|Home | About | Journals | Submit | Contact Us | Français|
The precise mitochondrial alterations that underlie the increased dependence of cancer cells on aerobic glycolysis for energy generation have remained a mystery. Recent evidence suggests that mitochondrial uncoupling – the abrogation of ATP synthesis in response to mitochondrial membrane potential – promotes the Warburg effect in leukemia cells, and may contribute to chemoresistance. Intriguingly, leukemia cells cultured on bone marrow-derived stromal feeder layers are more resistant to chemotherapy, increase the expression of uncoupling protein 2, and decrease the entry of pyruvate into the Krebs cycle – without compromising the consumption of oxygen, suggesting a shift to the oxidation of non-glucose carbon sources to maintain mitochondrial integrity and function. Since fatty acid oxidation has been linked to chemoresistance and mitochondrial uncoupling, it is tempting to speculate that Warburg’s observations may indeed be the result of the preferential oxidation of fatty acids by cancer cell mitochondria. Therefore, targeting fatty acid oxidation or anaplerotic pathways that support fatty acid oxidation may provide additional therapeutic tools for the treatment of hematopoietic malignancies.
More than half a century ago Otto Warburg proposed that cancer cells originated from non-neoplastic cells that acquired a permanent respiratory defect that bypassed the Pasteur effect, i.e. the inhibition of fermentation by oxygen (1). This hypothesis was based on results of extensive characterization of the fermentation and oxygen consumption capacity of normal and malignant tissues – including mouse ascites and Earle’s cells of different malignancies but same genetic origin – that conclusively demonstrated a higher ratio of fermentation to respiration in the neoplastic cells. Moreover, the data indicated that the more malignant Earle’s cancer cells displayed a higher ratio of fermentation to respiration than their less malignant counterparts, suggesting to Warburg and his colleagues that a gradual and cumulative decrease in mitochondrial activity was associated with malignant transformation. Interestingly, the precise nature of these gradual and cumulative changes has eluded investigators for nearly 80 years, albeit Warburg’s observations of an increased rate of aerobic glycolysis in cancer cells have been reproduced countless times – not to mention the wealth of positron emission tomography images that support an increased uptake of radiolabeled glucose in tumor tissues.
It is noteworthy that while Warburg’s hypothesis remains a topic of discussion amongst cancer biologists, Otto Warburg himself had alluded to an alternative hypothesis put forth by Feodor Lynen – one which did not necessitate permanent or transmissible alterations to the oxidative capacity of mitochondria – that suggested the possibility that the increased dependence of cancer cells on glycolysis stemmed not from their inability to reduce oxygen, but rather from their inability to synthesize ATP in response to the mitochondrial proton gradient (ΔΨM) (1). Lynen’s hypothesis was partly based on his work (2) and the previous work of Ronzoni and Ehrenfest utilizing the prototypical protonophore 2,4-dinitrophenol (DNP) which causes a “short circuit” in the electrochemical gradient that abolishes the mitochondrial synthesis of ATP, and decreases the entry of pyruvate into the Krebs cycle (3). Subsequent work demonstrated that mitochondrial uncoupling (i.e., the abrogation of ATP synthesis in response to ΔΨM) results in a metabolic shift to the utilization of non-glucose carbon sources to maintain mitochondrial function (4,5). Given the elusiveness of permanent transmissible alterations to the oxidative capacity of cancer cells that could broadly support Warburg’s hypothesis, could Lynen’s hypothesis better explain the dependence of cancer cells on glycolysis for ATP generation?
Over the past several decades it has become increasingly clear that mitochondrial uncoupling occurs under physiological conditions, such as during cold acclimation in mammals, and is mediated, at least in part, by uncoupling proteins (UCPs) (6,7). UCP1 was the first uncoupling protein identified, and was shown to play a role in energy dissipation as heat in mammalian brown fat (6). During cold acclimation, UCP1 accumulates in the inner mitochondrial membrane and short circuits ΔΨM (created by the mitochondrial respiratory chain) by sustaining an inducible proton conductance (7). Other UCPs have been identified in humans (UCP2-4), albeit their functions may be unrelated to the maintenance of core body temperature, and instead involved in the reprogramming of metabolic pathways. For instance, recent work demonstrates that UCP2 is necessary for efficient oxidation of glutamine (8), and may promote the metabolic shift from glucose oxidation to fatty acid oxidation (4). Likewise, UCP3 has also been shown to promote fatty acid oxidation in muscle tissue via, in part, an increased flux of fatty acid anions (9), however, like for UCP2, the nature of its proton conductance remains controversial (reviewed in (10). More interesting perhaps, are recent observations that UCP2 is overexpressed in various chemoresistant cancer cell lines and primary human colon cancer, and that overexpression of this uncoupling protein leads to an increased apoptotic threshold (11) – suggesting that in addition to metabolic reprogramming, uncoupling proteins may ipso facto provide a prosurvival advantage to malignant cells.
It is important to point out that physiological fatty acid oxidation has been shown to be associated with an uncoupling and/or thermogenic phenotype in various cell types (reviewed in (12)). In addition, it is also significant that glycolysis derived pyruvate – as well as α-ketoglutarate derived from glutaminolysis – may be necessary to provide anaplerotic substrates (i.e. those that replenish intermediates in metabolic cycles) for efficient Krebs cycle utilization of fatty acid-derived acetyl CoA (13) suggesting the possibility that in certain cell types high rates of aerobic glycolysis may be necessary for efficient mitochondrial oxidation of fatty acids (“fats burn in the fire of carbohydrates”). The above support the concept – and indirectly, Lynen’s hypothesis – that the Warburg effect may in fact be the result of fatty acid and/or glutamine oxidation in favor of pyruvate utilization.
We have recently reported that leukemia cells cultured on bone marrow-derived mesenchymal stromal cells (MSC) – cells that support both normal and malignant hematopoiesis by multiple mechanisms – demonstrate increased aerobic glycolysis and reduced ΔΨM (14), suggesting that cues from the bone marrow microenvironment may further enhance the Warburg effect in leukemia cells. A priori we hypothesized that MSC decreased mitochondrial function in leukemia cells; however, our experiments revealed that the oxygen consumption capacity of leukemia cells was not affected, and in fact, displayed a transient (~6–8 hr) increase after exposure to MSC. In addition, leukemia cells cultured on MSC were less (25%) sensitive to the ΔΨM dissipating effects of oligomycin, and as previously reported (15,16), more resistant to apoptosis induced by a variety of chemotherapeutic agents. This finding suggests that in leukemia cells cultured on MSC feeder layers ΔΨM is more dependent on oxygen consumption than glycolysis, and that the coculture phenotype represents a prosurvival mitochondrial metabolic shift, rather than a compromised mitochondrial function. Additionally, it was observed that in contrast to hypoxia (~6% oxygen) – which markedly increased the uptake of glucose, and a fluorescent glucose derivative from the medium – MSC feeder layers did not increase the uptake of glucose in leukemia cells, further supporting the notion that the increased accumulation of lactate in the medium of MSC-leukemia cocultures is indicative of reduced entry of pyruvate into the Krebs cycle of leukemia cells.
Since the above observations supported the possibility that MSC may induce mitochondrial uncoupling in leukemia cells, we investigated whether MSC feeder layers were modulating the expression of uncoupling proteins (UCP1 – 4). We observed that leukemia cells only expressed UCP2, and that MSC induced pronounced accumulation of this uncoupling protein. Surprisingly, siRNA silencing of UCP2 expression did not completely overcome the dissipation of ΔΨM induced by MSC, albeit decreased expression of this protein markedly decreased the accumulation of lactate in the medium of MSC-leukemia cocultures. Moreover, albeit leukemia cells rapidly lost ΔΨM when exposed to MSC feeder layers (~30 min), maximal expression of UCP2 did not occur until 24 to 48 hr after coculture, and conversely, the rapid dissipation of ΔΨM was insensitive to inhibition of protein synthesis with cycloheximide. Taken together, the above results suggest that while UCP2 expression may contribute to the observed loss of ΔΨM, it is likely that other factor(s) may initiate the dissipation of the electrochemical gradient; nonetheless, the data reported support the findings of Derdak et al. (11) that expression of UCP2 in colon cancer cells resulted in decreased ΔΨM, increased oxygen consumption, and increased dependence on aerobic glycolysis for ATP generation. It is thus likely that in multiple cancer types UCP2 may be involved in metabolic reprogramming away from the oxidation of pyruvate, a phenomenon that may in turn facilitate the maintenance of a reduced ΔΨM.
Interestingly, the study by Derdak et al. (11) demonstrated that dissipation of ΔΨM utilizing the protonophore FCCP opposed the onset of apoptosis in colon cancer cells after exposure to a variety of chemotherapeutic agents, and similarly, we also demonstrated that a related protonophore (CCCP) could partially protect leukemia cells against apoptosis induction by mitoxanthrone, suggesting one potential mechanism via which UCP2 may promote chemoresistance. Likewise, MSC feeder layers protected OCI-AML3 cells from apoptosis, but not the growth inhibitory effects of mitoxanthrone, AraC, and vincristine. It is noteworthy that leukemia cells that did not increase the expression of UCP2 when cultured with MSC feeder layers, did not increase lactate generation, did not dissipate ΔΨM, and were not protected from the cytotoxic effects of chemotherapy when cultured with MSC. The above suggests that the observed metabolic reprogramming and decreased ΔΨM in OCI-AML3 cells may be one part of the multifactorial response involved in stroma-mediated chemoresistance. As UCP2 expression in colon cancer cells inhibited ROS generation after exposure to chemotherapeutic agents (11), and since the generation of ROS is dependent on ΔΨM, it is conceivable that the antiapoptotic effects of mitochondrial uncoupling in leukemia cells may be mediated by decreased oxidative damage. On the other hand, the above study also demonstrated that UCP2 expression in colon cancer cells decreased the ROS-dependent multisite phosphorylation of the transactivation domain of p53 after exposure to camptothecin – resulting in decreased expression of the p53 target gene PUMA – supporting the notion that mitochondrial uncoupling may also modulate oncogenic and tumor suppressor pathways via ROS signaling. It is thus provoking to speculate that targeting UCP2 – as well as the metabolic reprogramming involved in initiating and maintaining the dissipation of ΔΨM (increased glutamine and/or fatty acid metabolism, decreased ROS, etc.) – could be exploited therapeutically to overcome chemoresistance.
The metabolic shift from the oxidation pyruvate to the uncoupled oxidation of glutamine or fatty acids highlights two critical concepts. First, glycolysis remains the critical pathway by which cancer cells meet their energy demands, not because of permanent transmissible alterations to the oxidative capacity of cells, but rather because of the inability of uncoupled mitochondria to generate ATP. Second, the continued reduction of oxygen, in the absence of pyruvate oxidation, suggests that anaplerotic reactions from non-glucose carbon skeletons must be replenishing critical intermediates from the Krebs cycle – reactions that may be amenable to therapeutic intervention, and that may critically depend on highly conserved uncoupling proteins – to in turn support the oxidation of fatty acids or glutamine (Figure 1). Curiously, anaplerotic reactions have recently been reported to support the activity of the Krebs cycle in glioma cells (17)– which utilize most of their glutamine carbon skeletons to regenerate α-ketoglutarate – while at the same time utilizing glucose carbon skeletons to synthesize fatty acids. Moreover, the required NADPH (the biosynthetic reducing equivalent) for fatty acid synthesis was provided by conversion of glutamate-derived malate to pyruvate, and to a lesser extent from the activity of the pentose phosphate shunt, further highlighting the importance of glutamine metabolism via the Krebs cycle (17). In the above study it was evident that the metabolism of glucose was largely anaerobic, even though the cells maintained the ability to consume oxygen, as well as an active Krebs cycle, suggesting the possibility that mitochondrial uncoupling and uncoupling proteins may promote the observed metabolic pattern.
Notably, a recent report demonstrated that the entry of pyruvate into the Krebs cycle, via pyruvate dehydrogenase (PDH), is supressed in cancer cells, and that the re-activation of PDH activity by dichloroacetate (DCA) induced cell death in several solid tumor cell lines and xenografts (18), supporting the notion that mitochondrial glucose oxidation may be incompatible with cancer cell survival. Likewise, it is interesting that pharmacological inhibition of fatty acid oxidation has been shown to potentiate apoptosis induced by a variety of chemotherapeutics in cancer cell lines (19), as well as palmitate-induced apoptosis in hematopoietic cells (20), suggesting a priori that the metabolism of fatty acids in the mitochondria may be linked to cell survival. In light of the above, it is intriguing to propose that targeting the mitochondrial metabolism of fatty acids and/or glutamine may hold therapeutic promise for the treatment of human malignancies. Conversely, given the important role of uncoupling proteins in the metabolic shift associated with increased fatty acid and glutamine metabolism in favor of glucose oxidation, it would be of great interest to develop therapeutic strategies that targeted these proteins.
It is important to point out that in the study by Bonnet et al. (18), solid tumor cell lines were found to display elevated ΔΨM and this was associated with increased aerobic glycolysis indicating that mitochondrial uncoupling may not be a universal phenomenon by which cancer cells activate the Warburg effect. Additionally, decreased ΔΨM per se, without reduction in the generation of ROS, may not be advantageous since this may trigger the opening of the mitochondrial transition pore with subsequent release of death promoting proteins. It is thus likely that the decrease in ROS generation produced by mitochondrial uncoupling increases the threshold for permeability transition in response to membrane potential drop – either via reduced oxidative damage to pore proteins, or alternatively, decreased proapoptotic signaling from p53 and/or other redox sensitive proteins like JNK, NFκB, and p38 MAPK.
In conclusion, recent investigations into the mechanisms that underlie the Warburg effect suggest that 1) mitochondrial uncoupling can promote aerobic glycolysis in the absence of permanent and transmissible alterations to the oxidative capacity of cells, 2) aerobic glycolysis may represent a shift to the oxidative metabolism of non-glucose carbon sources, and 3) mitochondrial uncoupling may be associated with increased resistance to chemotherapeutic insults. The above suggest the importance of understanding the mechanisms of mitochondrial uncoupling and their relation to metabolic alterations observed in cancer cells.
Supported by grants from the National Cancer Institute (PO1 CA55164) and the Paul and Mary Haas Chair in Genetics (to M.A.), and Leukemia Texas Young Investigator Award (to I.S.).