Emerging evidence indicates that impaired cellular energy metabolism is the defining characteristic of nearly all cancers regardless of cellular or tissue origin. In contrast to normal cells, which derive most of their usable energy from oxidative phosphorylation, most cancer cells become heavily dependent on substrate level phosphorylation to meet energy demands. Evidence is reviewed supporting a general hypothesis that genomic instability and essentially all hallmarks of cancer, including aerobic glycolysis (Warburg effect), can be linked to impaired mitochondrial function and energy metabolism. A view of cancer as primarily a metabolic disease will impact approaches to cancer management and prevention.

Introduction

Cancer is a complex disease involving numerous tempo-spatial changes in cell physiology, which ultimately lead to malignant tumors. Abnormal cell growth (neoplasia) is the biological endpoint of the disease. Tumor cell invasion of surrounding tissues and distant organs is the primary cause of morbidity and mortality for most cancer patients. The biological process by which normal cells are transformed into malignant cancer cells has been the subject of a large research effort in the biomedical sciences for many decades. Despite this research effort, cures or long-term management strategies for metastatic cancer are as challenging today as they were 40 years ago when President Richard Nixon declared a war on cancer.

Confusion surrounds the origin of cancer. Contradictions and paradoxes have plagued the field. Without a clear idea on cancer origins, it becomes difficult to formulate a clear strategy for effective management. Although very specific processes underlie malignant transformation, a large number of unspecific influences can initiate the disease including radiation, chemicals, viruses, inflammation, etc. Indeed, it appears that prolonged exposure to almost any provocative agent in the environment can potentially cause cancer. That a very specific process could be initiated in very unspecific ways was considered “the oncogenic paradox” by Szent-Gyorgyi. This paradox has remained largely unresolved.

In a landmark review, Hanahan and Weinberg suggested that six essential alterations in cell physiology could underlie malignant cell growth. These six alterations were described as the hallmarks of nearly all cancers and included, 1) self-sufficiency in growth signals, 2) insensitivity to growth inhibitory (antigrowth) signals, 3) evasion of programmed cell death (apoptosis), 4) limitless replicative potential, 5) sustained vascularity (angiogenesis), and 6) tissue invasion and metastasis. Genome instability, leading to increased mutability, was considered the essential enabling characteristic for manifesting the six hallmarks. However, the mutation rate for most genes is low making it unlikely that the numerous pathogenic mutations found in cancer cells would occur sporadically within a normal human lifespan. This then created another paradox. If mutations are such rare events, then how is it possible that cancer cells express so many different types and kinds of mutations?

The loss of genomic “caretakers” or “guardians”, involved in sensing and repairing DNA damage, was proposed to explain the increased mutability of tumor cells]. The loss of these caretaker systems would allow genomic instability thus enabling pre-malignant cells to reach the six essential hallmarks of cancer. It has been difficult, however, to define with certainty the origin of pre-malignancy and the mechanisms by which the caretaker/guardian systems themselves are lost during the emergent malignant state. In addition to the six recognized hallmarks of cancer, aerobic glycolysis or the Warburg effect is also a robust metabolic hallmark of most tumors. Although no specific gene mutation or chromosomal abnormality is common to all cancers, nearly all cancers express aerobic glycolysis, regardless of their tissue or cellular origin. Aerobic glycolysis in cancer cells involves elevated glucose uptake with lactic acid production in the presence of oxygen. This metabolic phenotype is the basis for tumor imaging using labeled glucose analogues and has become an important diagnostic tool for cancer detection and management. Genes for glycolysis are overexpressed in the majority of cancers examined.

The origin of the Warburg effect in tumor cells has been controversial. The discoverer of this phenomenon, Otto Warburg, initially proposed that aerobic glycolysis was an epiphenomenon of a more fundamental problem in cancer cell physiology, i.e., impaired or damaged respiration. An increased glycolytic flux was viewed as an essential compensatory mechanism of energy production in order to maintain the viability of tumor cells. Although aerobic glycolysis and anaerobic glycolysis are similar in that lactic acid is produced under both situations, aerobic glycolysis can arise in tumor cells from damaged respiration whereas anaerobic glycolysis arises from the absence of oxygen. As oxygen will reduce anaerobic glycolysis and lactic acid production in most normal cells (Pasteur effect), the continued production of lactic acid in the presence of oxygen can represent an abnormal Pasteur effect. This is the situation in most tumor cells. Only those body cells able to increase glycolysis during intermittent respiratory damage were considered capable of forming cancers. Cells unable to elevate glycolysis in response to respiratory insults, on the other hand, would perish due to energy failure. Cancer cells would therefore arise from normal body cells through a gradual and irreversible damage to their respiratory capacity. Aerobic glycolysis, arising from damaged respiration, is the single most common phenotype found in cancer.

Based on metabolic data collected from numerous animal and human tumor samples, Warburg proposed with considerable certainty and insight that irreversible damage to respiration was the prime cause of cancer. Warburg’s theory, however, was attacked as being too simplistic and not consistent with evidence of apparent normal respiratory function in some tumor cells. The theory did not address the role of tumor-associated mutations, the phenomenon of metastasis, nor did it link the molecular mechanisms of uncontrolled cell growth directly to impaired respiration. Indeed, Warburg’s biographer, Hans Krebs, mentioned that Warburg’s idea on the primary cause of cancer, i.e., the replacement of respiration by fermentation (glycolysis), was only a symptom of cancer and not the cause. The primary cause was assumed to be at the level of gene expression. The view of cancer as a metabolic disease was gradually displaced with the view of cancer as a genetic disease. While there is renewed interest in the energy metabolism of cancer cells, it is widely thought that the Warburg effect and the metabolic defects expressed in cancer cells arise primarily from genomic mutability selected during tumor progression. Emerging evidence, however, questions the genetic origin of cancer and suggests that cancer is primarily a metabolic disease.

Our goal is to revisit the argument of tumor cell origin and to provide a general hypothesis that genomic mutability and essentially all hallmarks of cancer, including the Warburg effect, can be linked to impaired respiration and energy metabolism. In brief, damage to cellular respiration precedes and underlies the genome instability that accompanies tumor development. Once established, genome instability contributes to further respiratory impairment, genome mutability, and tumor progression. In other words, effects become causes. This hypothesis is based on evidence that nuclear genome integrity is largely dependent on mitochondrial energy homeostasis and that all cells require a constant level of useable energy to maintain viability. While Warburg recognized the centrality of impaired respiration in the origin of cancer, he did not link this phenomenon to what are now recognize as the hallmarks of cancer. We review evidence that make these linkages and expand Warburg’s ideas on how impaired energy metabolism can be exploited for tumor management and prevention.

“The standard of care should never have been written in granite. It should be flexible. If you have something else that comes along that might be better, you’d think there would be enthusiasm.”

Professor Tom Seyfried

Mitochondrial function in cancer cells

Considerable controversy has surrounded the issue of mitochondrial function in cancer cells. Sidney Weinhouse and Britton Chance initiated much of this controversy through their critical evaluation of the Warburg theory and the role of mitochondrial function. Basically, Weinhouse felt that quantitatively and qualitatively normal carbon and electron transport could occur in cancer cells despite the presence of elevated glycolysis. Weinhouse assumed that oxygen consumption and CO₂production were indicative of coupled respiration. However, excessive amounts of Donnan active material (ATP) would be produced if elevated glycolysis were expressed together with coupled respiration. Accumulation of Donnan active material will induce cell swelling and produce a physiological state beyond the Gibbs-Donnan equilibrium. The occurrence of up-regulated glycolysis together with normal coupled respiration is incompatible with metabolic homeostasis and cell viability. Chance and Hess also argued against impaired respiration in cancer based on their spectrophotometric studies showing mostly normal electron transfer in ascites tumor cells. These studies, however, failed to assess the level of ATP production as a consequence of normal electron transfer and did not exclude the possibility of elevated ATP production through TCA cycle substrate level phosphorylation. As discussed below, mitochondrial uncoupling can give the false impression of functional respiratory capacity.

Oxygen uptake and CO₂ production can occur in mitochondria that are uncoupled and/or dysfunctional. While reduced oxygen uptake can be indicative of reduced oxidative phosphorylation, increased oxygen uptake may or may not be indicative of increased oxidative phosphorylation and ATP production. Ramanathan and co-workers showed that oxygen consumption was greater, but oxygen dependent (aerobic) ATP synthesis was less in cells with greater tumorigenic potential than in cells with lower tumorigenic potential. These findings are consistent with mitochondrial uncoupling in tumor cells. It was for these types of observations in other systems that Warburg considered the phenomenon of aerobic glycolysis as too capricious to serve as a reliable indicator of respiratory status. Heat production is also greater in poorly differentiated high glycolytic tumor cells than in differentiated low glycolytic cells. Heat production is consistent with mitochondrial uncoupling in these highly tumorigenic cells. Although Burk, Schade, Colowick and others convincingly dispelled the main criticisms of the Warburg theory, citations to the older arguments for normal respiration in cancer cells persist in current discussions of the subject.

Besides glucose, glutamine can also serve as a major energy metabolite for some cancers. Glutamine is often present in high concentrations in culture media and serum. Cell viability and growth can be maintained from energy generated through substrate level phosphorylation in the TCA cycle using glutamine as a substrate. Energy obtained through this pathway could give the false impression of normal oxidative phosphorylation, as oxygen consumption and CO₂ production can arise from glutaminolysis and uncoupled oxidative phosphorylation. Hence, evidence suggesting that mitochondrial function is normal in cancer cells should be considered with caution unless data are provided, which exclude substrate level phosphorylation through glutaminolysis or glycolysis as alternative sources of energy.

Mitochondrial dysfunction in cancer cells

Numerous studies show that tumor mitochondria are structurally and functionally abnormal and incapable of generating normal levels of energy. Recent evidence also shows that the in vitro growth environment alters the lipid composition of mitochondrial membranes and electron transport chain function. Moreover, the mitochondrial lipid abnormalities induced from the in vitro growth environment are different from the lipid abnormalities found between normal tissue and tumors that are grown in vivo. It appears that the in vitro growth environment reduces Complex I activity and obscures the boundaries of the Crabtree and the Warburg effects. The Crabtree effect involves the inhibition of respiration by high levels of glucose, whereas the Warburg effect involves inhibition of respiration from impaired oxidative phosphorylation. While the Crabtree effect is reversible, the Warburg effect is largely irreversible. Similarities in mitochondrial lipids found between lung epidermoid carcinoma and fetal lung cells are also consistent with respiratory defects in tumor cells. The bioenergetic capacity of mitochondria is dependent to a large extent on the content and composition of mitochondrial lipids.

Alterations in mitochondrial membrane lipids and especially the inner membrane enriched lipid, cardiolipin, disrupt the mitochondrial proton motive gradient (ΔΨ_m) thus inducing protein-independent uncoupling with concomitant reduction in respiratory energy production. Cancer cells contain abnormalities in cardiolipin content or composition, which are associated with electron transport abnormalities. Cardiolipin is the only lipid synthesized almost exclusively in the mitochondria. Proteins of the electron transport chain evolved to function in close association with cardiolipin. Besides altering the function of most electron transport chain complexes including the F1-ATPase, abnormalities in cardiolipin content and composition can also inhibit uptake of ADP through the adenine nucleotide transporter thus altering the efficiency of oxidative phosphorylation. Abnormalities in the content and composition of cardiolipin will also prevent oxidation of the coenzyme Q couple thus producing reactive oxygen species during tumor progression. Increased ROS production can impair genome stability, tumor suppressor gene function, and control over cell proliferation. Hence, abnormalities in CL can alter cancer cell respiration in numerous ways.

Cardiolipin abnormalities in cancer cells can arise from any number of unspecific influences to include damage from mutagens and carcinogens, radiation, low level hypoxia, inflammation, ROS, or from inherited mutations that alter mitochondrial energy homeostasis. Considering the dynamic behavior of mitochondria involving regular fusions and fissions, abnormalities in mitochondrial lipid composition and especially of cardiolipin could be rapidly disseminated throughout the cellular mitochondrial network and could even be passed along to daughter cells somatically, through cytoplasmic inheritance.

Besides lipidomic evidence supporting the Warburg cancer theory, recent studies from Cuezva and colleagues also provide compelling proteomic evidence supporting the theory. Their results showed a drop in the β-F1-ATPase/Hsp60 ratio concurrent with an upregulation of the glyceraldehyde-3-phosphate dehydrogenase potential in most common human tumors. These and other observations indicate that the bioenergetic capacity of tumor cells is largely defective. Viewed collectively, the bulk of the experimental evidence indicates that mitochondria structure and function is abnormal in cancer cells. Hence, mitochondrial dysfunction will cause cancer cells to rely more heavily than non-cancer cells on substrate level phosphorylation for energy production in order to maintain membrane pump function and cell viability.

Implications of the hypothesis to cancer prevention

If impaired mitochondrial energy metabolism underlies the origin of most cancers as proposed here, then protecting mitochondria from damage becomes a logical and simple approach for preventing cancer. It is well documented that the incidence of cancer can be significantly reduced by avoiding exposure to those agents or conditions that provoke tissue inflammation such as smoking, alcohol, carcinogenic chemicals, ionizing radiation, obesity etc. Chronic inflammation, regardless of origin, damages tissue morphogenetic fields that eventually produce neoplastic cells. Part of this tissue damage will involve injury to the mitochondria in the affected cells. The prevention of inflammation and damage to the tissue microenvironment will go far in reducing the incidence of most cancers. Vaccines against some oncogenic viruses can also reduce the incidence of cancers, as these viruses can damage mitochondria in infected tissues. Hence, simply reducing exposure to cancer risk factors, which produce chronic inflammation and mitochondrial damage, will reduce the incidence of at least 80% of all cancers. In principle, there are few chronic diseases more easily preventable than cancer.

In addition to avoiding exposure to established cancer risk factors, the metabolism of ketone bodies protects the mitochondria from inflammation and damaging ROS. ROS production increases naturally with age and damages cellular proteins, lipids, and nucleic acids. Accumulation of ROS decreases the efficiency of mitochondrial energy production. The origin of mitochondrial ROS comes largely from the spontaneous reaction of molecular oxygen (O₂) with the semiquinone radical of coenzyme Q, ^.QH, to generate the superoxide radical O₂–^.. Coenzyme Q is a hydrophobic molecule that resides in the inner mitochondrial membrane and is essential for electron transfer. Ketone body metabolism increases the ratio of the oxidized form to the fully reduced form of coenzyme Q (CoQ/CoQH₂). Oxidation of the coenzyme Q couple reduces the amount of the semiquinone radical, thus decreasing superoxide production.

Since the cytosolic free NADP⁺/NADPH concentration couple is in near equilibrium with the glutathione couple, ketone body metabolism will also increase the reduced form of glutathione thus facilitating destruction of hydrogen peroxide. The reduction of free radicals through ketone body metabolism will therefore reduce tissue inflammation provoked by ROS while enhancing the energy efficiency of mitochondria. Ketone bodies are not only a more efficient metabolic fuel than glucose, but also possess anti-inflammatory potential. Metabolism of ketone bodies for energy will maintain mitochondrial health and efficiency thus reducing the incidence of cancer.

The simplest means of initiating the metabolism of ketone bodies is through dietary energy restriction with adequate nutrition. It is important to emphasize adequate nutrition, as calorie restriction associated with malnutrition can potentially increase cancer incidence. Consequently, consumption of foods containing the active groups of respiratory enzymes (iron salts, riboflavin, nicotinamide, and pantothenic acid) could be effective in maintaining health when combined with dietary energy restriction. The lowering of circulating glucose levels through calorie restriction facilitates the uptake and metabolism of ketone bodies for use as an alternative respiratory fuel. The metabolism of ketone bodies increases succinate dehydrogenase activity while enhancing the overall efficiency of energy production through respiration. In essence, dietary energy restriction and ketone body metabolism delays entropy. As cancer is a disease of accelerated entropy, dietary energy restriction targets the very essence of the disease.

It is well documented that dietary energy restriction can reduce the incidence of both inherited and acquired cancers in experimental animals. Evidence also indicates that dietary energy restriction can reduce the incidence of several human cancers. The implementation of periodic dietary energy restriction, which targets multiple cancer provoking factors, can be a simple and cost effective life-style change that is capable of reducing the incidence of cancer. Dietary energy restriction in rodents, however, is comparable to water only therapeutic fasting or to very low caloric diets (500-600 kcal/day) in humans. In light of this fact, it remains to be determined if members of our species are willing or motivated enough to adopt the life style changes necessary to prevent cancer.

Conclusions

Evidence is reviewed supporting a general hypothesis that cancer is primarily a disease of energy metabolism. All of the major hallmarks of the disease can be linked to impaired mitochondrial function. In order to maintain viability, tumor cells gradually transition to substrate level phosphorylation using glucose and glutamine as energy substrates. While cancer causing germline mutations are rare, the abundance of somatic genomic abnormalities found in the majority of cancers can arise as a secondary consequence of mitochondrial dysfunction. Once established, somatic genomic instability can contribute to further mitochondrial defects and to the metabolic inflexibility of the tumor cells. Systemic metastasis is the predicted outcome following protracted mitochondrial damage to cells of myeloid origin. Tumor cells of myeloid origin would naturally embody the capacity to exit and enter tissues. Two major conclusions emerge from the hypothesis; first that many cancers can regress if energy intake is restricted and, second, that many cancers can be prevented if energy intake is restricted. Consequently, energy restricted diets combined with drugs targeting glucose and glutamine can provide a rational strategy for the longer-term management and prevention of most cancers.