imaging studies of mitochondrial dynamics have implicated a cycle of fusion, fission, and autophagy in the quality control of mitochondrial function by selectively increasing the membrane potential of some mitochondria at the expense of the turnover of others. This complex, dynamical system creates spatially distributed networks that are dependent on active transport along cytoskeletal networks and on protein import leading to biogenesis. To study the relative impacts of local interactions between neighboring mitochondria and their reorganization via transport, we have developed a spatiotemporal mathematical model encompassing all of these processes in which we focus on the dynamics of a health parameter meant to mimic the functional state of mitochondria. In agreement with previous models, we show that both autophagy and the generation of membrane potential asymmetry following a fusion/fission cycle are required for maintaining a healthy mitochondrial population. This health maintenance is affected by mitochondrial density and motility primarily through changes in the frequency of fusion events. Health is optimized when the selectivity thresholds for fusion and fission are matched, providing a mechanistic basis for the observed coupling of the two processes through the protein OPA1. We also demonstrate that the discreteness of the components exchanged during fusion is critical for quality control, and that the effects of limiting total amounts of autophagy and biogenesis have distinct consequences on health and population size, respectively. Taken together, our results show that several general principles emerge from the complexity of the quality control cycle that can be used to focus and interpret future experimental studies, and our modeling framework provides a road-map for deconstructing the functional importance of local interactions in communities of cells as well as organelles.     

Mitochondrial energy metabolism and redox state in dyslipidemias

Changes in mitochondrial function are intimately associated with metabolic diseases. Here, we review recent evidence relating alterations in mitochondrial energy metabolism, ion transport and redox state in hypercholesterolemia and hypertriglyceridemia. We focus mainly on changes in mitochondrial respiration, K(+) and Ca(2+) transport, reactive oxygen species generation and susceptibility to mitochondrial permeability transition.     

Influence of glyoxylic acid on properties of isolated mitochondria]

Glyoxalate is an effector of oxidative phosphorylation in isolated mitochondria : it slows down State 3 but does not affect State 4 respiration. This report presents the findings of our study on the mechanism of action of glyoxalate ; these findings are listed below. The inhibition of Stage 3 respiration by glyoxalate does not set in immediately, can be reversed in part by the addition of an uncoupling agent or a dithiol, is non-competitive against succinate and can be demonstrated with substrates requiring the involvement of other membrane transport systems. Glyoxalate prevents the increased oxygen uptake stimulated by 2,4-DNP or Sr++. Glyoxalate also inhibits phosphate transport and this inhibition can account for most of the effect observed. The inhibition of State 3 respiration is paralleled by a decrease in the mitochondrial accumulation of succinate : this decrease could arise from a direct effect of glyoxalate on dicarboxylic acid transport or could be the result of an inhibiton of the phosphate transport system, which is connected with the former. The decrease in the respiratory rate of uncoupled mitochondria placed in a phosphate free medium demonstrates that the effector acts directly at the substrate transport or/and electron transfer level. Phosphate, by delaying the respiratory inhibiton due to glyoxalate, has a protecting effect on mitochondrial functions. Glyoxalate is thus acting at several mitochondrial sites. It acts presumably by forming hemimercaptals, blocking sulfhydryl groups. Its effects can be accounted for by the unfolding of such (hemicercaptal) groups under the influence of ADP, Pi, uncoupling or others agents which bring about conformational changes in the internal mitochondrial membrane.