Posts Tagged ‘Lactate dehydrogenase’

Refined Warburg hypothesis -2.1.2

Writer and Curator: Larry H. Bernstein, MD, FCAP

Refined Warburg Hypothesis -2.1.2

The Warburg discoveries from 1922 on, and the influence on metabolic studies for the next 50 years was immense, and then the revelations of the genetic code took precedence.  Throughout this period, however, the brilliant work of Briton Chance, a giant of biochemistry at the University of Pennsylvania, opened new avenues of exploration that led to a recent resurgence in this vital need for answers in cancer research. The next two series of presentations will open up this resurgence of fundamental metabolic research in cancer and even neurodegenerative diseases. Cancer Cell Metabolism. Warburg and Beyond

Hsu PP, Sabatini DM
Cell, Sep 5, 2008; 134:703-707

Described decades ago, the Warburg effect of aerobic glycolysis is a key metabolic hallmark of cancer, yet its significance remains unclear. In this Essay, we re-examine the Warburg effect and establish a framework for understanding its contribution to the altered metabolism of cancer cells.

It is hard to begin a discussion of cancer cell metabolism without first mentioning Otto Warburg. A pioneer in the study of respiration, Warburg made a striking discovery in the 1920s. He found that, even in the presence of ample oxygen, cancer cells prefer to metabolize glucose by glycolysis, a seeming paradox as glycolysis, when compared to oxidative phosphorylation, is a less efficient pathway for producing ATP (Warburg, 1956). The Warburg effect has since been demonstrated in different types of tumors and the concomitant increase in glucose uptake has been exploited clinically for the detection of tumors by fluorodeoxyglucose positron emission tomography (FDG-PET). Although aerobic glycolysis has now been generally accepted as a metabolic hallmark of cancer, its causal relationship with cancer progression is still unclear. In this Essay, we discuss the possible drivers, advantages, and potential liabilities of the altered metabolism of cancer cells (Figure 1). Although our emphasis on the Warburg effect reflects the focus of the field, we would also like to encourage a broader approach to the study of cancer metabolism that takes into account the contributions of all interconnected small molecule pathways of the cell.

Figure 1. The Altered Metabolism of Cancer Cells

Drivers (A and B). The metabolic derangements in cancer cells may arise either from the selection of cells that have adapted to the tumor microenvironment or from aberrant signaling due to oncogene activation. The tumor microenvironment is spatially and temporally heterogeneous, containing regions of low oxygen and low pH (purple). Moreover, many canonical cancer-associated signaling pathways induce metabolic reprogramming. Target genes activated by hypoxia inducible factor (HIF) decrease the dependence of the cell on oxygen, whereas Ras, Myc, and Akt can also upregulate glucose consumption and glycolysis. Loss of p53 may also recapitulate the features of the Warburg effect, that is, the uncoupling of glycolysis from oxygen levels. Advantages (C–E). The altered metabolism of cancer cells is likely to imbue them with several proliferative and survival advantages, such as enabling cancer cells to execute the biosynthesis of macromolecules (C), to avoid apoptosis (D), and to engage in local metabolite-based paracrine and autocrine signaling (E). Potential Liabilities (F and G). This altered metabolism, however, may also confer several vulnerabilities on cancer cells. For example, an upregulated metabolism may result in the build up of toxic metabolites, including lactate and noncanonical nucleotides, which must be disposed of (F). Moreover, cancer cells may also exhibit a high energetic demand, for which they must either increase flux through normal ATP-generating processes, or else rely on an increased diversity of fuel sources (G).

The Tumor Microenvironment Selects for Altered Metabolism

One compelling idea to explain the Warburg effect is that the altered metabolism of cancer cells confers a selective advantage for survival and proliferation in the unique tumor microenvironment. As the early tumor expands, it outgrows the diffusion limits of its local blood supply, leading to hypoxia and stabilization of the hypoxia-inducible transcription factor, HIF. HIF initiates a transcriptional program that provides multiple solutions to hypoxic stress (reviewed in Kaelin and Ratcliffe, 2008). Because a decreased dependence on aerobic respiration becomes advantageous, cell metabolism is shifted toward glycolysis by the increased expression of glycolytic enzymes, glucose transporters, and inhibitors of mitochondrial metabolism. In addition, HIF stimulates angiogenesis (the formation of new blood vessels) by upregulating several factors, including most prominently vascular endothelial growth factor (VEGF).

The oxygen levels within a tumor vary both spatially and temporally, and the resulting rounds of fluctuating oxygen levels potentially select for tumors that constitutively upregulate glycolysis. Interestingly, with the possible exception of tumors that have lost the von Hippel-Lindau protein (VHL), which normally mediates degradation of HIF, HIF is still coupled to oxygen levels, as evident from the heterogeneity of HIF expression within the tumor microenvironment (Wiesener et al., 2001; Zhong et al., 1999). Therefore, the Warburg effect—that is, an uncoupling of glycolysis from oxygen levels—cannot be explained solely by upregulation of HIF.

Recent work has demonstrated that the key components of the Warburg effect—increased glucose consumption, decreased oxidative phosphorylation, and accompanying lactate production—are also distinguishing features of oncogene activation. The signaling molecule Ras, a powerful oncogene when mutated, promotes glycolysis (reviewed in Dang and Semenza, 1999; Samanathan et al., 2005). Akt kinase, a well-characterized downstream effector of insulin signaling, reprises its role in glucose uptake and utilization in the cancer setting (reviewed in Manning and Cantley, 2007), whereas the Myc transcription factor upregulates the expression of various metabolic genes (reviewed in Gordan et al., 2007). The most parsimonious route to tumorigenesis may be activation of key oncogenic nodes that execute a proliferative program, of which metabolism may be one important arm. Moreover, regulation of metabolism is not exclusive to oncogenes. Loss of the tumor suppressor protein p53 prevents expression of the gene encoding SCO2 (the synthesis of cytochrome c oxidase protein), which interferes with the function of the mitochondrial respiratory chain (Matoba et al., 2006). A second p53 effector, TIGAR (TP53-induced glycolysis and apoptosis regulator), inhibits glycolysis by decreasing levels of fructose-2,6-bisphosphate, a potent stimulator of glycolysis and inhibitor of gluconeogenesis (Bensaad et al., 2006). Other work also suggests that p53-mediated regulation of glucose metabolism may be dependent on the transcription factor NF-κB (Kawauchi et al., 2008).
It has been shown that inhibition of lactate dehydrogenase A (LDH-A) prevents the Warburg effect and forces cancer cells to revert to oxidative phosphorylation in order to reoxidize NADH and produce ATP (Fantin et al., 2006; Shim et al., 1997). While the cells are respiratory competent, they exhibit attenuated tumor growth, suggesting that aerobic glycolysis might be essential for cancer progression. In a primary fibroblast cell culture model of stepwise malignant transformation through overexpression of telomerase, large and small T antigen, and the H-Ras oncogene, increasing tumorigenicity correlates with sensitivity to glycolytic inhibition. This finding suggests that the Warburg effect might be inherent to the molecular events of transformation (Ramanathan et al., 2005). However, the introduction of similar defined factors into human mesenchymal stem cells (MSCs) revealed that transformation can be associated with increased dependence on oxidative phosphorylation (Funes et al., 2007). Interestingly, when introduced in vivo these transformed MSCs do upregulate glycolytic genes, an effect that is reversed when the cells are explanted and cultured under normoxic conditions. These contrasting models suggest that the Warburg effect may be context dependent, in some cases driven by genetic changes and in others by the demands of the microenvironment. Regardless of whether the tumor microenvironment or oncogene activation plays a more important role in driving the development of a distinct cancer metabolism, it is likely that the resulting alterations confer adaptive, proliferative, and survival advantages on the cancer cell.

Altered Metabolism Provides Substrates for Biosynthetic Pathways

Although studies in cancer metabolism have largely been energy-centric, rapidly dividing cells have diverse requirements. Proliferating cells require not only ATP but also nucleotides, fatty acids, membrane lipids, and proteins, and a reprogrammed metabolism may serve to support synthesis of macromolecules. Recent studies have shown that several steps in lipid synthesis are required for and may even actively promote tumorigenesis. Inhibition of ATP citrate lyase, the distal enzyme that converts mitochondrial-derived citrate into cytosolic acetyl coenzyme A, the precursor for many lipid species, prevents cancer cell proliferation and tumor growth (Hatzivassiliou et al., 2005). Fatty acid synthase, expressed at low levels in normal tissues, is upregulated in cancer and may also be required for tumorigenesis (reviewed in Menendez and Lupu, 2007). Furthermore, cancer cells may also enhance their biosynthetic capabilities by expressing a tumor-specific form of pyruvate kinase (PK), M2-PK. Pyruvate kinase catalyzes the third irreversible reaction of glycolysis, the conversion of phosphoenolpyruvate (PEP) to pyruvate. Surprisingly, the M2-PK of cancer cells is thought to be less active in the conversion of PEP to pyruvate and thus less efficient at ATP production (reviewed in Mazurek et al., 2005). A major advantage to the cancer cell, however, is that the glycolytic intermediates upstream of PEP might be shunted into synthetic processes.

Biosynthesis, in addition to causing an inherent increase in ATP demand in order to execute synthetic reactions, should also cause a decrease in ATP supply as various glycolytic and Krebs cycle intermediates are diverted. Lipid synthesis, for example, requires the cooperation of glycolysis, the Krebs cycle, and the pentose phosphate shunt. As pyruvate must enter the mitochondria in this case, it avoids conversion to lactate and therefore cannot contribute to glycolysis-derived ATP. Moreover, whereas increased biosynthesis may explain the glucose hunger of cancer cells, it cannot explain the increase in lactic acid production originally described by Warburg, suggesting that lactate must also result from the metabolism of non-glucose substrates. Recently, it has been demonstrated that glutamine may be metabolized by the citric acid cycle in cancer cells and converted into lactate, producing NADPH for lipid biosynthesis and oxaloacetate for replenishment of Krebs cycle intermediates (DeBerardinis et al., 2007).

Metabolic Pathways Regulate Apoptosis

In addition to involvement in proliferation, altered metabolism may promote another cancer-essential function: the avoidance of apoptosis. Loss of the p53 target TIGAR sensitizes cancer cells to apoptosis, most likely by causing an increase in reactive oxygen species (Bensaad et al., 2006). On the other hand, overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) prevents caspase-independent cell death, presumably by stimulating glycolysis, increasing cellular ATP levels, and promoting autophagy (Colell et al., 2007). Whether or not GAPDH plays a physiological role in the regulation of cell death remains to be determined. Intriguingly, Bonnet et al. (2007) have reported that treating cancer cells with dichloroacetate (DCA), a small molecule inhibitor of pyruvate dehydrogenase kinase, has striking effects on their survival and on xenograft tumor growth.

DCA, a currently approved treatment for congenital lactic acidosis, activates oxidative phosphorylation and promotes apoptosis by two mechanisms. First, increased flux through the electron transport chain causes depolarization of the mitochondrial membrane potential (which the authors found to be hyperpolarized specifically in cancer cells) and release of the apoptotic effector cytochrome c. Second, an increase in reactive oxygen species generated by oxidative phosphorylation upregulates the voltage-gated K+ channel, leading to potassium ion efflux and caspase activation. Their work suggests that cancer cells may shift their metabolism to glycolysis in order to prevent cell death and that forcing cancer cells to respire aerobically can counteract this adaptation.

Cancer Cells May Signal Locally in the Tumor Microenvironment

Cancer cells may rewire metabolic pathways to exploit the tumor microenvironment and to support cancer-specific signaling. Without access to the central circulation, it is possible that metabolites can be concentrated locally and reach suprasystemic levels, allowing cancer cells to engage in metabolite-mediated autocrine and paracrine signaling that does not occur in normal tissues. So called androgen-independent prostate cancers may only be independent from exogenous, adrenal-synthesized androgens. Androgen-independent prostate cancer cells still express the androgen receptor and may be capable of autonomously synthesizing their own androgens (Stanbrough et al., 2006).

Metabolism as an Upstream Modulator of Signaling Pathways

Not only is metabolism downstream of oncogenic pathways, but an altered upstream metabolism may affect the activity of signaling pathways that normally sense the state of the cell. Individuals with inherited mutations in succinate dehydrogenase and fumarate hydratase develop highly angiogenic tumors, not unlike those exhibiting loss of the VHL tumor suppressor protein that acts upstream of HIF (reviewed in Kaelin and Ratcliffe, 2008). The mechanism of tumorigenesis in these cancer syndromes is still contentious. However, it has been proposed that loss of succinate dehydrogenase and fumarate hydratase causes an accumulation of succinate or fumarate, respectively, leading to inhibition of the prolyl hydroxylases that mark HIF for VHL-mediated degradation (Isaacs et al., 2005; Pollard et al., 2005; Selak et al., 2005). In this rare case, succinate dehydrogenase and fumarate hydratase are acting as bona fide tumor suppressors.

There are many complex questions to be answered: Is it possible that cancer cells exhibit “metabolite addiction”? Are there unique cancer-specific metabolic pathways, or combinations of pathways, utilized by the cancer cell but not by normal cells? Are different stages of metabolic adaptations required for the cancer cell to progress from the primary tumor stage to invasion to metastasis? How malleable is cancer metabolism? Cancer metabolism. The Warburg effect today

Ferreira LMR
Exp Molec Pathol 2010; 89:372-383.

One of the first studies on the energy metabolism of a tumor was carried out, in 1922, in the laboratory of Otto Warburg. He established that cancer cells exhibited a specific metabolic pattern, characterized by a shift from respiration to fermentation, which has been later named the Warburg effect. Considerable work has been done since then, deepening our understanding of the process, with consequences for diagnosis and therapy. This review presents facts and perspectives on the Warburg effect for the 21st century.

Research highlights

► Warburg first established a tumor metabolic pattern in the 1920s. ► Tumors’ increased glucose uptake has been studied since then. ► Cancer bioenergetics’ study provides insights in all its hallmarks. ► New cancer diagnostic and therapeutic techniques focus on cancer metabolism.

Contestation to Warburg’s ideas
Glucose’s uptake and intracellular fates
Lactate production and induced acidosis
Impairment of mitochondrial function
Tumour microenvironment
Proliferating versus cancer cells
More on cancer bioenergetics – integration of metabolism
Perspectives New aspects of the Warburg effect in cancer cell biology

Bensinger SJ, Cristofk HR
Sem Cell Dev Biol 2012; 23:352-361

Altered cellular metabolism is a defining feature of cancer [1]. The best studied metabolic phenotype of cancer is aerobic glycolysis–also known as the Warburg effect–characterized by increased metabolism of glucose to lactate in the presence of sufficient oxygen. Interest in the Warburg effect has escalated in recent years due to the proven utility of FDG-PET for imaging tumors in cancer patients and growing evidence that mutations in oncogenes and tumor suppressor genes directly impact metabolism. The goals of this review are to provide an organized snapshot of the current understanding of regulatory mechanisms important for Warburg effect and its role in tumor biology. Since several reviews have covered aspects of this topic in recent years, we focus on newest contributions to the field and reference other reviews where appropriate.


► This review discusses regulatory mechanisms that contribute to the Warburg effect in cancer. ► We list cancers for which FDG-PET has established applications as well as those cancers for which FDG-PET has not been established. ► PKM2 is highlighted as an important integrator of diverse cellular stimuli to modulate metabolic flux and cancer cell proliferation. ► We discuss how cancer metabolism can directly influence gene expression programs. ► Contribution of aerobic glycolysis to the cancer microenvironment and chemotherapeutic resistance/susceptibility is also discussed.

Regulation of the Warburg effect

PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

Fig. 1. PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

Metabolism can directly influence gene expression programs

Metabolism can directly influence gene expression programs

Fig. 2. Metabolism can directly influence gene expression programs. A schematic representation of how metabolism can intrinsically influence epigenetics resulting in durable and heritable gene expression programs in progeny. Choosing between glycolysis and oxidative phosphorylation. A tumor’s dilemma

Jose C, Ballance N, Rossignal R
Biochim Biophys Acta 201; 1807(6): 552-561.

A considerable amount of knowledge has been produced during the last five years on the bioenergetics of cancer cells, leading to a better understanding of the regulation of energy metabolism during oncogenesis, or in adverse conditions of energy substrate intermittent deprivation. The general enhancement of the glycolytic machinery in various cancer cell lines is well described and recent analyses give a better view of the changes in mitochondrial oxidative phosphorylation during oncogenesis. While some studies demonstrate a reduction of oxidative phosphorylation (OXPHOS) capacity in different types of cancer cells, other investigations revealed contradictory modifications with the upregulation of OXPHOS components and a larger dependency of cancer cells on oxidative energy substrates for anabolism and energy production. This apparent conflictual picture is explained by differences in tumor size, hypoxia, and the sequence of oncogenes activated. The role of p53, C-MYC, Oct and RAS on the control of mitochondrial respiration and glutamine utilization has been explained recently on artificial models of tumorigenesis. Likewise, the generation of induced pluripotent stem cells from oncogene activation also showed the role of C-MYC and Oct in the regulation of mitochondrial biogenesis and ROS generation. In this review article we put emphasis on the description of various bioenergetic types of tumors, from exclusively glycolytic to mainly OXPHOS, and the modulation of both the metabolic apparatus and the modalities of energy substrate utilization according to tumor stage, serial oncogene activation and associated or not fluctuating microenvironmental substrate conditions. We conclude on the importance of a dynamic view of tumor bioenergetics.

Research Highlights

►The bioenergetics of cancer cells differs from normals. ►Warburg hypothesis is not verified in tumors using mitochondria to synthesize ATP. ►Different oncogenes can either switch on or switch off OXPHOS. ►Bioenergetic profiling is a prerequisite to metabolic therapy. ►Aerobic glycolysis and OXPHOS cooperate during cancer progression.

  1. Cancer cell variable bioenergetics

Cancer cells exhibit profound genetic, bioenergetic and histological differences as compared to their non-transformed counterpart. All these modifications are associated with unlimited cell growth, inhibition of apoptosis and intense anabolism. Transformation from a normal cell to a malignant cancer cell is a multi-step pathogenic process which includes a permanent interaction between cancer gene activation (oncogenes and/or tumor-suppressor genes), metabolic reprogramming and tumor-induced changes in microenvironment. As for the individual genetic mapping of human tumors, their metabolic characterization (metabolic–bioenergetic profiling) has evidenced a cancer cell-type bioenergetic signature which depends on the history of the tumor, as composed by the sequence of oncogenes activated and the confrontation to intermittent changes in oxygen, glucose and amino-acid delivery.

In the last decade, bioenergetic studies have highlighted the variability among cancer types and even inside a cancer type as regards to the mechanisms and the substrates preferentially used for deriving the vital energy. The more popular metabolic remodeling described in tumor cells is an increase in glucose uptake, the enhancement of glycolytic capacity and a high lactate production, along with the absence of respiration despite the presence of high oxygen concentration (Warburg effect) [1]. To explain this abnormal bioenergetic phenotype pioneering hypotheses proposed the impairment of mitochondrial function in rapidly growing cancer cells [2].

Although the increased consumption of glucose by tumor cells was confirmed in vivo by positron emission tomography (PET) using the glucose analog 2-(18F)-fluoro-2-deoxy-d-glucose (FDG), the actual utilization of glycolysis and oxidative phosphorylation (OXPHOS) cannot be evaluated with this technique. Nowadays, Warburg’s “aerobic-glycolysis” hypothesis has been challenged by a growing number of studies showing that mitochondria in tumor cells are not inactive per se but operate at low capacity [3] or, in striking contrast, supply most of the ATP to the cancer cells [4]. Intense glycolysis is effectively not observed in all tumor types. Indeed not all cancer cells grow fast and intense anabolism is not mandatory for all cancer cells. Rapidly growing tumor cells rely more on glycolysis than slowly growing tumor cells. This is why a treatment with bromopyruvate, for example is very efficient only on rapidly growing cells and barely useful to decrease the growth rate of tumor cells when their normal proliferation is slow. Already in 1979, Reitzer and colleagues published an article entitled “Evidence that glutamine, not sugar, is the major energy source for cultured Hela cells”, which demonstrated that oxidative phosphorylation was used preferentially to produce ATP in cervical carcinoma cells [5]. Griguer et al. also identified several glioma cell lines that were highly dependent on mitochondrial OXPHOS pathway to produce ATP [6]. Furthermore, a subclass of glioma cells which utilize glycolysis preferentially (i.e., glycolytic gliomas) can also switch from aerobic glycolysis to OXPHOS under limiting glucose conditions  [7] and [8], as observed in cervical cancer cells, breast carcinoma cells, hepatoma cells and pancreatic cancer cells [9][10] and [11]. This flexibility shows the interplay between glycolysis and OXPHOS to adapt the mechanisms of energy production to microenvironmental changes as well as differences in tumor energy needs or biosynthetic activity. Herst and Berridge also demonstrated that a variety of human and mouse leukemic and tumor cell lines (HL60, HeLa, 143B, and U937) utilize mitochondrial respiration to support their growth [12]. Recently, the measurement of OXPHOS contribution to the cellular ATP supply revealed that mitochondria generate 79% of the cellular ATP in HeLa cells, and that upon hypoxia this contribution is reduced to 30% [4]. Again, metabolic flexibility is used to survive under hypoxia. All these studies demonstrate that mitochondria are efficient to synthesize ATP in a large variety of cancer cells, as reviewed by Moreno-Sanchez [13]. Despite the observed reduction of the mitochondrial content in tumors [3][14][15][16][17][18] and [19], cancer cells maintain a significant level of OXPHOS capacity to rapidly switch from glycolysis to OXPHOS during carcinogenesis. This switch is also observed at the level of glutamine oxidation which can occur through two modes, “OXPHOS-linked” or “anoxic”, allowing to derive energy from glutamine or serine regardless of hypoxia or respiratory chain reduced activity [20].
While glutamine, glycine, alanine, glutamate, and proline are typically oxidized in normal and tumor mitochondria, alternative substrate oxidations may also contribute to ATP supply by OXPHOS. Those include for instance the oxidation of fatty-acids, ketone bodies, short-chain carboxylic acids, propionate, acetate and butyrate (as recently reviewed in [21]).

  1. Varying degree of mitochondrial utilization during tumorigenesis

In vivo metabolomic analyses suggest the existence of a continuum of bioenergetic remodeling in rat tumors according to tumor size and its rate of growth [22]. Peter Vaupel’s group showed that small tumors were characterized by a low conversion of glucose to lactate whereas the conversion of glutamine to lactate was high. In medium sized tumors the flow of glucose to lactate as well as oxygen utilization was increased whereas glutamine and serine consumption were reduced. At this stage tumor cells started with glutamate and alanine production. Large tumors were characterized by a low oxygen and glucose supply but a high glucose and oxygen utilization rate. The conversion of glucose to glycine, alanine, glutamate, glutamine, and proline reached high values and the amino acids were released [22]. Certainly, in the inner layers constituting solid tumors, substrate and oxygen limitation is frequently observed. Experimental studies tried to reproduce these conditions in vitro and revealed that nutrients and oxygen limitation does not affect OXPHOS and cellular ATP levels in human cervix tumor [23]. Furthermore, the growth of HeLa cells, HepG2 cells and HTB126 (breast cancer) in aglycemia and/or hypoxia even triggered a compensatory increase in OXPHOS capacity, as discussed above. Yet, the impact of hypoxia might be variable depending on cell type and both the extent and the duration of oxygen limitation.
In two models of sequential oncogenesis, the successive activation of specific oncogenes in non-cancer cells evidenced the need for active OXPHOS to pursue tumorigenesis. Funes et al. showed that the transformation of human mesenchymal stem cells increases their dependency on OXPHOS for energy production [24], while Ferbeyre et al. showed that cells expressing oncogenic RAS display an increase in mitochondrial mass, mitochondrial DNA, and mitochondrial production of reactive oxygen species (ROS) prior to the senescent cell cycle arrest [25]. Such observations suggest that waves of gene regulation could suppress and then restore OXPHOS in cancer cells during tumorigenesis [20]. Therefore, the definition of cancer by Hanahan and Weinberg [26] restricted to six hallmarks (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 angiogenesis, and 6—tissue invasion and metastases) should also include metabolic reprogramming, as the seventh hallmark of cancer. This amendment was already proposed by Tennant et al. in 2009 [27]. In 2006, the review Science published a debate on the controversial views of Warburg theory [28], in support of a more realistic description of cancer cell’s variable bioenergetic profile. The pros think that high glycolysis is an obligatory feature of human tumors, while the cons propose that high glycolysis is not exclusive and that tumors can use OXPHOS to derive energy. A unifying theory closer to reality might consider that OXPHOS and glycolysis cooperate to sustain energy needs along tumorigenesis [20]. The concept of oxidative tumors, against Warburg’s proposal, was introduced by Guppy and colleagues, based on the observation that breast cancer cells can generate 80% of their ATP by the mitochondrion [29]. The comparison of different cancer cell lines and excised tumors revealed a variety of cancer cell’s bioenergetic signatures which raised the question of the mechanisms underlying tumor cell metabolic reprogramming, and the relative contribution of oncogenesis and microenvironment in this process. It is now widely accepted that rapidly growing cancer cells within solid tumors suffer from a lack of oxygen and nutrients as tumor grows. In such situation of compromised energy substrate delivery, cancer cell’s metabolic reprogramming is further used to sustain anabolism (Fig. 1), through the deviation of glycolysis, Krebs cycle truncation and OXPHOS redirection toward lipid and protein synthesis, as needed to support uncontrolled tumor growth and survival [30] and [31]. Again, these features are not exclusive to all tumors, as Krebs cycle truncation was only observed in some cancer cells, while other studies indicated that tumor cells can maintain a complete Krebs cycle [13] in parallel with an active citrate efflux. Likewise, generalizations should be avoided to prevent over-interpretations.
Fig. 1. Energy metabolism at the crossroad between catabolism and anabolism.

Energy metabolism at the crossroad between catabolism and anabolism.

Energy metabolism at the crossroad between catabolism and anabolism.

The oncogene C-MYC participate to these changes via the stimulation of glutamine utilization through the coordinate expression of genes necessary for cells to engage in glutamine catabolism [30]. According to Newsholme EA and Board M [32] both glycolysis and glutaminolysis not only serve for ATP production, but also provide precious metabolic intermediates such as glucose-6-phosphate, ammonia and aspartate required for the synthesis of purine and pyrimidine nucleotides (Fig. 1). In this manner, the observed apparent excess in the rates of glycolysis and glutaminolysis as compared to the requirement for energy production could be explained by the need for biosynthetic processes. Yet, one should not reduce the shift from glycolysis to OXPHOS utilization to the sole activation of glutaminolysis, as several other energy substrates can be used by tumor mitochondria to generate ATP [21]. The contribution of these different fuels to ATP synthesis remains poorly investigated in human tumors.

  1. The metabolism of pre-cancer cells and its ongoing modulation by carcinogenesis

At the beginning of cancer, there might have been a cancer stem cell hit by an oncogenic event, such as alterations in mitogen signaling to extracellular growth factor receptors (EGFR), oncogenic activation of these receptors, or oncogenic alterations of downstream targets in the pathways that leads to cell proliferation (RAS–Raf–ERK and PI3K–AKT, both leading to m-TOR activation stimulating cell growth). Alterations of checkpoint genes controlling the cell cycle progression like Rb also participate in cell proliferation (Fig. 2) and this re-entry in the cell cycle implies three major needs to fill in: 1) supplying enough energy to grow and 2) synthesize building blocks de novo and 3) keep vital oxygen and nutrients available. However, the bioenergetic status of the pre-cancer cell could determine in part the evolution of carcinogenesis, as shown on mouse embryonic stem cells. In this study, Schieke et al. showed that mitochondrial energy metabolism modulates both the differentiation and tumor formation capacity of mouse embryonic stem cells [37]. The idea that cancer derives from a single cell, known as the cancer stem cell hypothesis, was introduced by observations performed on leukemia which appeared to be organized as origination from a primitive hematopoietic cell [38]. Nowadays cancer stem cells were discovered for all types of tumors [39][40][41] and [42], but little is known of their bioenergetic properties and their metabolic adaptation to the microenvironment. This question is crucial as regards the understanding of what determines the wide variety of cancer cell’s metabolic profile.

Impact of different oncogenes on tumor progression and energy metabolism remodeling.

Impact of different oncogenes on tumor progression and energy metabolism remodeling.

Fig. 2. Impact of different oncogenes on tumor progression and energy metabolism remodeling.

The analysis of the metabolic changes that occur during the transformation of adult mesenchymal stem cells revealed that these cells did not switch to aerobic glycolysis, but their dependency on OXPHOS was even increased [24]. Hence, mitochondrial energy metabolism could be critical for tumorigenesis, in contrast with Warburg’s hypothesis. As discussed above, the oncogene C-MYC also stimulates OXPHOS [30]. Furthermore, it was recently demonstrated that cells chronically treated with oligomycin repress OXPHOS and produce larger tumors with higher malignancy [19]. Likewise, alteration of OXPHOS by mutations in mtDNA increases tumorigenicity in different types of cancer cells [43][44] and [45].

Recently, it was proposed that mitochondrial energy metabolism is required to generate reactive oxygen species used for the carcinogenetic process induced by the K-RAS mutation [46]. This could explain the large number of mitochondrial DNA mutations found in several tumors. The analysis of mitochondria in human embryonic cells which derive energy exclusively from anaerobic glycolysis have demonstrated an immature mitochondrial network characterized by few organelles with poorly developed cristae and peri-nuclear distribution [47] and [48]. The generation of human induced pluripotent stem cell by the introduction of different oncogenes as C-MYC and Oct4 reproduced this reduction of mitochondrial OXPHOS capacity[49] and [50]. This indicates again the impact of oncogenes on the control of OXPHOS and might explain the existence of pre-cancer stem cells with different bioenergetic backgrounds, as modeled by variable sequences of oncogene activation. Accordingly, the inhibition of mitochondrial respiratory chain has been recently found associated with enhancement of hESC pluripotency [51].

Based on the experimental evidence discussed above, one can argue that 1) glycolysis is indeed a feature of several tumors and associates with faster growth in high glucose environment, but 2) active OXPHOS is also an important feature of (other) tumors taken at a particular stage of carcinogenesis which might be more advantageous than a “glycolysis-only” type of metabolism in conditions of intermittent shortage in glucose delivery. The metabolic apparatus of cancer cells is not fixed during carcinogenesis and might depend both on the nature of the oncogenes activated and the microenvironment. It was indeed shown that cancer cells with predominant glycolytic metabolism present a higher malignancy when submitted to carcinogenetic induction and analysed under fixed experimental conditions of high glucose [19]. Yet, if one grows these cells in a glucose-deprived medium they shift their metabolism toward predominant OXPHOS, as shown in HeLa cells and other cell types [9]. Therefore, one might conclude that glycolytic cells have a higher propensity to generate aggressive tumors when glucose availability is high. However, these cells can become OXPHOS during tumor progression [24] and [52]. All these observations indicate again the importance of maintaining an active OXPHOS metabolism to permit evolution of both embryogenesis and carcinogenesis, which emphasizes the importance of targeting mitochondria to alter this malignant process.

  1. Oncogenes and the modulation of energy metabolism

Several oncogenes and associated proteins such as HIF-1α, RAS, C-MYC, SRC, and p53 can influence energy substrate utilization by affecting cellular targets, leading to metabolic changes that favor cancer cell survival, independently of the control of cell proliferation. These oncogenes stimulate the enhancement of aerobic glycolysis, and an increasing number of studies demonstrate that at least some of them can also target directly the OXPHOS machinery, as discussed in this article (Fig. 2). For instance, C-MYC can concurrently drive aerobic glycolysis and/or OXPHOS according to the tumor cell microenvironment, via the expression of glycolytic genes or the activation of mitochondrial oxidation of glutamine [53]. The oncogene RAS has been shown to increase OXPHOS activity in early transformed cells [24][52] and [54] and p53 modulates OXPHOS capacity via the regulation of cytochrome c oxidase assembly [55]. Hence, carcinogenic p53 deficiency results in a decreased level of COX2 and triggers a shift toward anaerobic metabolism. In this case, lactate synthesis is increased, but cellular ATP levels remain stable [56]. The p53-inducible isoform of phosphofructokinase, termed TP53-induced glycolysis and apoptotic regulator, TIGAR, a predominant phosphatase activity isoform of PFK-2, has also been identified as an important regulator of energy metabolism in tumors [57].

  1. Tumor specific isoforms (or mutated forms) of energy genes

Tumors are generally characterized by a modification of the glycolytic system where the level of some glycolytic enzymes is increased, some fetal-like isozymes with different kinetic and regulatory properties are produced, and the reverse and back-reactions of the glycolysis are strongly reduced [60]. The GAPDH marker of the glycolytic pathway is also increased in breast, gastric, lung, kidney and colon tumors [18], and the expression of glucose transporter GLUT1 is elevated in most cancer cells. The group of Cuezva J.M. developed the concept of cancer bioenergetic signature and of bioenergetic index to describe the metabolic profile of cancer cells and tumors [18], [61], [64], [65]. This signature describes the changes in the expression level of proteins involved in glycolysis and OXPHOS, while the BEC index gives a ratio of OXPHOS protein content to glycolytic protein content, in good correlation with cancer prognostic[61]. Recently, this group showed that the beta-subunit of the mitochondrial F1F0-ATP synthase is downregulated in a large number of tumors, thus contributing to the Warburg effect [64] and [65]. It was also shown that IF1 expression levels were increased in hepatocellular carcinomas, possibly to prevent the hydrolysis of glytolytic ATP [66]. Numerous changes occur at the level of OXPHOS and mitochondrial biogenesis in human tumors, as we reviewed previously [67]. Yet the actual impact of these changes in OXPHOS protein expression level or catalytic activities remains to be evaluated on the overall fluxes of respiration and ATP synthesis. Indeed, the metabolic control analysis and its extension indicate that it is often required to inhibit activity beyond a threshold of 70–85% to affect the metabolic fluxes [68] and [69]. Another important feature of cancer cells is the higher level of hexokinase II bound to mitochondrial membrane (50% in tumor cells). A study performed on human gliomas (brain) estimated the mitochondrial bound HK fraction (mHK) at 69% of total, as compared to 9% for normal brain [70]. This is consistent with the 5-fold amplification of the type II HK gene observed by Rempel et al. in the rapidly growing rat AS-30D hepatoma cell line, relative to normal hepatocytes [71]. HKII subcellular fractionation in cancer cells was described in several studies [72][73] and [74]. The group led by Pete Pedersen explained that mHK contributes to (i) the high glycolytic capacity by utilizing mitochondrially regenerated ATP rather than cytosolic ATP (nucleotide channelling) and (ii) the lowering of OXPHOS capacity by limiting Pi and ADP delivery to the organelle [75] and [76].

All these observations are consistent with the increased rate of FDG uptake observed by PET in living tumors which could result from both an increase in glucose transport, and/or an increase in hexokinase activity. However, FDG is not a complete substrate for glycolysis (it is only transformed into FDG-6P by hexokinase before to be eliminated) and cannot be used to evidence a general increase in the glycolytic flux. Moreover, FDG-PET scan also gives false positive and false negative results, indicating that some tumors do not depend on, or do not have, an increased glycolytic capacity. The fast glycolytic system described above is further accommodated in cancer cells by an increase in the lactate dehydrogenase isoform A (LDH-A) expression level. This isoform presents a higher Vmax useful to prevent the inhibition of high glycolysis by its end product (pyruvate) accumulation. Recently, Fantin et al. showed that inhibition of LDH-A in tumors diminishes tumorigenicity and was associated with the stimulation of mitochondrial respiration [79]. The preferential expression of the glycolytic pyruvate kinase isoenzyme M2 (PKM2) in tumor cells, determines whether glucose is converted to lactate for regeneration of energy (active tetrameric form, Warburg effect) or used for the synthesis of cell building blocks (nearly inactive dimeric form) [80]. In the last five years, mutations in proteins of the respiratory system (SDH, FH) and of the TCA cycle (IDH1,2) leading to the accumulation of metabolite and the subsequent activation of HIF-1α were reported in a variety of human tumors [81], [82] and [83].

  1. Tumor microenvironment modulates cancer cell’s bioenergetics

It was extensively described how hypoxia activates HIF-1α which stimulates in turn the expression of several glycolytic enzymes such as HK2, PFK, PGM, enolase, PK, LDH-A, MCT4 and glucose transporters Glut 1 and Glut 3. It was also shown that HIF-1α can reduce OXPHOS capacity by inhibiting mitochondrial biogenesis [14] and [15], PDH activity [87] and respiratory chain activity [88]. The low efficiency and uneven distribution of the vascular system surrounding solid tumors can lead to abrupt changes in oxygen (intermittent hypoxia) but also energy substrate delivery. .. The removal of glucose, or the inhibition of glycolysis by iodoacetate led to a switch toward glutamine utilization without delay followed by a rapid decrease in acid release. This illustrates once again how tumors and human cancer cell lines can utilize alternative energy pathway such as glutaminolysis to deal with glucose limitation, provided the presence of oxygen. It was also observed that in situations of glucose limitation, tumor derived-cells can adapt to survive by using exclusively an oxidative energy substrate [9] and [10]. This is typically associated with an enhancement of the OXPHOS system. … In summary, cancer cells can survive by using exclusively OXPHOS for ATP production, by altering significantly mitochondrial composition and form to facilitate optimal use of the available substrate (Fig. 3). Yet, glucose is needed to feed the pentose phosphate pathway and generate ribose essential for nucleotide biosynthesis. This raises the question of how cancer cells can survive in the growth medium which do not contain glucose (so-called “galactose medium” with dialysed serum [9]). In the OXPHOS mode, pyruvate, glutamate and aspartate can be derived from glutamine, as glutaminolysis can replenish Krebs cycle metabolic pool and support the synthesis of alanine and NADPH [31]. Glutamine is a major source for oxaloacetate (OAA) essential for citrate synthesis. Moreover, the conversion of glutamine to pyruvate is associated with the reduction of NADP+ to NADPH by malic enzyme. Such NADPH is a required electron donor for reductive steps in lipid synthesis, nucleotide metabolism and GSH reduction. In glioblastoma cells the malic enzyme flux was estimated to be high enough to supply all of the reductive power needed for lipid synthesis [31].

Fig. 3. Interplay between energy metabolism, oncogenes and tumor microenvironment during tumorigenesis (the “metabolic wave model”).

Interplay between energy metabolism, oncogenes and tumor microenvironment

Interplay between energy metabolism, oncogenes and tumor microenvironment

While the mechanisms leading to the enhancement of glycolytic capacity in tumors are well documented, less is known about the parallel OXPHOS changes. Both phenomena could result from a selection of pre-malignant cells forced to survive under hypoxia and limited glucose delivery, followed by an adaptation to intermittent hypoxia, pseudo-hypoxia, substrate limitation and acidic environment. This hypothesis was first proposed by Gatenby and Gillies to explain the high glycolytic phenotype of tumors [91], [92] and [93], but several lines of evidence suggest that it could also be used to explain the mitochondrial modifications observed in cancer cells.

  1. Aerobic glycolysis and mitochondria cooperate during cancer progression

Metabolic flexibility considers the possibility for a given cell to alternate between glycolysis and OXPHOS in response to physiological needs. Louis Pasteur found that in most mammalian cells the rate of glycolysis decreases significantly in the presence of oxygen (Pasteur effect). Moreover, energy metabolism of normal cell can vary widely according to the tissue of origin, as we showed with the comparison of five rat tissues[94]. During stem cell differentiation, cell proliferation induces a switch from OXPHOS to aerobic glycolysis which might generate ATP more rapidly, as demonstrated in HepG2 cells [95] or in non-cancer cells[96] and [97]. Thus, normal cellular energy metabolism can adapt widely according to the activity of the cell and its surrounding microenvironment (energy substrate availability and diversity). Support for this view came from numerous studies showing that in vitro growth conditions can alter energy metabolism contributing to a dependency on glycolysis for ATP production [98].

Yet, Zu and Guppy analysed numerous studies and showed that aerobic glycolysis is not inherent to cancer but more a consequence of hypoxia[99].

Table 1. Impact of different oncogenes on energy metabolism

Impact of different oncogenes on energy metabolism.

Impact of different oncogenes on energy metabolism. Mitohormesis

Yun J, Finkel T
Cell Metab May 2014; 19(5):757–766

For many years, mitochondria were viewed as semiautonomous organelles, required only for cellular energetics. This view has been largely supplanted by the concept that mitochondria are fully integrated into the cell and that mitochondrial stresses rapidly activate cytosolic signaling pathways that ultimately alter nuclear gene expression. Remarkably, this coordinated response to mild mitochondrial stress appears to leave the cell less susceptible to subsequent perturbations. This response, termed mitohormesis, is being rapidly dissected in many model organisms. A fuller understanding of mitohormesis promises to provide insight into our susceptibility for disease and potentially provide a unifying hypothesis for why we age.

Figure 1. The Basis of Mitohormesis. Any of a number of endogenous or exogenous stresses can perturb mitochondrial function. These perturbations are relayed to the cytosol through, at present, poorly understood mechanisms that may involve mitochondrial ROS as well as other mediators. These cytoplasmic signaling pathways and subsequent nuclear transcriptional changes induce various long-lasting cytoprotective pathways. This augmented stress resistance allows for protection from a wide array of subsequent stresses.

Figure 2. Potential Parallels between the Mitochondrial Unfolded Protein Response and Quorum Sensing in Gram-Positive Bacteria. In the C. elegans UPRmt response, mitochondrial proteins (indicated by blue swirls) are degraded by matrix proteases, and the oligopeptides that are generated are then exported through the ABC transporter family member HAF-1. Once in the cytosol, these peptides can influence the subcellular localization of the transcription factor ATFS-1. Nuclear ATFS-1 is capable of orchestrating a broad transcriptional response to mitochondrial stress. As such, this pathway establishes a method for mitochondrial and nuclear genomes to communicate. In some gram-positive bacteria, intracellularly generated peptides can be similarly exported through an ABC transporter protein. These peptides can be detected in the environment by a membrane-bound histidine kinases (HK) sensor. The activation of the HK sensor leads to phosphorylation of a response regulator (RR) protein that, in turn, can alter gene expression. This program allows communication between dispersed gram-positive bacteria and thus coordinated behavior of widely dispersed bacterial genomes.

Figure 3. The Complexity of Mitochondrial Stresses and Responses. A wide array of extrinsic and intrinsic mitochondrial perturbations can elicit cellular responses. As detailed in the text, genetic or pharmacological disruption of electron transport, incorrect folding of mitochondrial proteins, stalled mitochondrial ribosomes, alterations in signaling pathways, or exposure to toxins all appear to elicit specific cytoprotective programs within the cell. These adaptive responses include increased mitochondrial number (biogenesis), alterations in metabolism, increased antioxidant defenses, and augmented protein chaperone expression. The cumulative effect of these adaptive mechanisms might be an extension of lifespan and a decreased incidence of age-related pathologies. Mitochondrial function and energy metabolism in cancer cells. Past overview and future perspectives

Mayevsky A
Mitochondrion. 2009 Jun; 9(3):165-79

The involvements of energy metabolism aspects of mitochondrial dysfunction in cancer development, proliferation and possible therapy, have been investigated since Otto Warburg published his hypothesis. The main published material on cancer cell energy metabolism is overviewed and a new unique in vivo experimental approach that may have significant impact in this important field is suggested. The monitoring system provides real time data, reflecting mitochondrial NADH redox state and microcirculation function. This approach of in vivo monitoring of tissue viability could be used to test the efficacy and side effects of new anticancer drugs in animal models. Also, the same technology may enable differentiation between normal and tumor tissues in experimental animals and maybe also in patients.

 Energy metabolism in mammalian cells

Fig. 1. Schematic representation of cellular energy metabolism and its relationship to microcirculatory blood flow and hemoglobin oxygenation.

Fig. 2. Schematic representation of the central role of the mitochondrion in the various processes involved in the pathology of cancer cells and tumors. Six issues marked as 1–6 are discussed in details in the text.

In vivo monitoring of tissue energy metabolism in mammalian cells

Fig. 3. Schematic presentation of the six parameters that could be monitored for the evaluation of tissue energy metabolism (see text for details).

Optical spectroscopy of tissue energy metabolism in vivo

Multiparametric monitoring system

Fig. 4. (A) Schematic representation of the Time Sharing Fluorometer Reflectometer (TSFR) combined with the laser Doppler flowmeter (D) for blood flow monitoring. The time sharing system includes a wheel that rotates at a speed of3000 rpm wit height filters: four for the measurements of mitochondrial NADH(366 nm and 450 nm)and four for oxy-hemoglobin measurements (585 nm and 577 nm) as seen in (C). The source of light is a mercury lamp. The probe includes optical fibers for NADH excitation (Ex) and emission (Em), laser Doppler excitation (LD in), laser Doppler emission (LD out) as seen in part E The absorption spectrum of Oxy- and Deoxy- Hemoglobin indicating the two wave length used (C).

Fig. 7. Comparison between mitochondrial metabolic states in vitro and the typical tissue metabolic states in vivo evaluated by NADH redox state, tissue blood flow and hemoglobin oxygenation as could be measured by the suggested monitoring system.

(very important) Metabolic Reprogramming. Cancer Hallmark Even Warburg Did Not Anticipate

Ward PS, Thompson CB.
Cancer Cell 2012; 21(3):297-308

Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-associated alterations in metabolism be viewed as an indirect response to cell proliferation and survival signals. We contend that altered metabolism has attained the status of a core hallmark of cancer.

The propensity for proliferating cells to secrete a significant fraction of glucose carbon through fermentation was first elucidated in yeast. Otto Warburg extended these observations to mammalian cells, finding that proliferating ascites tumor cells converted the majority of their glucose carbon to lactate, even in oxygen-rich conditions. Warburg hypothesized that this altered metabolism was specific to cancer cells, and that it arose from mitochondrial defects that inhibited their ability to effectively oxidize glucose carbon to CO2. An extension of this hypothesis was that dysfunctional mitochondria caused cancer (Koppenol et al., 2011). Warburg’s seminal finding has been observed in a wide variety of cancers. These observations have been exploited clinically using 18F-deoxyglucose positron emission tomography (FDG-PET). However, in contrast to Warburg’s original hypothesis, damaged mitochondria are not at the root of the aerobic glycolysis exhibited by most tumor cells. Most tumor mitochondria are not defective in their ability to carry out oxidative phosphorylation. Instead, in proliferating cells mitochondrial metabolism is reprogrammed to meet the challenges of macromolecular synthesis. This possibility was never considered by Warburg and his contemporaries.

Advances in cancer metabolism research over the last decade have enhanced our understanding of how aerobic glycolysis and other metabolic alterations observed in cancer cells support the anabolic requirements associated with cell growth and proliferation. It has become clear that anabolic metabolism is under complex regulatory control directed by growth factor signal transduction in non-transformed cells. Yet despite these advances, the repeated refrain from traditional biochemists is that altered metabolism is merely an indirect phenomenon in cancer, a secondary effect that pales in importance to the activation of primary proliferation and survival signals (Hanahan and Weinberg, 2011). Most proto-oncogenes and tumor suppressor genes encode components of signal transduction pathways. Their roles in carcinogenesis have traditionally been attributed to their ability to regulate the cell cycle and sustain proliferative signaling while also helping cells evade growth suppression and/or cell death (Hanahan and Weinberg, 2011). But evidence for an alternative concept, that the primary functions of activated oncogenes and inactivated tumor suppressors are to reprogram cellular metabolism, has continued to build over the past several years. Evidence is also developing for the proposal that proto-oncogenes and tumor suppressors primarily evolved to regulate metabolism.

We begin this review by discussing how proliferative cell metabolism differs from quiescent cell metabolism on the basis of active metabolic reprogramming by oncogenes and tumor suppressors. Much of this reprogramming depends on utilizing mitochondria as functional biosynthetic organelles. We then further develop the idea that altered metabolism is a primary feature selected for during tumorigenesis. Recent advances have demonstrated that altered metabolism in cancer extends beyond adaptations to meet the increased anabolic requirements of a growing and dividing cell. Changes in cancer cell metabolism can also influence cellular differentiation status, and in some cases these changes arise from oncogenic alterations in metabolic enzymes themselves.

Metabolism in quiescent vs. proliferating cells nihms-360138-f0001

Metabolism in quiescent vs. proliferating cells: both use mitochondria.
(A) In the absence of instructional growth factor signaling, cells in multicellular organisms lack the ability to take up sufficient nutrients to maintain themselves. Neglected cells will undergo autophagy and catabolize amino acids and lipids through the TCA cycle, assuming sufficient oxygen is available. This oxidative metabolism maximizes ATP production. (B) Cells that receive instructional growth factor signaling are directed to increase their uptake of nutrients, most notably glucose and glutamine. The increased nutrient uptake can then support the anabolic requirements of cell growth: mainly lipid, protein, and nucleotide synthesis (biomass). Excess carbon is secreted as lactate. Proliferating cells may also use strategies to decrease their ATP production while increasing their ATP consumption. These strategies maintain the ADP:ATP ratio necessary to maintain glycolytic flux. Green arrows represent metabolic pathways, while black arrows represent signaling.

Metabolism is a direct, not indirect, response to growth factor signaling nihms-360138-f0002

Metabolism is a direct, not indirect, response to growth factor signaling nihms-360138-f0002

Metabolism is a direct, not indirect, response to growth factor signaling.
(A) The traditional demand-based model of how metabolism is altered in proliferating cells. In response to growth factor signaling, increased transcription and translation consume free energy and decrease the ADP:ATP ratio. This leads to enhanced flux of glucose carbon through glycolysis and the TCA cycle for the purpose of producing more ATP. (B) Supply-based model of how metabolism changes in proliferating cells. Growth factor signaling directly reprograms nutrient uptake and metabolism. Increased nutrient flux through glycolysis and the mitochondria in response to growth factor signaling is used for biomass production. Metabolism also impacts transcription and translation through mechanisms independent of ATP availability.

Alterations in classic oncogenes directly reprogram cell metabolism to increase nutrient uptake and biosynthesis. PI3K/Akt signaling downstream of receptor tyrosine kinase (RTK) activation increases glucose uptake through the transporter GLUT1, and increases flux through glycolysis. Branches of glycolytic metabolism contribute to nucleotide and amino acid synthesis. Akt also activates ATP-citrate lyase (ACL), promoting the conversion of mitochondria-derived citrate to acetyl-CoA for lipid synthesis. Mitochondrial citrate can be synthesized when glucose-derived acetyl-CoA, generated by pyruvate dehydrogenase (PDH), condenses with glutamine-derived oxaloacetate (OAA) via the activity of citrate synthase (CS). mTORC1 promotes protein synthesis and mitochondrial metabolism. Myc increases glutamine uptake and the conversion of glutamine into a mitochondrial carbon source by promoting the expression of the enzyme glutaminase (GLS). Myc also promotes mitochondrial biogenesis. In addition, Myc promotes nucleotide and amino acid synthesis, both through direct transcriptional regulation and through increasing the synthesis of mitochondrial metabolite precursors.

Pyruvate kinase M2 (PKM2) expression in proliferating cells is regulated by signaling and mitochondrial metabolism to facilitate macromolecular synthesis. PKM2 is a less active isoform of the terminal glycolytic enzyme pyruvate kinase. It is also uniquely inhibited downstream of tyrosine kinase signaling. The decreased enzymatic activity of PKM2 in the cytoplasm promotes the accumulation of upstream glycolytic intermediates and their shunting into anabolic pathways. These pathways include the serine synthetic pathway that contributes to nucleotide and amino acid production. When mitochondrial metabolism is excessive, reactive oxygen species (ROS) from the mitochondria can feedback to inhibit PKM2 activity. Acetylation of PKM2, dependent on acetyl-CoA availability, may also promote PKM2 degradation and further contribute to increased flux through anabolic synthesis pathways branching off glycolysis.

IDH1 and IDH2 mutants convert glutamine carbon to the oncometabolite 2-hydroxyglutarate to dysregulate epigenetics and cell differentiation. (A) α-ketoglutarate, produced in part by wild-type isocitrate dehydrogenase (IDH), can enter the nucleus and be used as a substrate for dioxygenase enzymes that modify epigenetic marks. These enzymes include the TET2 DNA hydroxylase enzyme which converts 5-methylcytosine to 5-hydroxymethylcytosine, typically at CpG dinucleotides. 5-hydroxymethylcytosine may be an intermediate in either active or passive DNA demethylation. α-ketoglutarate is also a substrate for JmjC domain histone demethylase enzymes that demethylate lysine residues on histone tails. (B) The common feature of cancer-associated mutations in cytosolic IDH1 and mitochondrial IDH2 is the acquisition of a neomorphic enzymatic activity. This activity converts glutamine-derived α-ketoglutarate to the oncometabolite 2HG. 2HG can competitively inhibit α-ketoglutarate-dependent enzymes like TET2 and the JmjC histone demethylases, thereby impairing normal epigenetic regulation. This results in altered histone methylation marks, in some cases DNA hypermethylation at CpG islands, and dysregulated cellular differentiation.

Hypoxia and HIF-1 activation promote an alternative pathway for citrate synthesis through reductive metabolism of glutamine. (A) In proliferating cells under normoxic conditions, citrate is synthesized from both glucose and glutamine. Glucose carbon provides acetyl-CoA through the activity of PDH. Glutamine carbon provides oxaloacetate through oxidative mitochondrial metabolism dependent on NAD+. Glucose-derived acetyl-CoA and glutamine-derived oxaloacetate condense to form citrate via the activity of citrate synthase (CS). Citrate can be exported to the cytosol for lipid synthesis. (B) In cells proliferating in hypoxia and/or with HIF-1 activation, glucose is diverted away from mitochondrial acetyl-CoA and citrate production. Citrate can be maintained through an alternative pathway of reductive carboxylation, which we propose to rely on reverse flux of glutamine-derived α-ketoglutarate through IDH2. This reverse flux in the mitochondria would promote electron export from the mitochondria when the activity of the electron transport chain is inhibited because of the lack of oxygen as an electron acceptor. Mitochondrial reverse flux can be accomplished by NADH conversion to NADPH by mitochondrial transhydrogenase and the resulting NADPH use in α-ketoglutarate carboxylation. When citrate/isocitrate is exported to the cytosol, some may be metabolized in the oxidative direction by IDH1 and contribute to a shuttle that produces cytosolic NADPH.

A major paradox remaining with PKM2 is that cells expressing PKM2 produce more glucose-derived pyruvate than PKM1-expressing cells, despite having a form of the pyruvate kinase enzyme that is less active and more sensitive to inhibition. One way to get around the PKM2 bottleneck and maintain/enhance pyruvate production may be through an proposed alternative glycolytic pathway, involving an enzymatic activity not yet purified, that dephosphorylates PEP to pyruvate without the generation of ATP (Vander Heiden et al., 2010). Another answer to this paradox may emanate from the serine synthetic pathway. The decreased enzymatic activity of PKM2 can promote the accumulation of the 3-phosphoglycerate glycolytic intermediate that serves as the entry point for the serine synthetic pathway branch off glycolysis. The little studied enzyme serine dehydratase can then directly convert serine to pyruvate. A third explanation may lie in the oscillatory activity of PKM2 from the inactive dimer to active tetramer form. Regulatory inputs into PKM2 like tyrosine phosphorylation and ROS destabilize the tetrameric form of PKM2 (Anastasiou et al., 2011; Christofk et al., 2008b; Hitosugi et al., 2009), but other inputs present in glycolytic cancer cells like fructose-1,6-bisphosphate and serine can continually allosterically activate and/or promote reformation of the PKM2 tetramer (Ashizawa et al., 1991; Eigenbrodt et al., 1983). Thus, PKM2 may be continually switching from inactive to active forms in cells, resulting in an apparent upregulation of flux through anabolic glycolytic branching pathways while also maintaining reasonable net flux of glucose carbon through PEP to pyruvate. With such an oscillatory system, small changes in the levels of any of the above-mentioned PKM2 regulatory inputs can cause exquisite, rapid, adjustments to glycolytic flux. This would be predicted to be advantageous for proliferating cells in the setting of variable extracellular nutrient availability. The capability for oscillatory regulation of PKM2 could also provide an explanation for why tumor cells do not select for altered glycolytic metabolism upstream of PKM2 through deletions and/or loss of function mutations of other glycolytic enzymes.

IDH1 mutations at R132 are not simply loss-of-function for isocitrate and α-ketoglutarate interconversion, but also acquire a novel reductive activity to convert α-ketoglutarate to 2-hydroxyglutarate (2HG), a rare metabolite found at only trace amounts in mammalian cells under normal conditions (Dang et al., 2009). However, it still remained unclear if 2HG was truly a pathogenic “oncometabolite” resulting from IDH1 mutation, or if it was just the byproduct of a loss of function mutation. Whether 2HG production or the loss of IDH1 normal function played a more important role in tumorigenesis remained uncertain.

A potential answer to whether 2HG production was relevant to tumorigenesis arrived with the study of mutations in IDH2, the mitochondrial homolog of IDH1. Up to this point a small fraction of gliomas lacking IDH1 mutations were known to harbor mutations at IDH2 R172, the analogous residue to IDH1 R132 (Yan et al., 2009). However, given the rarity of these IDH2 mutations, they had not been characterized for 2HG production. The discovery of IDH2 R172 mutations in AML as well as glioma samples prompted the study of whether these mutations also conferred the reductive enzymatic activity to produce 2HG. Enzymatic assays and measurement of 2HG levels in primary AML samples confirmed that these IDH2 R172 mutations result in 2HG elevation (Gross et al., 2010; Ward et al., 2010).

It was then investigated if the measurement of 2HG levels in primary tumor samples with unknown IDH mutation status could serve as a metabolite screening test for both cytosolic IDH1 and mitochondrial IDH2 mutations. AML samples with low to undetectable 2HG were subsequently sequenced and determined to be IDH1 and IDH2 wild-type, and several samples with elevated 2HG were found to have neomorphic mutations at either IDH1 R132 or IDH2 R172 (Gross et al., 2010). However, some 2HG-elevated AML samples lacked IDH1 R132 or IDH2 R172 mutations. When more comprehensive sequencing of IDH1 and IDH2 was performed, it was found that the common feature of this remaining subset of 2HG-elevated AMLs was another mutation in IDH2, occurring at R140 (Ward et al., 2010). This discovery provided additional evidence that 2HG production was the primary feature being selected for in tumors.

In addition to intensifying efforts to find the cellular targets of 2HG, the discovery of the 2HG-producing IDH1 and IDH2 mutations suggested that 2HG measurement might have clinical utility in diagnosis and disease monitoring. While much work is still needed in this area, serum 2HG levels have successfully correlated with IDH1 R132 mutations in AML, and recent data have suggested that 1H magnetic resonance spectroscopy can be applied for 2HG detection in vivo for glioma (Andronesi et al., 2012; Choi et al., 2012; Gross et al., 2010; Pope et al., 2012). These methods may have advantages over relying on invasive solid tumor biopsies or isolating leukemic blast cells to obtain material for sequencing of IDH1 and IDH2. Screening tumors and body fluids by 2HG status also has potentially increased applicability given the recent report that additional IDH mutations can produce 2HG (Ward et al., 2011). These additional alleles may account for the recently described subset of 2HG-elevated chondrosarcoma samples that lacked the most common IDH1 or IDH2 mutations but were not examined for other IDH alterations (Amary et al., 2011). Metabolite screening approaches can also distinguish neomorphic IDH mutations from SNPs and sequencing artifacts with no effect on IDH enzyme activity, as well as from an apparently rare subset of loss-of-function, non 2HG-producing IDH mutations that may play a secondary tumorigenic role in altering cellular redox (Ward et al., 2011).

Will we find other novel oncometabolites like 2HG? We should consider basing the search for new oncometabolites on those metabolites already known to cause disease in pediatric inborn errors of metabolism (IEMs). 2HG exemplifies how advances in research on IEMs can inform research on cancer metabolism, and vice versa. Methods developed by those studying 2HG aciduria were used to demonstrate that R(-)-2HG (also known as D-2HG) is the exclusive 2HG stereoisomer produced by IDH1 and IDH2 mutants (Dang et al., 2009; Ward et al., 2010). Likewise, following the discovery of 2HG-producing IDH2 R140 mutations in leukemia, researchers looked for and successfully found germline IDH2 R140 mutations in D-2HG aciduria. IDH2 R140 mutations now account for nearly half of all cases of this devastating disease (Kranendijk et al., 2010). While interest has surrounded 2HG due to its apparent novelty as a metabolite not found in normal non-diseased cells, there are situations where 2HG appears in the absence of metabolic enzyme mutations. For example, in human cells proliferating in hypoxia, α-ketoglutarate can accumulate and be metabolized through an enhanced reductive activity of wild-type IDH2 in the mitochondria, leading to 2HG accumulation in the absence of IDH mutation (Wise et al., 2011). The ability of 2HG to alter epigenetics may reflect its evolutionary ancient status as a signal for elevated glutamine/glutamate metabolism and/or oxygen deficiency.

With this broadened view of what constitutes an oncometabolite, one could argue that the discoveries of two other oncometabolites, succinate and fumarate, preceded that of 2HG. Loss of function mutations in the TCA cycle enzymes succinate dehydrogenase (SDH) and fumarate hydratase (FH) have been known for several years to occur in pheochromocytoma, paraganglioma, leiomoyoma, and renal carcinoma. It was initially hypothesized that these mutations contribute to cancer through mitochondrial damage producing elevated ROS (Eng et al., 2003). However, potential tumorigenic effects were soon linked to the elevated levels of succinate and fumarate arising from loss of SDH and FH function, respectively. Succinate was initially found to impair PHD2, the α-ketoglutarate-dependent enzyme regulating HIF stability, through product inhibition (Selak et al., 2005). Subsequent work confirmed that fumarate could inhibit PHD2 (Isaacs et al., 2005), and that succinate could also inhibit the related enzyme PHD3 (Lee et al., 2005). These observations linked the elevated HIF levels observed in SDH and FH deficient tumors to the activity of the succinate and fumarate metabolites. Recent work has suggested that fumarate may have other important roles that predominate in FH deficiency. For example, fumarate can modify cysteine residues to inhibit a negative regulator of the Nrf2 transcription factor. This post-translational modification leads to the upregulation of antioxidant response genes (Adam et al., 2011; Ooi et al., 2011).

There are still many unanswered questions regarding the biology of SDH and FH deficient tumors. In light of the emerging epigenetic effects of 2HG, it is intriguing that succinate has been shown to alter histone demethylase activity in yeast (Smith et al., 2007). Perhaps elevated succinate and fumarate resulting from SDH and FH mutations can promote tumorigenesis in part through epigenetic modulation.

Despite rapid technological advances in studying cell metabolism, we remain unable to reliably distinguish cytosolic metabolites from those in the mitochondria and other compartments. Current fractionation methods often lead to metabolite leakage. Even within one subcellular compartment, there may be distinct pools of metabolites resulting from channeling between metabolic enzymes. A related challenge lies in the quantitative measurement of metabolic flux; i.e., measuring the movement of carbon, nitrogen, and other atoms through metabolic pathways rather than simply measuring the steady-state levels of individual metabolites. While critical fluxes have been quantified in cultured cancer cells and methods for these analyses continue to improve (DeBerardinis et al., 2007; Mancuso et al., 2004; Yuan et al., 2008), many obstacles remain such as cellular compartmentalization and the reliance of most cell culture on complex, incompletely defined media.

Over the past decade, the study of metabolism has returned to its rightful place at the forefront of cancer research. Although Warburg was wrong about mitochondria, he was prescient in his focus on metabolism. Data now support the concepts that altered metabolism results from active reprogramming by altered oncogenes and tumor suppressors, and that metabolic adaptations can be clonally selected during tumorigenesis. Altered metabolism should now be considered a core hallmark of cancer. There is much work to be done. A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth

Schell JC, Olson KA, …, Xie J, Egnatchik RA, Earl EG, DeBerardinis RJ, Rutter J.
Mol Cell. 2014 Nov 6; 56(3):400-13

Cancer cells are typically subject to profound metabolic alterations, including the Warburg effect wherein cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis. We show herein that the mitochondrial pyruvate carrier (MPC), composed of the products of the MPC1 and MPC2 genes, modulates fractional pyruvate oxidation. MPC1 is deleted or underexpressed in multiple cancers and correlates with poor prognosis. Cancer cells re-expressing MPC1 and MPC2 display increased mitochondrial pyruvate oxidation, with no changes in cell growth in adherent culture. MPC re-expression exerted profound effects in anchorage-independent growth conditions, however, including impaired colony formation in soft agar, spheroid formation, and xenograft growth. We also observed a decrease in markers of stemness and traced the growth effects of MPC expression to the stem cell compartment. We propose that reduced MPC activity is an important aspect of cancer metabolism, perhaps through altering the maintenance and fate of stem cells.

Figure 2. Re-Expressed MPC1 and MPC2 Form a Mitochondrial Complex (A and B) (A) Western blot and (B) qRT-PCR analysis of the indicated colon cancer cell lines with retroviral expression of MPC1 (or MPC1-R97W) and/or MPC2. (C) Western blots of human heart tissue, hematologic cancer cells, and colon cancer cell lines with and without MPC1 and MPC2 re-expression. (D) Fluorescence microscopy of MPC1-GFP and MPC2-GFP overlaid with Mitotracker Red in HCT15 cells. Scale bar: 10 mm. (E) Blue-native PAGE analysis of mitochondria from control and MPC1/2-expressing cells. (F) Western blots of metabolic and mitochondrial proteins across four colon cancer cell lines with or without MPC1/2 expression

Figure 3. MPC Re-Expression Alters Mitochondrial Pyruvate Metabolism (A) OCR at baseline and maximal respiration in HCT15 (n = 7) and HT29 (n = 13) with pyruvate as the sole carbon source (mean ± SEM). (B and C) Schematic and citrate mass isotopomer quantification in cells cultured with D-[U-13C]glucose and unlabeled glutamine for 6 hr (mean ± SD, n = 2). (D) Glucose uptake and lactate secretion normalized to protein concentration (mean ± SD, n = 3). (E–G) (E) Western blots of PDH, phospho-PDH, and PDK1; (F) PDH activity assay and (G) CS activity assay with or without MPC1 and MPC2 expression (mean ± SD, n = 4). (H and I) Effects of MPC1/2 re-expression on mitochondrial membrane potential and ROS production (mean ± SD, n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4. MPC Re-Expression Alters Growth under Low-Attachment Conditions (A) Cell number of control and MPC1/2 re-expressing cell lines in adherent culture (mean ± SD, n = 7). (B) Cell viability determined by trypan blue exclusion and Annexin V/PI staining (mean ± SD, n = 3). (C–F) (C) EdU incorporation of MPC re-expressing cell lines at 3 hr post EdU pulse. Growth in 3D culture evaluated by (D) soft agar colony formation (mean ± SD, n = 12, see also Table S1) and by ([E] and [F]) spheroid formation ± MPC inhibitor UK5099 (mean ± SEM, n = 12). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 7. MPC Re-Expression Alters the Cancer Initiating Cell Population (A) Western blot quantification of ALDHA and Lin28A from control or MPC re-expressing HT29 xenografts (mean ± SEM, n = 10). (B and C) Percentage of ALDHhi (n = 3) and CD44hi (n = 5) cells as determined by flow cytometry (mean ± SEM). (D) Western blot analysis of stem cell markers in control and MPC re-expressing cell lines. (E) Relative MPC1 and MPC2 mRNA levels in ALDH sorted HCT15 cells (n = 4,mean ± SEM). 2D growth of (F) whole-population HCT15 cells and (G) ALDH sorted cells. Area determined by ImageJ after crystal violet staining (mean ± SD, n = 6). (H and I) (H) Adherent and (I) spheroid growth of main population (MP) versus side population (SP) HCT15 cells. (mean ± SD, n = 6). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Our demonstration that the MPC is lost or underexpressed in many cancers might provide clarifying context for earlier attempts to exploit metabolic regulation for cancer therapeutics. The PDH kinase inhibitor dichloroacetate, which impairs PDH phosphorylation and increases pyruvate oxidation, has been explored extensively as a cancer therapy (Bonnet et al., 2007; Olszewski et al., 2010). It has met with mixed results, however, and has typically failed to dramatically decrease tumor burden as a monotherapy (Garon et al., 2014;
Sanchez-Arago et al., 2010; Shahrzadetal.,2010). Is one possible reason for these failures that the MPC has been lost or inactivated, thereby limiting the metabolic effects of PDH activity? The inclusion of the MPC adds additional complexity to targeting cancer metabolism for therapy but has the potential to explain why treatments may be more effective in some studies than in others (Fulda et al., 2010; Hamanaka and Chandel, 2012; Tennant et al., 2010; Vander Heiden, 2011). The redundant measures to limit pyruvate oxidation make it easy to understand why expression of the MPC leads to relatively modest metabolic changes in cells grown in adherent culture conditions. While subtle, we observed a number of changes in metabolic parameters, all of which are consistent with enhanced mitochondrial pyruvate entry and oxidation. There are at least two possible explanations for the discrepancy that we observed between the impact on adherent and nonadherent cell proliferation. One hypothesis is that the stress of nutrient deprivation and detachment combines with these subtle metabolic effects to impair survival and proliferation.  ECM1 promotes the Warburg effect through EGF-mediated activation of PKM2

Lee KM, Nam K, Oh S, Lim J, Lee T, Shin I.
Cell Signal. 2015 Feb; 27(2):228-35

The Warburg effect is an oncogenic metabolic switch that allows cancer cells to take up more glucose than normal cells and favors anaerobic glycolysis. Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein that is overexpressed in various types of carcinoma. Using two-dimensional digest-liquid chromatography-mass spectrometry (LC-MS)/MS, we showed that the expression of proteins associated with the Warburg effect was upregulated in trastuzumab-resistant BT-474 cells that overexpressed ECM1 compared to control cells. We further demonstrated that ECM1 induced the expression of genes that promote the Warburg effect, such as glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and hypoxia-inducible factor 1 α (HIF-1α). The phosphorylation status of pyruvate kinase M2 (PKM-2) at Ser37, which is responsible for the expression of genes that promote the Warburg effect, was affected by the modulation of ECM1 expression. Moreover, EGF-dependent ERK activation that was regulated by ECM1 induced not only PKM2 phosphorylation but also gene expression of GLUT1 and LDHA. These findings provide evidence that ECM1 plays an important role in promoting the Warburg effect mediated by PKM2.

Fig. 1.ECM1 induces a metabolic shift toward promoting Warburg effect. (A) The levels of glucose uptake were examined with a cell-based assay. (B) Levels of lactate production were measured using a lactate assay kit. (C) Cellular ATP content was determined with a Cell Titer-Glo luminescent cell viability assay. Error bars represent mean ± SD of triplicate experiments (*p b 0.05, ***p b 0.0005).

Fig.2. ECM1 up-regulates expression of gene sassociated with the Warburg effect. (A) Cell lysates were analyzed by western blotting using antibodies specific for ECM1, LDHA, GLUT1,and actin (as a loading control). The intensities of the bands were quantified using 1D Scan software and plotted. (BandC) mRNA levels of each gene were determined by real-time PCR using specific primers. (D) HIF-1α-dependent transcriptional activities were examined using a hypoxia response element (HRE) reporter indual luciferase assays. Error bars represent mean ± SD of triplicate experiments (*p b 0.05, **p b 0.005, ***p b 0.0005).

Fig.3. ECM1-dependent upregulation of gene expression is not mediated byEgr-1.

Fig.4. ECM1 activates PKM2 via EGF-mediated ERK activation

Fig. 5. TheWarburg effect is attenuated by silencing of PKM2 in breast cancer cells

Recently, a non-glycolytic function of PKM2 was reported. Phosphorylated PKM2 at Ser37 is translocated into the nucleus after EGFR and ERK activation and regulates the expression of cyclin D1, c-Myc, LDHA, and GLUT1[19,37]. Here, we showed that ECM1 regulates the phosphorylation level and translocation of PKM2 via the EGFR/ ERK pathway. As we previously showed that ECM1 enhances the EGF response and increases EGFR expression through MUC1-dependent stabilization [17], it seemed likely that activation of the EGFR/ERK pathway by ECM1 is linked to PKM2 phosphorylation. Indeed, we show here that ECM1 regulates the phosphorylation of PKM2 at Ser37 and enhances the Warburg effect through the EGFR/ERK pathway. HIF-1α is known to be responsible for alterations in cancer cell metabolism [38] and our current studies showed that the expression level of HIF-1α is up-regulated by ECM1 (Fig. 2C and D). To determine the mechanism by which ECM1 upregulated HIF-1α expression, we focused on the induction of Egr-1 by EGFR/ERK signaling [39]. However, although Egr-1 expression was regulated by ECM1 we failed to find evidence that Egr-1 affected the expression of genes involved in the Warburg effect (Fig. 3C). Moreover, ERK-dependent PKM2 activation did not regulate HIF-1α expression in BT-474 cells (Fig. 4D and5B). These results suggested that the upregulation of HIF-1α by ECM1 is not mediated by the EGFR/ERK pathway.


In the current study we showed that ECM1 altered metabolic phenotypes of breast cancer cells toward promoting the Warburg effect.

Phosphorylation and nuclear translocation of PKM2 were induced by ECM1 through the EGFR/ERK pathway. Moreover, phosphorylated PKM2 increased the expression of metabolic genes such as LDHA and GLUT1, and promoted glucose uptake and lactate production. These findings provide a new perspective on the distinct functions of ECM1 in cancer cell metabolism. Supplementary data to this article can be found online at


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[8] G.L. Semenza, Cancer 3 (2003) 721–732. Glutamine Oxidation Maintains the TCA Cycle and Cell Survival during impaired Mitochondrial Pyruvate Transport

Chendong Yang, B Ko, CT. Hensley,…, J Rutter, ME. Merritt, RJ. DeBerardinis
Molec Cell  6 Nov 2014; 56(3):414–424


  • Mitochondria produce acetyl-CoA from glutamine during MPC inhibition
    •Alanine synthesis is suppressed during MPC inhibition
    •MPC inhibition activates GDH to supply pools of TCA cycle intermediates
    •GDH supports cell survival during periods of MPC inhibition


Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

Yang et al, Graphical Abstract

Yang et al, Graphical Abstract

Graphical abstract

Figure 1. Pyruvate Depletion Redirects Glutamine Metabolism to Produce AcetylCoA and Citrate (A) Top: Anaplerosis supplied by [U-13C]glutamine. Glutamine supplies OAA via a-KG, while acetylCoA is predominantly supplied by other nutrients, particularly glucose. Bottom: Glutamine is converted to acetyl-CoA in the absence of glucosederived pyruvate. Red circles represent carbons arising from [U-13C]glutamine, and gray circles are unlabeled. Reductive carboxylation is indicated by the green dashed line. (B) Fraction of succinate, fumarate, malate, and aspartate containing four 13C carbons after culture of SFxL cells for 6 hr with [U-13C]glutamine in the presence or absence of 10 mM unlabeled glucose (Glc). (C) Mass isotopologues of citrate after culture of SFxL cells for 6 hr with [U-13C]glutamine and 10 mM unlabeled glucose, no glucose, or no glucose plus 6 mM unlabeled pyruvate (Pyr). (D) Citrate m+5 and m+6 after culture of HeLa or Huh-7 cells for 6 hr with [U-13C]glutamine and 10 mM unlabeled glucose, no glucose, or no glucose plus 6 mM unlabeled pyruvate. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2. Isolated Mitochondria Convert Glutamine to Citrate (A) Western blot of whole-cell lysates (Cell) and preparations of isolated mitochondria (Mito) or cytosol from SFxL cells. (B) Oxygen consumption in a representative mitochondrial sample. Rates before and after addition of ADP/GDP are indicated. (C) Mass isotopologues of citrate produced by mitochondria cultured for 30 min with [U-13C] glutamine and with or without pyruvate.

Figure 3. Blockade of Mitochondrial Pyruvate Transport Activates Glutamine-Dependent Citrate Formation (A) Dose-dependent effects of UK5099 on citrate labeling from [U-13C]glucose and [U-13C]glutamine in SFxL cells. (B) Time course of citrate labeling from [U-13C] glutamine with or without 200 mM UK5099. (C) Abundance of total citrate and citrate m+6 in cells cultured in [U-13C]glutamine with or without 200 mM UK5099. (D) Mass isotopologues of citrate in cells cultured for 6 hr in [U-13C]glutamine with or without 10 mM CHC or 200 mM UK5099. (E) Effect of silencing ME2 on citrate m+6 after 6 hr of culture in [U-13C]glutamine. Relative abundances of citrate isotopologues were determined by normalizing total citrate abundance measured by mass spectrometry against cellular protein for each sample then multiplying by the fractional abundance of each isotopologue. (F) Effect of silencing MPC1 or MPC2 on formation of citrate m+6 after 6 hr of culture in [U-13C]glutamine. (G) Citrate isotopologues in primary human fibroblasts of varying MPC1 genotypes after culture in [U-13C]glutamine. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S1.

Figure 4. Kinetic Analysis of the Metabolic Effects of Blocking Mitochondrial Pyruvate Transport (A) Summation of 13C spectra acquired over 2 min of exposure of SFxL cells to hyperpolarized [1-13C] pyruvate. Resonances are indicated for [1-13C] pyruvate (Pyr1), the hydrate of [1-13C]pyruvate (Pyr1-Hydr), [1-13C]lactate (Lac1), [1-13C]alanine (Ala1), and H[13C]O3 (Bicarbonate). (B) Time evolution of appearance of Lac1, Ala1, and bicarbonate in control and UK5099-treated cells. (C) Relative 13C NMR signals for Lac1, Ala1, and bicarbonate. Each signal is summed over the entire acquisition and expressed as a fraction of total 13C signal. (D) Quantity of intracellular and secreted alanine in control and UK5099-treated cells. Data are the average and SD of three independent cultures. *p < 0.05; ***p < 0.001. See also Figure S2.

Figure 5. Inhibiting Mitochondrial Pyruvate Transport Enhances the Contribution of Glutamine to Fatty Acid Synthesis (A) Mass isotopologues of palmitate extracted from cells cultured with [U-13C] glucose or [U-13C]glutamine, with or without 200 mM UK5099. For simplicity, only even-labeled isotopologues (m+2, m+4, etc.) are shown. (B) Fraction of lipogenic acetyl-CoA derived from glucose or glutamine with or without 200 mM UK5099. Data are the average and SD of three independent cultures. ***p < 0.001. See also Figure S3.

Figure 6. Blockade of Mitochondrial Pyruvate Transport Induces GDH (A) Two routes by which glutamate can be converted to AKG. Blue and green symbols are the amide (g) and amino (a) nitrogens of glutamine, respectively. (B) Utilization and secretion of glutamine (Gln), glutamate (Glu), and ammonia (NH4+) by SFxL cells with and without 200 mM UK5099. (C) Secretion of 15N-alanine and 15NH4+ derived from [a-15N]glutamine in SFxL cells expressing a control shRNA (shCtrl) or either of two shRNAs directed against GLUD1 (shGLUD1-A and shGLUD1-B). (D) Left: Phosphorylation of AMPK (T172) and acetyl-CoA carboxylase (ACC, S79) during treatment with 200 mM UK5099. Right: Steady-state levels of ATP 24 hr after addition of vehicle or 200 mM UK5099. (E) Fractional contribution of the m+6 isotopologue to total citrate in shCtrl, shGLUD1-A, and shGLUD1-B SFxL cells cultured in [U-13C]glutamine with or without 200 mM UK5099. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.

Figure 7. GDH Sustains Growth and Viability during Suppression of Mitochondrial Pyruvate Transport (A) Relative growth inhibition of shCtrl, shGLUD1A, and shGLUD1-B SFxL cells treated with 50 mM UK5099 for 3 days. (B) Relative growth inhibition of SFxL cells treated with combinations of 50 mM of the GDH inhibitor EGCG, 10 mM of the GLS inhibitor BPTES, and 200 mM UK5099 for 3 days. (C) Relative cell death assessed by trypan blue staining in SFxL cells treated as in (B). (D) Relative cell death assessed by trypan blue staining in SF188 cells treated as in (B) for 2 days. (E) (Left) Growth of A549-derived subcutaneous xenografts treated with vehicle (saline), EGCG, CHC, or EGCG plus CHC (n = 4 for each group). Data are the average and SEM. Right: Lactate abundance in extracts of each tumor harvested at the end of the experiment. Data in (A)–(D) are the average and SD of three independent cultures. NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S5.

Mitochondrial metabolism complements glycolysis as a source of energy and biosynthetic precursors. Precursors for lipids, proteins, and nucleic acids are derived from the TCA cycle. Maintaining pools of these intermediates is essential, even under circumstances of nutrient limitation or impaired supply of glucose-derived pyruvate to the mitochondria. Glutamine’s ability to produce both acetyl-CoA and OAA allows it to support TCA cycle activity as a sole carbon source and imposes a greater cellular dependence on glutamine metabolism when MPC function or pyruvate supply is impaired. Other anaplerotic amino acids could also supply both OAA and acetyl-CoA, providing flexible support for the TCA cycle when glucose is limiting. Although fatty acids are an important fuel in some cancer cells (Caro et al., 2012), and fatty acid oxidation is induced upon MPC inhibition, this pathway produces acetyl-CoA but not OAA. Thus, fatty acids would need to be oxidized along with an anaplerotic nutrient in order to enable the cycle to function as a biosynthetic hub. Notably, enforced MPC overexpression also impairs growth of some tumors (Schell et al., 2014), suggesting that maximal growth may require MPC activity to be maintained within a narrow window. After decades of research on mitochondrial pyruvate transport, molecular components of the MPC were recently reported (Halestrap, 2012; Schell and Rutter, 2013). MPC1 and MPC2 form a heterocomplex in the inner mitochondrial membrane, and loss of either component impairs pyruvate import, leading to citrate depletion (Bricker et al., 2012; Herzig et al., 2012). Mammalian cells lacking functional MPC1 display normal glutamine-supported respiration (Bricker et al., 2012), consistent with our observation that glutamine supplies the TCA cycle in absence of pyruvate import. We also observed that isolated mitochondria produce fully labeled citrate from glutamine, indicating that this pathway operates as a self-contained mechanism to maintain TCA cycle function. Recently, two well-known classes of drugs have unexpectedly been shown to inhibit MPC. First, thiazolidinediones, commonly used as insulin sensitizers, impair MPC function in myoblasts (Divakaruni et al.,2013). Second, the phosphodiesterase inhibitor Zaprinast inhibits MPC in the retina and brain (Du et al., 2013b). Zaprinast also induced accumulation of aspartate, suggesting that depletion of acetyl-CoA impaired the ability of a new turn of the TCA cycle to be initiated from OAA; as a consequence, OAA was transaminated to aspartate. We noted a similar phenomenon in cancer cells, suggesting that UK5099 elicits a state in which acetyl-CoA supply is insufficient to avoid OAA accumulation. Unlike UK5099, Zaprinast did not induce glutamine-dependent acetyl-CoA formation. This may be related to the reliance of isolated retinas on glucose rather than glutamine to supply TCA cycle intermediates or the exquisite system used by retinas to protect glutamate from oxidation (Du et al., 2013a). Zaprinast was also recently shown to inhibit glutaminase (Elhammali et al., 2014), which would further reduce the contribution of glutamine to the acetyl-CoA pool.

Comment by reader –

The results from these studies served as a good
reason to attempt the vaccination of patients using p53-
derived peptides, and a several clinical trials are currently
in progress. The most advanced work used a long
synthetic peptide mixture derived from p53 (p53-SLP; ISA
Pharmaceuticals, Bilthoven, the Netherlands) (Speetjens
et al., 2009; Shangary et al., 2008; Van der Burg et al.,
2001). The vaccine is delivered in the adjuvant setting
and induces T helper type cells.


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Diagnostic Value of Cardiac Biomarkers

Diagnostic Value of Cardiac Biomarkers

Author and Curator: Larry H Bernstein, MD, FCAP 

These presentations covered several views of the utilization of cardiac markers that have evolved for over 60 years.  The first stage was the introduction of enzymatic assays and isoenzyme measurements to distinguish acute hepatitis and acute myocardial infarction, which included lactate dehydrogenase (LD isoenzymes 1, 2) at a time that late presentation of the patient in the emergency rooms were not uncommon, with the creatine kinase isoenzyme MB declining or disappeared from the circulation.  The world health organization (WHO) standard definition then was the presence of two of three:

1. Typical or atypical precordial pressure in the chest, usually with radiation to the left arm

2. Electrocardiographic changes of Q-wave, not previously seen, definitive; ST- elevation of acute myocardial injury with repolarization;
T-wave inversion.

3. The release into the circulation of myocardial derived enzymes –
creatine kinase – MB (which was adapted to measure infarct size), LD-1,
both of which were replaced with troponins T and I, which are part of the actomyosin contractile apparatus.

The research on infarct size elicited a major research goal for early diagnosis and reduction of infarct size, first with fibrinolysis of a ruptured plaque, and this proceeded into the full development of a rapidly evolving interventional cardiology as well as cardiothoracic surgery, in both cases, aimed at removal of plaque or replacement of vessel.  Surgery became more imperative for multivessel disease, even if only one vessel was severely affected.

So we have clinical history, physical examination, and emerging biomarkers playing a large role for more than half a century.  However, the role of biomarkers broadened.  Patients were treated with antiplatelet agents, and a hypercoagulable state coexisted with myocardial ischemic injury.  This made the management of the patient reliant on long term followup for Warfarin with the international normalized ratio (INR) for a standardized prothrombin time (PT), and reversal of the PT required transfusion with thawed fresh frozen plasma (FFP).  The partial thromboplastin test (PPT) was necessary in hospitalization to monitor the heparin effect.

Thus, we have identified the use of traditional cardiac biomarkers for:

1. Diagnosis
2. Therapeutic monitoring

The story is only the beginning.  Many patients who were atypical in presentation, or had cardiovascular ischemia without plaque rupture were problematic.  This led to a concerted effort to redesign the troponin assays for high sensitivity with the concern that the circulation should normally be free of a leaked structural marker of myocardial damage. But of course, there can be a slow leak or a decreased rate of removal of such protein from the circulation, and the best example of this would be the patient with significant renal insufficiency, as TnT is clear only through the kidney, and TNI is clear both by the kidney and by vascular endothelium.  The introduction of the high sensitivity assay has been met with considerable confusion, and highlights the complexity of diagnosis in heart disease.  Another test that is used for the diagnosis of heart failure is in the class of natriuretic peptides (BNP, pro NT-BNP, and ANP), the last of which has been under development.

While there is an exponential increase in the improvement of cardiac devices and discovery of pharmaceutical targets, the laboratory support for clinical management is not mature.  There are miRNAs that may prove valuable, matrix metalloprotein(s), and potential endothelial and blood cell surface markers, they require

1. codevelopment with new medications
2. standardization across the IVD industry
3. proficiency testing applied to all laboratories that provide testing
4. the measurement  on multitest automated analyzers with high capability in proteomic measurement  (MS, time of flight, MS-MS)

nejmra1216063_f1   Atherosclerotic Plaques Associated with Various Presentations               nejmra1216063_f2     Inflammatory Pathways Predisposing Coronary Arteries to Rupture and Thrombosis.        atherosclerosis progression

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Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?

Author: Larry H. Bernstein, MD, FCAP  

A Critical Review

What is the Warburg effect?

“Warburg Effect” describes the preference of glycolysis and lactate fermentation rather than oxidative phosphorylation for energy production in cancer cells. Mitochondrial metabolism is an important and necessary component in the functioning and maintenance of the organelle, and accumulating evidence suggests that dysfunction of mitochondrial metabolism plays a role in cancer. Progress has demonstrated the mechanisms of the mitochondrial metabolism-to-glycolysis switch in cancer development and how to target this metabolic switch.
In vertebrates, food is digested and supplied to cells mainly in the form of glucose. Glucose is broken down further to make Adenosine Triphosphate (ATP) by two pathways. One is via anaerobic metabolism occurring in the cytoplasm, also known as glycolysis. The major physiological significance of glycolysis lies in making ATP quickly, but in a minuscule amount. The breakdown process continues in the mitochondria via the Krebs’s cycle coupled with oxidative phosphorylation, which is more efficient for ATP production. Cancer cells seem to be well-adjust to glycolysis. In the 1920s, Otto Warburg first proposed that cancer cells show increased levels of glucose consumption and lactate fermentation even in the presence of ample oxygen (known as “Warburg Effect”). Based on this theory, oxidative phosphorylation switches to glycolysis which promotes the proliferation of cancer cells. Many studies have demonstrated glycolysis as the main metabolic pathway in cancer cells.
Why cancer cells prefer glycolysis, an inefficient metabolic pathway?

It is now accepted that glycolysis provides cancer cells with the most abundant extracellular nutrient, glucose, to make ample ATP metabolic intermediates, such as ribose sugars, glycerol and citrate, nonessential amino acids, and the oxidative pentose phosphate pathway, which serve as building blocks for cancer cells.
Since, cancer cells have increased rates of aerobic glycolysis, investigators argue over the function of mitochondria in cancer cells. Mitochondrion, a one of the smaller organelles, produces most of the energy in the form of ATP to supply the body. In Warburg’s theory, the function of cellular mitochondrial respiration is dampened and mitochondria are not fully functional. There are many studies backing this theory. A recent review on hypoxia nicely summarizes some current studies and speculates that the “Warburg Effect” provides a benefit to the tumor not by increasing glycolysis but by decreasing mitochondrial activity.
Glycolysis is enhanced and beneficial to cancer cells. The mammalian target of rapamycin (mTOR) has been well discussed in its role to promote glycolysis; recent literature has revealed some new mechanisms of how glycolysis is promoted during skin cancer development.
On the other hand, Akt is not only involved in the regulation of mitochondrial metabolism in skin cancer but also of glycolysis. Activation of Akt has been found to phosphorylate FoxO3a, a downstream transcription factor of Akt, which promotes glycolysis by inhibiting apoptosis in melanoma. In addition, activated Akt is also associated with stabilized c-Myc and activation of mTOR, which both increase glycolysis for cancer cells.
Nevertheless, ras mutational activation prevails in skin cancer. Oncogenic ras induces glycolysis. In human squamous cell carcinoma, the c-Jun NH(2)-terminal Kinase (JNK) is activated as a mediator of ras signaling, and is essential for ras-induced glycolysis, since pharmacological inhibitors if JNK suppress glycolysis. CD147/basigin, a member of the immunoglobulin superfamily, is high expressed in melanoma and other cancers.
Glyoxalase I (GLO1) is a ubiquitous cellular defense enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis. In human melanoma tissue, GLO1 is upregulated at both the mRNA and protein levels.
Knockdown of GLO1 sensitizes A375 and G361 human metastatic melanoma cells to apoptosis.
The transcription factor HIF-1 upregulates a number of genes in low oxygen conditions including glycolytic enzymes, which promotes ATP synthesis in an oxygen independent manner. Studies have demonstrated that hypoxia induces HIF-1 overexpression and its transcriptional activity increases in parallel with the progression of many tumor types. A recent study demonstrated that in malignant melanoma cells, HIF-1 is upregulated, leading to elevated expression of Pyruvate Dehydrogenase Kinase 1 (PDK1), and downregulated mitochondrial oxygen consumption.
The M2 isoform of Pyruvate Kinase (PKM2), which is required for catalyzing the final step of aerobic glycolysis, is highly expressed in cancer cells; whereas the M1 isoform (PKM1) is expressed in normal cells. Studies using the skin cell promotion model (JB6 cells) demonstrated that PKM2 is activated whereas PKM1 is inactivated upon tumor promoter treatment. Acute increases in ROS inhibited PKM2 through oxidation of Cys358 in human lung cancer cells. The levels of ROS and stage of tumor development may be pivotal for the role of PKM2.

Mitochondrial metabolism and glycolysis targeting for cancer drug delivery
In cancer cells including skin cancer cells, the metabolic shift is composed of increased glycolysis, activation of anabolic pathways including amino acid and pentose phosphate production, and increased fatty acid biosynthesis. More and more studies have converged on particular glycolytic and mitochondrial metabolic targets for cancer drug discovery.
A marker for increased glycolysis in melanoma is the elevated levels of Lactate Dehydrogenase (LDH) in the blood of patients with melanoma, which has proven to be an accurate predictor of prognosis and response to treatments. LDH converts pyruvate, the final product of glycolysis, to lactate when oxygen is absent. High concentrations of lactate, in turn, negatively regulate LDH. Therefore, targeting acid excretion may provide a feasible and effective therapeutic approach for melanoma. For instance, JugloSne, a main active component in walnut, has been used in traditional medicines. Studies have shown that Juglone causes cell membrane damage and increased LDH levels in a concentration-dependent manner in cultured melanoma cells. As one of the rate-limiting enzyme of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozyme 3 (PFKFB3) is activated in neoplastic cells. Studies have confirmed that an inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), suppresses glycolysis in neoplastic cells. In melanoma cell lines, the concentrations of Fru-2, 6-BP, lactate, ATP, NAD+, and NADH are diminished by 3PO. Therefore, targeting PFKFB3 using 3PO and other PFKFB3 specific inhibitors could be effective in melanoma chemotherapy.
A new NO (nitric oxide) donating compound [(S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid–nitric oxide (GIT-27NO)] has been tested in treating melanoma cells. The results suggest that GIT-27/NO causes a dose-dependent reduction of mitochondrial respiration in treated A375 human melanoma cells.

At least two mitochondrial enzymes are affected by angiostatin which include malate dehydrogenase, a member of the Kreb’s cycle enzymes; and adenosine triphosphate synthase. Both are identified potential angiostatin-binding partners. Treated with angiostatin, the ATP concentrations of A2058 cells were decreased. Meanwhile, using siRNA of these two enzymes also inhibited the ATP production. PKM2 is up regulated in the early stage of skin carcinogenesis, therefore, targeting PKM2 could serve as a new approach for skin cancer prevention and therapy.
The signaling pathways critical for this glycolytic activation could serve as preventive and therapeutic targets for human skin cancer.

The Historical Challenge posed by the Warburg Hypothesis.

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.
In a landmark review, 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,

  • self-sufficiency in growth signals,
  • insensitivity to growth inhibitory (antigrowth) signals,
  • evasion of programmed cell death (apoptosis),
  • limitless replicative potential,
  • sustained vascularity (angiogenesis), and
  • tissue invasion and metastasis.

Genome instability, leading to increased mutability, was considered the essential enabling characteristic for manifesting the six hallmarks. 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.
In addition to the six recognized hallmarks of cancer, aerobic glycolysis or the Warburg effect is also a robust metabolic hallmark of most tumors. 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.
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.
Warburg proposed with considerable certainty and insight that irreversible damage to respiration was the prime cause of cancer. 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. 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.
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.
Abnormal metabolism of tumors, a selective advantage
The initial observation of Warburg 1916 on tumor glycolysis with lactate production is still a crucial observation. Two fundamental findings complete the metabolic picture:

  • the discovery of the M2 pyruvate kinase (PK) typical of tumors
  • and the implication of tyrosine kinase signals and subsequent phosphorylations in the M2 PK blockade.

A typical feature of tumor cells is a glycolysis associated to an inhibition of apoptosis. Tumors overexpress the high affinity hexokinase 2, which strongly interacts with the mitochondrial ANT-VDAC-PTP complex. In this position, close to the ATP/ADP exchanger (ANT), the hexokinase receives efficiently its ATP substrate. As long as hexokinase occupies this mitochondria site, glycolysis is efficient. However, this has another consequence, hexokinase pushes away from the mitochondria site the permeability transition pore (PTP), which inhibits the release of cytochrome C, the apoptotic trigger. The site also contains a voltage dependent anion channel (VDAC) and other proteins. The repulsion of PTP by hexokinase would reduce the pore size and the release of cytochrome C. Thus, the apoptosome-caspase proteolytic structure does not assemble in the cytoplasm. The liver hexokinase or glucokinase, is different it has less interaction with the site, has a lower affinity for glucose; because of this difference, glucose goes preferentially to the brain.
Further, phosphofructokinase gives fructose 1-6 bisphosphate; glycolysis is stimulated if an allosteric analogue, fructose 2-6 bis phosphate increases in response to a decrease of cAMP. The activation of insulin receptors in tumors has multiple effects, among them; a decrease of cAMP, which will stimulate glycolysis. Another control point is glyceraldehyde P dehydrogenase that requires NAD+ in the glycolytic direction. If the oxygen supply is normal, the mitochondria malate/aspartate (MAL/ASP) shuttle forms the required NAD+ in the cytosol and NADH in the mitochondria. In hypoxic conditions, the NAD+ will essentially come via lactate dehydrogenase converting pyruvate into lactate. This reaction is prominent in tumor cells; it is the first discovery of Warburg on cancer.
At the last step of glycolysis, pyruvate kinase (PK) converts phospho-enolpyruvate (PEP) into pyruvate, which enters in the mitochondria as acetyl- CoA, starting the citric acid cycle and oxidative metabolism. To explain the PK situation in tumors we must recall that PK only works in the glycolytic direction, from PEP to pyruvate, which implies that gluconeogenesis uses other enzymes for converting pyruvate into PEP. In starvation, when cells need glucose, one switches from glycolysis to gluconeogenesis and ketogenesis; PK and pyruvate dehydrogenase (PDH) are off, in a phosphorylated form, presumably following a cAMP-glucagon-adrenergic signal. In parallel, pyruvate carboxylase (Pcarb) becomes active.
Moreover, in starvation, much alanine comes from muscle protein proteolysis, and is transaminated into pyruvate. Pyruvate carboxylase first converts pyruvate to OAA and then, PEP carboxykinase converts OAA to PEP etc…, until glucose. The inhibition of PK is necessary, if not one would go back to pyruvate. Phosphorylation of PK, and alanine, inhibit the enzyme.
PK and a PDH of tumors are inhibited by phosphorylation and alanine, like for gluconeogenesis, in spite of an increased glycolysis! Moreover, in tumors, one finds a particular PK, the M2 embryonic enzyme [2,9,10] the dimeric, phosphorylated form is inactive, leading to a “bottleneck “. The M2 PK has to be activated by fructose 1-6 bis P its allosteric activator, whereas the M1 adult enzyme is a constitutive active form. The M2 PK bottleneck between glycolysis and the citric acid cycle is a typical feature of tumor cell glycolysis.
Above the bottleneck, the massive entry of glucose accumulates PEP, which converts to OAA via mitochondria PEP carboxykinase, an enzyme requiring biotine-CO2-GDP. This source of OAA is abnormal, since Pcarb, another biotin-requiring enzyme, should have provided OAA. Tumors may indeed contain “morule inclusions” of biotin-enzyme suggesting an inhibition of Pcarb, presumably a consequence of the maintained citrate synthase activity, and decrease of ketone bodies that normally stimulate Pcarb. The OAA coming via PEP carboxykinase and OAA coming from aspartate transamination or via malate dehydrogenase condenses with acetyl CoA, feeding the elevated tumoral citric acid condensation starting the Krebs cycle.
Thus, tumors have to find large amounts of acetyl CoA for their condensation reaction; it comes essentially from lipolysis and β oxidation of fatty acids, and enters in the mitochondria via the carnitine transporter. This is the major source of acetyl CoA. It is as if the mechanism switching from gluconeogenesis to glycolysis was jammed in tumors, PK and PDH are at rest, like for gluconeogenesis, but citrate synthase is on. Thus, citric acid condensation pulls the glucose flux in the glycolytic direction, which needs NAD+; it will come from the pyruvate to lactate conversion by lactate dehydrogenase (LDH) no longer in competition with a quiescent Pcarb.
Since the citrate condensation consumes acetyl CoA, ketone bodies do not form; while citrate will support the synthesis of triglycerides via ATP citrate lyase and fatty acid synthesis… The cytosolic OAA drives the transaminases in a direction consuming amino acid. The result of these metabolic changes is that tumors burn glucose while consuming muscle protein and lipid stores of the organism. In a normal physiological situation, one mobilizes stores for making glucose or ketone bodies, but not while burning glucose!
The 21st Century Genomic Challenge?
According to the modern understanding of cancer, it is a disease caused by genetic and epigenetic alterations. Although this is now widely accepted, perhaps more emphasis has been given to the fact that cancer is a genetic disease. Numerous studies, including our earlier works, have supported the notion that carcinogenesis involves the activation of tumor-promoting oncogenes and the inactivation of growth-inhibiting tumor suppressor genes. It should be noted that in the post-genome sequencing project period of the 21st century, an in depth investigation of the factors associated with tumorigenesis is required for achieving it. Extensive research is warranted in two areas, namely, tumor bioenergetics and the cancer stem cell (CSC) hypothesis, neither of which received the required attention after the success of the genome sequencing project. An investigation of these two concepts would give rise to a new era in the study of cancer biology. Indeed, recent studies have indicated that the two apparently distinct fields might be related to each other and can converge more rapidly than previously recognized.
Warburg Effect Revisited
Cancer cells rarely depend on mitochondria for respiration and obtain almost half of their ATP by directly metabolizing glucose to lactic acid, even in the presence of oxygen. However, with the discovery that tumors do not show any shift to glycolysis, Warburg’s cancer theory (high lactate production and low mitochondrial respiration in tumor under normal oxygen pressure) was gradually discredited. Otto Warburg won a Nobel Prize in 1931 for the discovery of tumor bioenergetics, which is now commonly used as the basis of positron emission tomography (PET), a highly sensitive noninvasive technique used in cancer diagnosis. The increasing number of recent reports on the Warburg effect has reestablished the significance of this effect in tumorigenesis, indicating that bioenergetics may play a critical role in malignant transformation. Furthermore, it has been reported that TP53, which is one of the most commonly mutated genes in cancer, can trigger the Warburg effect. Glycolytic conversion is initiated in the early stages in cells that are genetically engineered to become cancerous, and the conversion was enhanced as the cells became more malignant. Therefore, the Warburg effect might directly contribute to the initiation of cancer formation not only by enhanced glycolysis but also via decreased respiration in the presence of oxygen, which suppresses apoptosis. This effect may also produce a metabolic shift to enhanced glycolysis and play a role in the early stages of multistep tumorigenesis in vivo.

Cancer Stem Cells (CSC) and Embryonic Stem Cells (ESC)
The importance of the cancer stem cell (CSC) hypothesis in therapy-related resistance and metastasis has been recognized during the past 2 decades. Accumulating evidence suggests that tumor bioenergetics plays a critical role in CSC regulation; this finding has opened up a new era of cancer medicine, which goes beyond cancer genomics.

Embryonic stem (ES) cells and immortalized primary and cancerous cells show a common concerted metabolic shift, including:

  • enhanced glycolysis,
  • decreased apoptosis, and
  • reduced mitochondrial respiration.

This finding reinforces the use of somatic stem cells or metastatic tumor cells in hypoxic niches. Hypoxia appears to regulate the functions of hematopoietic stem cells in the bone marrow and metastatic tumor cells by preserving important stem cell functions, such as:

  • cell cycle control,
  • survival,
  • metabolism, and
  • protection against oxidative stress.

Several companies and laboratories are now attempting to evaluate the bioenergetics associated with tumorigenesis by testing and challenging the available anticancer drugs.

A small population of cancer-initiating cells plays a very important role in current investigations. These CSCs may cause resistance to chemotherapy or radiation therapy or lead to post-therapy recurrence even when most of the cancer cells appear to be dead. In addition to their genetic alterations, CSCs are believed to mimic normal adult stem cells with regard to properties like self-renewal and undifferentiated status, which eventually leads to the formation of differentiated cells. Unlike well-differentiated daughter cells, small populations of CSCs are believed to be more resistant to toxic injuries and chemoradiotherapy. Indeed, the conventional cancer therapies have always been targeted toward proliferating cells. The control of CSCs, which is often exercised in the dormant phase of the cell cycle, can now be applied to achieve complete tumor regression.
Identification of cancer-specific markers
Due to their potential use in clinical applications, the surface markers of CSCs have been studied and identified. Adult stem cells and their malignant counterparts share similar intrinsic and extrinsic factors that regulate the

  • self renewal,
  • differentiation, and
  • proliferation pathways.

The following are the examples of candidate markers: musashi-1 (Msi-1), hairy and enhancer of split homolog-1 (Hes-1), CD133 (prominin-1, Prom1), epithelial cellular adhesion molecule (EpCam), claudin-7,29 CD44 variant isoforms, Lgr5,30Hedgehog (Hh), bone morphogenic protein (Bmp), Notch, and Wnt.
Is cancer a metabolic disease and genomic instability a secondary effect?
Bioenergetics of Cancer Stem Cells
The bioenergetics associated with the adaptation of CSCs to their micro-environment still requires extensive research. Although numerous studied suggested the association between Warburg effect and reduced oxidative stress in cancer, the relevant molecular mechanism was not known until very recently when Ruckenstuhl, et al. reported their findings in a yeast model.

How cancer cells achieve one of the most common phenotypes, namely, the “Warburg effect,” i.e., elevated glycolysis in the presence of oxygen, is still a topic of hypothesis, unless the involvement of glycolysis genes is considered.
The Warburg effect has been observed in differentiating cancer cells (e.g., cells that undergo epithelial-to-mesenchymal and mesenchymal-to-amoeboid transition), cells resistant to anoikis, and cells which interact with the stromal components of the metastatic niche. The epithelial-to-mesenchymal transition is involved in the resistance to chemotherapy in gastrointestinal cancer cells.

Cancer metastasis can be regarded as an integrated “escape program” triggered by redox changes. These alterations might be associated with avoiding oxidative stress in the niche of the tumor cells, or presumably with the response to treatments aimed at genetic targets, such as chemotherapy and radiation.
The introduction of induced pluripotent stem (iPS) cell genes was necessary for inducing the expression of immature status-related proteins in gastrointestinal cancer cells, and that the induced pluripotent cancer (iPC) cells were distinct from natural cancer cells with regard to their sensitivity to differentiation inducing treatment. For the complete eradication of cancer, however, future efforts should be directed toward improving translational research.
Cancer metabolism.
Glycolysis is elevated in tumors, but a pyruvate kinase (PK) “bottleneck” interrupts phosphoenol pyruvate (PEP) to pyruvate conversion. Thus, alanine following muscle proteolysis transaminates to pyruvate, feeding lactate dehydrogenase, converting pyruvate to lactate, (Warburg effect) and NAD+ required for glycolysis. Cytosolic malate dehydrogenase also provides NAD+ (in OAA to MAL direction). Malate moves through the shuttle giving back OAA in the mitochondria. Below the PK-bottleneck, pyruvate dehydrogenase (PDH) is phosphorylated (second bottleneck). However, citrate condensation increases: acetyl-CoA, will thus come from fatty acids b-oxydation and lipolysis, while OAA sources are via PEP carboxy kinase, and malate dehydrogenase, (pyruvate carboxylase is inactive). Citrate quits the mitochondria, (note interrupted Krebs cycle). In the cytosol, ATP citrate lyase cleaves citrate into acetyl CoA and OAA.
Acetyl CoA will make fatty acids-triglycerides. Above all, OAA pushes transaminases in a direction usually associated to gluconeogenesis! This consumes protein stores, providing alanine (ALA); like glutamine, it is essential for tumors. The transaminases output is aspartate (ASP) it joins with ASP from the shuttle and feeds ASP transcarbamylase, starting pyrimidine synthesis. ASP in not processed by argininosuccinate synthetase, which is blocked, interrupting the urea cycle.
Arginine gives ornithine via arginase, ornithine is decarboxylated into putrescine by ornithine decarboxylase. Putrescine and SAM form polyamines (spermine spermidine) via SAM decarboxylase. The other product 5-methylthioadenosine provides adenine. Arginine deprivation should affect tumors. The SAM destruction impairs methylations, particularly of PP2A, removing the “signaling kinase brake”, PP2A also fails to dephosphorylate PK and PDH, forming the “bottlenecks”.

Insulin or IGF actions boost the cellular influx of glucose and glycolysis. However, if the signaling pathway gets out of control, the tyrosine kinase phosphorylations may lead to a parallel PK blockade explaining the tumor bottleneck at the end of glycolysis. Since an activation of enyme kinases may indeed block essential enzymes (PK, PDH and others); in principle, the inactivation of phosphatases may also keep these enzymes in a phosphorylated form and lead to a similar bottleneck and we do know that oncogenes bind and affect PP2A phosphatase. In sum, a perturbed MAP kinase pathway, elicits metabolic features that would give to tumor cells their metabolic advantage.

Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment
Cancer cells show a broad spectrum of bioenergetic states, with some cells using aerobic glycolysis while others rely on oxidative phosphorylation as their main source of energy. In addition, there is mounting evidence that metabolic coupling occurs in aggressive tumors, between epithelial cancer cells and the stromal compartment, and between well-oxygenated and hypoxic compartments. We recently showed that oxidative stress in the tumor stroma, due to aerobic glycolysis and mitochondrial dysfunction, is important for cancer cell mutagenesis and tumor progression. More specifically, increased autophagy/mitophagy in the tumor stroma drives a form of parasitic epithelial-stromal metabolic coupling. These findings explain why it is effective to treat tumors with either inducers or inhibitors of autophagy, as both would disrupt this energetic coupling. We also discuss evidence that glutamine addiction in cancer cells produces ammonia via oxidative mitochondrial metabolism.

Ammonia production in cancer cells, in turn, could then help maintain autophagy in the tumor stromal compartment. In this vicious cycle, the initial glutamine provided to cancer cells would be produced by autophagy in the tumor stroma. Thus, we believe that parasitic epithelial-stromal metabolic coupling has important implications for cancer diagnosis and therapy, for example, in designing novel metabolic imaging techniques and establishing new targeted therapies. In direct support of this notion, we identified a loss of stromal caveolin-1 as a marker of oxidative stress, hypoxia, and autophagy in the tumor microenvironment, explaining its powerful predictive value. Loss of stromal caveolin-1 in breast cancers is associated with early tumor recurrence, metastasis, and drug resistance, leading to poor clinical outcome.
The conventional ‘Warburg effect’ versus oxidative mitochondrial metabolism
Warburg’s original work indicated that while glucose uptake and lactate production are greatly elevated, a cancer cell’s rate of mitochondrial respiration is similar to that of normal cells. He, however, described it as a ‘respiratory impairment’ due to the fact that, in cancer cells, mitochondrial respiration is smaller, relative to their glycolytic power, but not smaller relative to normal cells. He recognized that oxygen consumption is not diminished in tumor cells, but that respiration is disturbed because glycolysis persists in the presence of oxygen. Unfortunately, the perception of his original findings was simplified over the years, and most subsequent papers validated that cancer cells undergo aerobic glycolysis and produce lactate, but did not measure mitochondrial respiration, and just presumed decreased tricarboxylic acid (TCA) cycle activity and reduced oxidative phosphorylation [1,2]. It is indeed well documented that, as a consequence of intra-tumoral hypoxia, the hypoxia-inducible factor (HIF)1α pathway is activated in many tumors cells, resulting in the direct up-regulation of lactate dehydrogenase (LDH) and increased glucose consumption.
It is now clear that cancer cells utilize both glycolysis and oxidative phosphorylation to satisfy their metabolic needs. Experimental assessments of ATP production in cancer cells have demonstrated that oxidative pathways play a signifi cant role in energy generation, and may be responsible for about 50 to 80% of the ATP generated. several studies now clearly indicate that mitochondrial activity and oxidative phosphorylation support tumor growth. Loss-of-function mutations in the TCA cycle gene IDH1 (isocitrate dehydrogenase 1) are found in about 70% of gliomas, but, interestingly, correlate with a better prognosis and improved survival, suggesting that severely decreased activity in one of the TCA cycle enzymes does not favor tumor aggressiveness. The mitochondrial protein p32 was shown to maintain high levels of oxidative phosphorylation in human cancer cells and to sustain tumorigenicity in vivo. In addition, STAT3 is known to enhance tumor growth and to predict poor prognosis in human cancers. Interestingly, a pool of STAT3 localizes to the mitochondria, to sustain high levels of mitochondrial respiration and to augment transformation by oncogenic Ras. Similarly, the mitochondrial transcription factor A (TFAM), which is required for mitochondrial DNA replication and oxidative phosphorylation, is also required for K-Ras induced lung tumorigenesis.
There is also evidence that pro-oncogenic molecules regulate mitochondrial function. Cyclin D1 inhibits mitochondrial function in breast cancer cells. Overexpression of cyclin D1 is observed in about 50% of invasive breast cancers and is associated with a good clinical outcome, indicating that inhibition of mitochondrial activity correlates with favorable prognosis. Importantly, it was shown that the oncogene c-Myc stimulates mitochondrial biogenesis, and enhances glutamine metabolism by regulating the expression of mitochondrial glutaminase, the first enzyme in the glutamine utilization pathway. Glutamine is an essential metabolic fuel that is converted to alpha-ketoglutarate and serves as a substrate for the TCA cycle or for glutathione synthesis, to promote energy production and cellular biosynthesis, and to protect against oxidative stress. Interestingly, pharmacological targeting of mitochondrial glutaminase inhibits cancer cell transforming activity, suggesting that glutamine metabolism and its role in fueling and replenishing the TCA cycle are required for neoplastic transformation.
Reverse Warburg Effect.
It is increasingly apparent that the tumor microenvironment regulates neoplastic growth and progression. Activation of the stroma is a critical step required for tumor formation. Among the stromal players, cancer associated fi broblasts (CAFs) have recently taken center stage [25]. CAFs are activated, contractile        fibroblasts that display features of myo-fibroblasts, express muscle specific actin, and show an increased ability to secrete and remodel the extracellular matrix. They are not just neutral spectators, but actively support malignant transformation and metastasis, as compared to normal resting fibroblasts.

Importantly, the tumor stroma dictates clinical outcome and constitutes a source of potential biomarkers. Expression profiling has identified a cancer-associated stromal signature that predicts good and poor clinical prognosis in breast cancer patients, independently of other factors.

A loss of caveolin-1 (Cav-1) in the stromal compartment is a novel biomarker for predicting poor clinical outcome in all of the most common subtypes of human breast cancer, including the more lethal triple negative subtype. A loss of stromal Cav-1 predicts early tumor recurrence, lymph node metastasis, tamoxifen-resistance, and poor survival.

Overall, breast cancer patients with a loss of stromal Cav-1show a 20% 5-year survival rate, compared to the 80% 5-year survival of patients with high stromal Cav-1 expression. In triple negative patients, the 5-year survival rate is 75.5% for high stromal Cav-1 versus 9.4% for absent stromal Cav-1. A loss of stromal Cav-1 also predicts progression to invasive disease in ductal carcinoma in situ patients, suggesting that a loss of Cav-1 regulates tumor progression. Similarly, a loss of stromal Cav-1 is associated with advanced disease and metastasis, as well as a high Gleason score, in prostate cancer patients.

The autophagic tumor stroma model of cancer metabolism.
Cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts (CAFs). This activates reactive oxygen species (ROS) production and autophagy. ROS production in CAFs, via the bystander eff ect, serves to induce random mutagenesis in epithelial cancer cells, leading to double-strand DNA breaks and aneuploidy. Cancer cells mount an anti-oxidant defense and upregulate molecules that protect them against ROS and autophagy, preventing them from undergoing apoptosis. So, stromal fibroblasts conveniently feed and mutagenize cancer cells, while protecting them against death. See the text for more details. A+, autophagy positive; A-, autophagy negative; AR, autophagy resistant.

1. Recycled Nutrients
2. Random Mutagenesis
3. Protection Against Apoptosis
The clinical use of PET is well established in Hodgkin’s lymphomas which are composed of less than 10% tumor cells, the rest being stromal and inflammatory cells. Yet, Hodgkin’s lymphomas are very PET avid tumors, suggesting that 2-deoxy-glucose uptake may be associated with the tumor stroma. That the fibrotic component may be glucose avid is further supported by the notion that PET is clinically used to assess the therapeutic response in gastrointestinal stromal tumors (GIST), which are a subset of tumors of mesenchymal origin.
The reverse Warburg effect can be described as ‘metabolic coupling’ between supporting glycolytic stromal cells and oxidative tumor cells. Metabolic cooperativity between adjacent cell-compartments is observed in several normal physiological settings.
The reverse Warburg effect.
Via oxidative stress, cancer cells activate two major transcription factors in adjacent stromal fibroblasts (hypoxia-inducible factor (HIF)1α and NFκB).
This leads to the onset of both autophagy and mitophagy, as well as aerobic glycolysis, which then produces recycled nutrients (such as lactate, ketones, and glutamine).
These high-energy chemical building blocks can then be transferred and used as fuel in the tricarboxylic acid cycle (TCA) in adjacent cancer cells.
The outcome is high ATP production in cancer cells, and protection against cell death. ROS, reactive oxygen species.
The methylation hypothesis and the role of PP2A phosphatase
Diethanolamine decreased choline derivatives and methyl donors in the liver, like seen in a choline deficient diet. Such conditions trigger tumors in mice, particularly in the B6C3F1 strain. Again, the historical perspective recalled by Newberne’s comment brings us back to insulin. Indeed, after the discovery of insulin in 1922, Banting and Best were able to keep alive for several months depancreatized dogs, treated with pure insulin. However, these dogs developed a fatty liver and died. Unlike pure insulin, the total pancreatic extract contained a substance that prevented fatty liver: a lipotropic substance identified later as being choline. Like other lipotropes, (methionine, folate, B12) choline supports transmethylation reactions, of a variety of substrates, that would change their cellular fate, or action, after methylation. In the particular case concerned here, the removal of triglycerides from the liver, as very low-density lipoprotein particles (VLDL), requires the synthesis of lecithin, which might decrease if choline and S-adenosyl methionine (SAM) are missing. Hence, a choline deficient diet decreases the removal of triglycerides from the liver; a fatty liver and tumors may then form. In sum, we have seen that pathways exemplified by the insulin-tyrosine kinase signaling pathway, which control anabolic processes, mitosis, growth and cell death, are at each step targets for oncogenes; we now find that insulin may also provoke fatty liver and cancer, when choline is not associated to insulin.

We know that after the tyrosine kinase reaction, serine-threonine kinases take over along the signaling route. It is thus highly probable that serine-threonine phosphatases will counteract the kinases and limit the intensity of the insulin or insulin like signals. One of the phosphatases involved is PP2A, itself the target of DNA viral oncogenes (Polyoma or SV40 antigens react with PP2A subunits and cause tumors). We found a possible link between the PP2A phosphatase brake and choline. the catalytic C subunit of PP2A is associated to a structural subunit A. When C receives a methyl, the dimer recruits a regulatory subunit B. The trimer then targets specific proteins that are dephosphorylated. choline, via SAM, methylates PP2A, which is targeted toward the serine-threonine kinases that are counteracted along the insulin-signaling pathway.

The choline dependent methylation of PP2A is the brake, the “antidote”, which limits “the poison” resulting from an excess of insulin signaling. Moreover, it seems that choline deficiency is involved in the L to M2 transition of PK isoenzymes. The negative regulation of Ras/MAP kinase signals mediated by PP2A phosphatase seems to be complex. The serine-threonine phosphatase does more than simply counteracting kinases; it binds to the intermediate Shc protein on the signaling cascade, which is inhibited. The targeting of PP2A towards proteins of the signaling pathway depends of the assembly of the different holoenzymes.

The relative decrease of methylated PP2A in the cytosol, not only cancels the brake over the signaling kinases, but also favors the inactivation of PK and PDH, which remain phosphorylated, contributing to the metabolic anomaly of tumor cells. In order to prevent tumors, one should then favor the methylation route rather than the phosphorylation route for choline metabolism. This would decrease triglycerides, promote the methylation of PP2A and keep it in the cytosol, reestablishing the brake over signaling kinases. Moreover, PK, and PDH would become active after the phosphatase action. One would also gain to inhibit their kinases as recently done with dichloroacetate for PDH kinase. The nuclear or cytosolic targeting of PP2A isoforms is a hypothesis also inspired by several works.
Hypoxic adaptations in the presence of oxygen
Through different biochemical and biophysical pathways, which are characteristic to cancer cells, tumor cells adopt this phenotype, i.e., high glycolysis and decreased respiration, in the presence of oxygen. It has been shown that although the induction of hypoxia and cellular proliferation engage entirely different cellular pathways, they often coexist during tumor growth. The ability of cells to grow during hypoxia results, in part, from the crosstalk between hypoxia-inducible factors (Hifs) and the proto-oncogene c-Myc. These genes partially regulate the development of complex adaptations of tumor cells growing in low O2, and contribute to fine tuning the adaptive responses of cells to hypoxic environments.

Hypoxic conditions seem to trigger back the expression of the fetal gene packet via HIF1-Von-Hippel signals. The mechanism would depend of a double switch since not all fetal genes become active after hypoxia. First, the histones have to be in an acetylated form, opening the way to transcription factors, this depends either of histone eacetylase (HDAC) inhibition or of histone acetyltransferase (HAT) activation, and represents the main switch

Growth hormone-IGF actions, the control of asymmetrical mitosis
When IGF – Growth hormone operate, the fatty acid source of acetyl CoA takes over. Indeed, GH stimulates a triglyceride lipase in adipocytes, increasing the release of fatty acids and their b oxidation. In parallel, GH would close the glycolytic source of acetyl CoA, perhaps inhibiting the hexokinase interaction with the mitochondrial ANT site. This effect, which renders apoptosis possible, does not occur in tumor cells. GH mobilizes the fatty acid source of acetyl CoA from adipocytes, which should help the formation of ketone bodies. Since citrate synthase activity is elevated in tumors, ketone bodies do not form. This result silences several genes like PETEN, P53, or methylase inhibitory genes. It is probable that the IGFBP gene gets silent as well.

Uncoupling Proteins in Cancer
Uncoupling proteins (UCPs) are a family of inner mitochondrial membrane proteins whose function is to allow the re-entry of protons to the mitochondrial matrix, by dissipating the proton gradient and, subsequently, decreasing membrane potential and production of reactive oxygen species (ROS). Due to their pivotal role in the intersection between energy efficiency and oxidative stress, UCPs are being investigated for a potential role in cancer.

Mitochondria have been shown to be key players in numerous cellular events tightly related with the biology of cancer. Although energy production relies on the glycolytic pathway in cancer cells, these organelles also participate in many other processes essential for cell survival and proliferation such as ROS production, apoptotic and necrotic cell death, modulation of oxygen concentration, calcium and iron homeostasis, and certain metabolic and biosynthetic pathways. Many of these mitochondrial-dependent processes are altered in cancer cells, leading to a phenotype characterized, among others, by higher oxidative stress, inhibition of apoptosis, enhanced cell proliferation, chemoresistance, induction of angiogenic genes and aggressive fatty acid oxidation. Uncoupling proteins, a family of inner mitochondrial membrane proteins specialized in energy-dissipation, has aroused enormous interest in cancer due to their relevant impact on such processes and their potential for the development of novel therapeutic strategies.
Briefly, oxidation of reduced nutrient molecules, such as carbohydrates, lipids, and proteins, through cellular metabolism yields electrons in the form of reduced hydrogen carriers NADH+ and FADH2. These reduced cofactors donate electrons to a series of protein complexes embedded in the inner mitochondrial membrane known as the electron transport chain (ETC). These complexes use the energy released from electron transport for active pumping of protons across the inner membrane, generating an electrochemical gradient. Mitochondria orchestrate conversions between different forms of energy, coupling aerobic respiration to phosphorylation.
Conversion of metabolic fuel into ATP is not a fully efficient process. Some of the energy of the electrochemical gradient is not coupled to ATP production due to a phenomenon known as proton leak, which consists of the return of protons to the mitochondrial matrix through alternative pathways that bypass ATP synthase. Although this apparently futile cycle of protons is physiologically important, accounting for 20-25% of basal metabolic rate, its function is still a subject of debate. Several different functions have been suggested for proton leak, including thermogenesis, regulation of energy metabolism, and control of body weight and attenuation of reactive oxygen species (ROS) production. Although a part of the proton leak may be attributed to biophysical properties of the inner membrane, such as protein/lipid interfaces, the bulk of the proton conductance is linked to the action of a family of mitochondrial proteins termed uncoupling proteins.

Mitochondria are the major sources of reactive oxygen species (ROS). Aerobic respiration involves the complete reduction of oxygen to water, which is catalysed by complex IV (or cytochrome c oxidase). Nevertheless, during the transfer of electrons along the electron transport complexes, single electrons sometimes escape and result in a single electron reduction of molecular oxygen to form a superoxide anion, which, in turn is the precursor of other ROS.

One of the most interesting functions attributed to UCPs is their ability to decrease the formation of mitochondrial ROS. Mitochondria are the main source of ROS in cells. Superoxide formation is strongly activated under resting (state 4) conditions when the membrane potential is high and the rate of electron transport is limited by lack of ADP and Pi. Thus, there is a well established strong positive correlation between membrane potential and ROS production.
A small increase in membrane potential gives rise to a large stimulation of ROS production, whereas a small decrease in membrane potential (10 mV) is able to inhibit ROS production by 70% . Therefore, mild uncoupling, i.e., a small decrease in membrane potential, has been suggested to have a natural antioxidant effect.

Consistent with such a proposal, the inhibition of UCPs by GDP in mitochondria has been shown to increase membrane potential and mitochondrial ROS production. The loss of UCP2 or UCP3 in knockouts yielded increased ROS production concurrent with elevated membrane potential specifically in those tissues normally expressing the missing protein.
The hypothesis of UCPs as an antioxidant defense has been strongly supported by the fact that these proteins have been shown to be activated by ROS or by-products of lipid peroxidation, showing that UCPs would form part of a negative feed-back mechanism aimed to mitigate excessive ROS production and oxidative damage.
ROS and Cancer
ROS are thought to play multiple roles in tumor initiation, progression and maintenance, eliciting cellular responses that range from proliferation to cell death. In normal cells, ROS play crucial roles in several biological mechanisms including phagocytosis, proliferation, apoptosis, detoxification and other biochemical reactions. Low levels of ROS regulate cellular signaling and play an important role in normal cell proliferation. During initiation of cancer, ROS may cause DNA damage and mutagenesis, while ROS acting as second messengers stimulate proliferation and inhibit apoptosis, conferring growth advantage to established cancer cells. Cancer cells have been found to have increased ROS levels.

One of the functional roles of these elevated ROS levels during tumor progression is constant activation of transcription factors such as NF-kappaB and AP-1 which induce genes that promote proliferation and inhibit apoptosis. In addition, oxidative stress can induce DNA damage which leads to genomic instability and the acquisition of new mutations, which may contribute to cancer progression.

Role of ROS in control of proliferation and apoptosis
ROS are also essential mediators of apoptosis which eliminates cancer and other cells that threaten our health [81–86]. Many chemotherapeutic drugs and radiotherapy are aimed at increasing ROS levels to promote apoptosis by stimulating pro-apoptotic singaling molecules such as ASK1, JNK and p38. Because of the pivotal role of ROS in triggering apoptosis, antioxidants can inhibit this protective mechanism by depleting ROS. Thus, antioxidant mechanisms are thought to interfere with the therapeutic activity of anticancer drugs that kill advanced stage cancer cells by apoptosis.

Effect of uncoupling proteins on proliferation and apoptosis in relation to ROS levels

Uncoupling-to-survive hypothesis (proposed by Brand)

  • the ability of UCP2 to increase lifespan is mediated by decreased ROS production and oxidative stress.
  • the ability of mild uncoupling to avoid ROS formation, gives a reasonable argument to hypothesize about a role for UCPs in cancer prevention

Consistently, Derdák et al. showed that Ucp2−/− mice treated with the carcinogen azoxymethane were found to develop more aberrant crypt foci and colon tumours than Ucp2+/+ in relation with increased oxidative stress and enhanced NF-kappaB activation.

Roles of UCPs in Cancer Progression
The growth of a tumor from a single genetically altered cell is a stepwise progression requiring the alterations of several genes which contribute to the acquisition of a malignant phenotype. Such genetic alterations are positively selected when in the tumor, they confer a proliferative, survival or treatment resistance advantage for the host cell. In addition, several mutations, such as those silencing tumour suppressor genes, trigger the probability of accumulating new mutations, so the process of malignant transformation is progressively self-accelerated.

Considering the ability of UCPs to modulate mutagenic ROS, as well as mitochondrial bioenergetics and membrane potential, both involved in regulation of cell survival, an interesting question is whether UCPs can be involved in the progression of cancer.

Increased uncoupled respiration may be a mechanism to lower cellular oxygen concentration and, thus, alter molecular pathways of oxygen sensing such as those regulated by hypoxia-inducible factor (HIF). In normoxia, the alpha subunit of HIF-1 is a target for prolyl hydroxylase, which makes HIF-1alpha a target for degradation by the proteasome. During hypoxia, prolyl hydroxylase is inhibited since it requires oxygen as a cosubstrate. Thus, hypoxia allows HIF to accumulate and translocate into the nucleus for induction of target genes regulating glycolysis, angiogenesis and hematopoiesis. By this mechanism, UCPs activity may contribute to increase the expression of genes related to the formation of blood vessels, and thus promote tumor growth.

Roles of UCPs in Cancer Energy Metabolism
Lynen and colleagues proposed that the root of the Warburg effect is not in the inability of mitochondria to carry out respiration, but rather would rely on their incapacity to synthesize ATP in response to membrane potential.

The ability of UCPs to uncouple ATP synthesis from respiration and the fact that UCP2 is overexpressed in several chemoresistant cancer cell lines and primary human colon cancers have lead to speculate about the existence of a link between UCPs and the Warburg effect. As mentioned above, uncoupling induced by overexpression of UCP2 has been shown to prevent ROS formation, and, in turn, increase apoptotic threshold in cancer cells, providing a pro-survival advantage and a resistance mechanism to cope with ROS-inducing chemo-therapeutic agents.

Mitochondrial Krebs cycle is one of the sources for these anabolic precursors. The export of these metabolites to cytoplasm for anabolic purposes involves the replenishment of the cycle intermediates by anaplerotic substrates such as pyruvate and glutamate. Thus, glycolysis-derived pyruvate, as well as alpha-ketoglutarate derived from glutaminolysis, may be necessary to sustain anaplerotic reactions. At the same time, to keep Krebs cycle functional, the reduced cofactors NADH and FADH2 would have to be re-oxidized, a function which relies on the mitochondrial respiratory chain. Once again, uncoupling may be crucial for cancer cell mitochondrial metabolism, allowing Krebs cycle to be kept functional to meet the vigorous biosynthetic demand of cancer cells.

Several cancer cells resistant to chemotherapeutics and radiation often exhibit higher rates of fatty acid oxidation and it has been observed that inhibition of fatty acid oxidation potentiates apoptotic death induced by chemotherapeutic agents. These findings are in agreement with the proposed need of fatty acid for the activity of UCPs, suggesting that the lack of these potential substrates or activators would decrease uncoupling activity, subsequently increasing membrane potential, ROS production and therefore lowering apoptotic threshold.

Roles of UCPs in Cancer Cachexia
Cachexia is a wasting syndrome characterized by weakness, weight and fat loss, and muscle atrophy which is often seen in patients with advanced cancer or AIDS. Cachexia has been suggested to be responsible for at least 20 % of cancer deaths and also plays an important part in the compromised immunity leading to death from infection. The imbalance between energy intake and energy expenditure underlying cachexia cannot be reversed nutritionally.

Alterations leading to high energy expenditure, such as excessive proton leak or mitochondrial uncoupling, are likely mechanisms underlying cachexia. In fact, increased expression of UCP1 in BAT and UCP2 and UCP3 in skeletal muscle have been shown in several murine models of cancer cachexia

Roles of UCPs in Chemoresistance

Cancer cells acquire drug resistance as a result of selection pressure dictated by unfavorable microenvironments. Although mild uncoupling may clearly be useful under normal conditions or under severe or chronic metabolic stress such as hypoxia or anoxia, it may be a mechanism to elude oxidative stress-induced apoptosis in advanced cancer cells. Several anti-cancer treatments are based on promotion of ROS formation, to induce cell growth arrest and apoptosis. Thus, increased UCP levels in cancer cells, rather than a marker of oxidative stress, may be a mechanisms conferring anti-apoptotic advantages to the malignant cell, increasing their ability to survive in adverse microenvironments, radiotherapy and chemotherapy. UCPs appear to play a permissive role in tumor cell survival and growth.

Expression of UCPs promote bioenergetics adaptation and cell survival. UCPs appear to be critical to determine the sensitivity of cancer cells to several chemotherapeutic agents and radiotherapy, interfering with the activation of mitochondria driven apoptosis.

From a therapeutic viewpoint, inhibition of glycolysis in UCP2 expressing tumours or specific inhibition of UCP2 are, respectively, attractive strategies to target the specific metabolic signature of cancer cells.

Hypoxia-inducible factor-1 in tumour angiogenesis
HIF-b subunits, is a heterodimeric transcriptional activator. In response to

  • stimulation of growth factors, and
  • activation of oncogenes as well as carcinogens,

HIF-1a is overexpressed and/or activated and targets those genes which are required for angiogenesis, metabolic adaptation to low oxygen and promotes survival.

Several dozens of putative direct HIF-1 target genes have been identified on the basis of one or more cis-acting hypoxia-response elements that contain an HIF-1 binding site. Activation of HIF-1 in combination with activated signaling pathways and regulators is implicated in tumour progression and prognosis.
In order for a macroscopic tumour to grow, adequate oxygen delivery must be effected via tumor angiogenesis that results from an increased synthesis of angiogenic factors and a decreased synthesis of anti-angiogenic factors. The metabolic adaptation of tumor cells to reduced oxygen availability by increasing glucose transport and glycolysis to promote survival are important consequences in response to hypoxia.

Hypoxia and HIF-1
Hypoxia is one of the major drivers to tumour progression as hypoxic areas form in human tumours when the growth of tumour cells in a given area outstrips local neovascularization, thereby creating areas of inadequate perfusion. Although several transcriptional factors have been reported to be involved
in the response to hypoxic stress such as AP-1, NF-kB and HIF-1, HIF-1 is the most potent inducer of the expression of genes such as those encoding for glycolytic enzymes, VEGF and erythropoietin.

HIF-a subunit exists as at least three isoforms, HIF-1a, HIF-2a and HIF-3a. HIF-1a and HIF-2a can form heterodimers with HIF-b. Although HIF-b subunits are constitutive nuclear proteins, both HIF-1a andHIF-2a subunits are strongly induced by hypoxia in a similar manner. HIF-1a is up-regulated in hypoxic tumour cells and activates the transcription of target genes by binding to cis-acting enhancers, hypoxic responsive element (HRE) close to the promoters of these genes with a result of tumour cellular adaptation to hypoxia and tumour angiogenesis, and promotion of further growth of the primary tumour. Studies have shown HIF-1a to be over-expressed by both tumour cells and such stromal cells as macrophages in many forms of human malignancy.

Regulation of HIF-1
The first regulator of HIF-1 is oxygen. HIF-1α appears to be the HIF-1 subunit regulated by hypoxia. The oxygen sensors in the HIF-1α pathway are two kinds of oxygen dependent hydroxylases. One is prolyl hydroxylase which could hydroxylize the proline residues 402 and 564 at the oxygen dependent domain (ODD) of HIF-1 in the presence of oxygen and iron with a result of HIF-α degradation. The other is hydroxylation of Asn803 at the C-terminal transactivation domain (TAD-C) by FIH-1, which could inhibit the interaction of HIF-1α with co-activator p300 with a subsequent inhibition of HIF-1α transactivity. The hydroxylation of proline 564 at ODD of HIF-1α under normoxia was shown using a novel hydroxylation-specific antibody to detect hydroxylized HIF-1α.

Oncogene comes as the second regulator. Many oncogenes have effects on HIF-1α. Among them, some function in regulation of HIF-1α protein stability or degradation, others play roles in several activated signaling pathways. Tumor suppressor genes as p53 and von Hippel-Lindau (VHL) influence the levels and functions of HIF-1. The wild type (wt) form of p53 protein was involved in inhibiting HIF-1 activity by targeting the HIF-1a subunit for Mdm2-mediated ubiquitination and proteasomal degradation, and in inducing inhibitors of angiogenesis such as thrombospondin-1, while loss of wt p53 (by gene deletion or mutation) could enhance HIF-1α accumulation in hypoxia.

The third regulator is a battery of growth factors and cytokines from stromal and parenchymal cells such as

  • EGF,
  • transforming growth factor-α,
  • insulin-like growth factors 1 and 2,
  • heregulin, and interleukin-1b

via autocrine and paracrine pathways. These regulators not only induce the expression of HIF-1α protein, HIF-1 DNA binding activity and transactivity, but also make HIF-1 target gene expression under normoxia or hypoxia.
The fourth one is a group of reactive oxygen species (ROS) resulting from carcinogens such as Vanadate and Cr (VI) or stimulation of cytokines such as angiotensin and TNFa. However, it seems controversial when it comes to the production of ROS under hypoxia and their individual role in regulation of HIF-1a. It is well known that ROS plays an important role in carcinogenesis induced by a variety of carcinogens.

Signaling Pathways Involved in Regulation of HIF-1α
HIF-1 is a phosphorylated protein and its phosphorylation is involved in HIF-1a subunit expression and/or stabilization as well as in the regulation of HIF-1 transcriptional activity. Three signaling pathways involved in the regulation of HIF-1α have been reported to date.

  • The PI-3k pathway has been mainly and frequently implicated in regulation of HIF-1α protein expression and stability.
  • Akt is also activated by hypoxia. Activated Akt initiates two different pathways in regulation of HIF-1α. The function of these two pathways appears to show consistent impact on HIF-1α activation.
  • Signal transduction pathway in HIF-1α regulation.Oncogenes, growth factors and hypoxia have been documented to regulate HIF-1α protein and increase its transactivity. GSK and mTOR were two target events of Akt and could contribute to decreasing HIF-1α degradation and increasing HIF-1α protein synthesis. Activated ERK1/2 could mainly up-regulate.

HIF-1a, Angiogenesis and Tumour Prognosis
Hypoxia, oncogenes and a variety of growth factors and cytokines increase HIF-1α stability and/or synthesis and transactivation to initiate tumour angiogenesis, metabolic adaptation to hypoxic situation and promote cell survival or anti-apoptosis resulting from a consequence of more than sixty putative direct HIF-1 target gene expressions.

The crucial role of HIF-1 in tumour angiogenesis has sparked scientists and clinical researchers to try their best to understand the whole diagram of HIF-1 so as to find out novel approaches to inhibit HIF-1 overexpression. Indeed, the combination of anti-angiogenic agent and inhibitor of HIF-1 might be particularly efficacious, as the angiogenesis inhibitor would cut off the tumour’s blood supply and HIF-1 inhibitor would reduce the ability of tumour adaptation to hypoxia and suppress the proliferation and promote apoptosis. Screens for small-molecule inhibitors of HIF-1 are underway and several agents that inhibit HIF-1, angiogenesis and xenograft growth have been identified.

Hypoxia, autophagy, and mitophagy in the tumor stroma
Metabolomic profiling reveals that Cav-1(-/-) null mammary fat pads display a highly catabolic metabolism, with the increased release of several metabolites, such as amino acids, ribose and nucleotides, and a shift towards gluconeogenesis, as well as mitochondrial dysfunction. These changes are consistent with increased autophagy, mitophagy and aerobic glycolysis, all processes that are induced by oxidative stress. Autophagy or ‘self-eating’ is the process by which cells degrade their own cellular components to survive during starvation or to eliminate damaged organelles after oxidative stress. Mitophagy, or mitochondrial-autophagy, is particularly important to remove damaged ROS-generating mitochondria.

An autophagy/mitophagy program is also triggered by hypoxia. Hypoxia is a common feature of solid tumors, and promotes cancer progression, invasion and metastasis. Interestingly, via induction of autophagy, hypoxia is sufficient to induce a dramatic loss of Cav-1 in fibroblasts. The hypoxia-induced loss of Cav-1 can be inhibited by the autophagy inhibitor chloroquine, or by pharmacological inhibition of HIF1α. Conversely, small interfering RNA-mediated Cav-1 knock-down is sufficient to induce pseudo-hypoxia, with HIF1α and NFκB activation, and to promote autophagy/mitophagy, as well as a loss of mitochondrial membrane potential in stromal cells. These results indicate that a loss of stromal Cav-1 is a marker of hypoxia and oxidative stress.

In a co-culture model, autophagy in cancer-associated fibroblasts was shown to promote tumor cell survival via the induction of the pro-autophagic HIF1α and NFκB pathways in the tumor stromal microenvironment. Finally, the mitophagy marker Bnip3L is selectively upregulated in the stroma of human breast cancers lacking Cav-1, but is notably absent from the adjacent breast cancer epithelial cells.

Metabolome profiling of several types of human cancer tissues versus corresponding normal tissues have consistently shown that cancer tissues are highly catabolic, with the significant accumulation of many amino acids and TCA cycle metabolites. The levels of reduced glutathione were decreased in primary and metastatic prostate cancers compared to benign adjacent prostate tissue, suggesting that aggressive disease is associated with increased oxidative stress. Also, these data show that the tumor microenvironment has increased oxidative-stress-induced autophagy and increased catabolism.

Taken together, all these findings suggest an integrated model whereby
A loss of stromal Cav-1 induces autophagy/mitophagy in the tumor stroma, via oxidative stress.

This creates a catabolic micro-environment with the local accumulation of chemical building blocks and recycled nutrients (such as amino acids and nucleotides), directly feeding cancer cells to sustain their survival and growth.
This novel idea is termed the ‘autophagic tumor stroma model of cancer’ .
This new paradigm may explain the ‘autophagy paradox’, which is based on the fact that both the systemic inhibition and systemic stimulation of autophagy prevent tumor formation.

What is presented suggests that vectorial energy transfer from the tumor stroma to cancer cells directly sustains tumor growth, and that interruption of such metabolic coupling will block tumor growth. Autophagy inhibitors (such as chloroquine) functionally block the catabolic transfer of metabolites from the stroma to the tumor, inducing cancer cell starvation and death. Conversely, autophagy inducers (such as rapamycin) promote autophagy in tumor cells and induce cell death. Thus, both inhibitors and inducers of autophagy will have a similar effect by severing the metabolic coupling of the stroma and tumor cells, resulting in tumor growth inhibition (cutting ‘off ’ the fuel supply).

This model may also explain why enthusiasm for antiangiogenic therapy has been dampened. In most cases, the clinical benefits are short term, and more importantly, new data suggest an unexpected link between anti-angiogenic treatments and metastasis. In pre-clinical models, anti-vascular endothelial growth factor (anti-VEGF) drugs (sunitinib and anti-VEGFR2 blocking antibodies) were shown to inhibit localized tumor formation, but potently induced relapse and metastasis. Thus, by inducing hypoxia in the tumor microenvironment, antiangiogenic drugs may create a more favorable metastatic niche.
Glutamine, glutaminolysis.
In direct support that cancer cells use mitochondrial oxidative metabolism, many investigators have shown that cancer cells are ‘addicted’ to glutamine. Glutamine is a non-essential amino acid that is metabolized to glutamate and enters the TCA cycle as α-ketoglutarate, resulting in high ATP generation via oxidative phosphorylation. Recent studies also show that ammonia is a by-product of glutaminolysis. In addition, ammonia can act as a diffusible inducer of autophagy. Given these observations, glutamine addiction in cancer cells provides another mechanism for driving and maintaining autophagy in the tumor micro-environment .

In support of this idea, a loss of Cav-1 in the stroma is sufficient to drive autophagy, resulting in increased glutamine production in the tumor micro-environment. Thus, this concept defines a new vicious cycle in which autophagy in the tumor stroma transfers glutamine to cancer cells, and the by-product of this metabolism, ammonia, maintains autophagic glutamine production. This model fits well with the ‘autophagic tumor stroma model of cancer metabolism’, in which energy rich recycled nutrients (lactate, ketones, and glutamine) fuel oxidative mitochondrial metabolism in cancer cells.

Glutamine utilization in cancer cells and the tumor stroma. Oxidative mitochondrial metabolism of glutamine in cancer cells produces ammonia. Ammonia production is sufficient to induce autophagy. Thus, autophagy in cancer-associated fibroblasts provides cancer cells with an abundant source of glutamine. In turn, the ammonia produced maintains the autophagic phenotype of the adjacent stromal fibroblasts.

Lessons from other paradigms

an infectious parasitic cancer cell that metastasizes and captures mitochondrial DNA from host cells
Cancer cells behave like ‘parasites’, by inducing oxidative stress in normal host fibroblasts, resulting in the production of recycled nutrients via autophagy.

This is exactly the same mechanism by which infectious parasites (such as malaria) obtain nutrients and are propagated by inducing oxidative stress and autophagy in host cells. In this regard, malaria is an ‘intracellular’ parasite, while cancer cells may be thought of as ‘extracellular’ parasites. This explains why chloroquine is both an effective antimalarial drug and an effective anti-tumor agent, as it functions as an autophagy inhibitor, cutting off the ‘fuel supply’ in both disease states.

Human cancer cells can ‘steal’ live mitochondria or mitochondrial DNA from adjacent mesenchymal stem cells in culture, which then rescues aerobic glycolysis in these cancer cells. This is known as mitochondrial transfer. Interestingly, metastatic breast cancer cells show the up-regulation of numerous mitochondrial proteins, specifically associated with oxidative phosphorylation, as seen by unbiased proteomic analysis.

Thus, increased mitochondrial oxidative metabolism may be a key driver of tumor cell metastasis. In further support of this argument, treatment of MCF7 cancer cells with lactate is indeed sufficient to induce mitochondrial biogenesis in these cells.

To determine if these findings may be clinically relevant, a lactate-induced gene signature was recently generated using MCF7 cells. This gene signature shows that lactate induces ‘stemness’ in cancer cells, and this lactate induced gene signature predicts poor clinical outcome (including tumor recurrence and metastasis) in breast cancer patients.


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English: Glycolysis pathway overview.

English: Glycolysis pathway overview. (Photo credit: Wikipedia)

Adenosine triphosphate





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Warburg Effect, glycolysis, pyruvate kinase, PKM2, PIM2, mtDNA, complex I, NSCLC

Ribeirão Preto Area, Brazil|Government Relations
Current- University, USP-FMRP Physiology – Biochemistry
Previous- Imperial Cancer Research Fund Lab, Instituto de Investigaciones Bioquimicas, Luta armada e CIA
Education – FMRP-USP
While exile interrupted ny graduation in Medicine, started a port grade at Leloir´s Instituto Investigaciones bioquímicas Buenos Aires Argentina Jan 1970 – jan 1972. PhD from 1972- jan 1976 FMRP-USP . Post Doc ICRF London England 1979 -1980

“Those…..whose acquitance with scientific research is derived chiefly from its practical results easily develop a completely false notion of the { scientific } mentality.”

My goal and previous experience besides laboratory work was dedicated to solve this apparent conflict.

When Pasteur did his work studying the chemistry outside yeast-cells, he was able to perceive that in anaerobiosis yeast cells were able to convert sugar at a great velocity to its end products. While the same yeast cells, therefore with the same genome did it at a very slow speed in aerobiosis. Warburg tested tumor cells for the same and found that while normal cells from the organ in which he has found tumors presented a similar metabolic regulatory response to anaerobic/aerobic transition tumor cells did not. Tumos cells continued to display strong acidification (producing great amounts of lactate in the culture media) in aerobiosis.

Tumor cells displayed a failure in this regulatory mechanism that, he O. Warburg, named Pasteur effect. He also noticed that this defect continues from old cancer cells to newer ones. Therefore, for him, it is a genetic defect. Furthermore, as mitochondria generates newer mitochondria in the cells, the genetic component is in the mitochondria that were the site of core metabolism in aerobiosis.

Larry Bernstein
Part of what you describe is in Warburg biography by Hans Krebs (out of print). He also refers to a Meyerhof Quotient to express the degree of metabolic anaerobiosis. I don’t recall a reference to a mitochondrial “genetic” defect. That is farsighted. He did conclude that once the cancer cells were truly anaplastic, metastatic behavior was irreversible. It was only later that another Nobelist who described fatty acid synthesis, I think, concluded that the synthetic process tied up the same aerobic pathway so that anaerobic glycolysis became essential for the cells energetics. You can correct me if I’m in error. Where does the substrate come from? Lean body mass breaks down to provide gluconeogenic precursors. Points of concern are – deamination of branch chain AAs, the splitting of a 6 carbon sugar into 2 3-carbon chains, and the conversion of pyruvate to lactate with the reverse reaction blocked. There is also evidence that there is an impairment in the TCA cycle at the point of fumarase. Then there is never any consideration of the flow of substrates back and forth across the mitochondrial membrane (malate, aspartate), and the redox potentials.

Jose: For reasons independent of my will, the copy of Science,123(3191):309-14,1956 translation of O Warburg original article is not in my hands. Some that do not find any alternative form to respond to my critics concerning molecular biology distortion of biochemistry left on purpose, almost all of my older reprints on the rain. Anyway, O. Warburg refers to mitochondria using the expression of “grana” or “grains” if my recollections are correct. The original work of O Warburg is one of 1924 – Biochemisch ZeiTschrift.,152:51-60.1924. “Weberssert method zur messing der atmung un glycolyse, drese zeitschr.” Other aspects of your interesting comment I will try to comment latter.

By Jose Eduardo de Salles Roselino
Larry Bernstein, not as a correction but , following my line of reasoning about carbono fluxes…It is almost impossible to figure out what was really inside the mind-set of a scientific researcher at the time he has performed his work. Anyway, by the knowledge available at his time, we may conjecture about, even when we acknowledge that, what he may have had in mind at the start could be quite different from what he have latter published from his results.
In case, we try to present the ideas behind Warburg´s works we may take into account the following pre-existing knowledge: Lavoisier (done by 1779-1784) measured very carefully the amount of heat released by respiration and chemical oxidative processes. Reached the conclusion that respiration was slower but essentially similar to carbon combustion by chemical oxidative processes. By early XIX caloric values for gram of sugar, lipids and proteins where made clear. T Schwann recognized that yeast cells convert sugar in ethanol plus a volatile acid (carbon dioxide). Pasteur-effect was seen just as a change in the velocity of product production from a same sugar and/or a decrease in sugar concentration in the growth medium. This is a change in carbon flux velocity in the oxidative process calorimetrically measured by Lavoisier.
In Germany, however, the preponderance of organic chemical point of view has great scientists as Berzelius, Liebig etc. to consider that yeast where similar to inorganic catalysts. The oxidative process was caused by oxygen only. You may amuse yourself reading, how they attack T. Schwann proposal in Annalen, 29: 100 (1839). When O Warburg paid tribute to L. Pasteur, we can see clearly on which side he was. Furthermore, his apparatus, in which I was introduced to this line of research, a rather modern version at that time (1960s) that look like a very futuristic robotic version of the original with a pair of blinking red and green round lights as two eyes on a metallic head, offers a very important clue about the change in carbon flow.
Inside a Warburg glass flask, kept at very finely controlled temperature to avoid changes in pressure derived from changes in the temperature, you would certainly find a central well in which the volatile acid must be trapped in order to determine a pressure reduction only due to the decrease in oxygen pressure. Inside this central well, you could determine one product (carbon dioxide) and in the outside of it where tissues or cells where in the media you could also determine the other end product of the oxidative combustion of sugar (lactate). This has provide him with the result that indicate not only a change in the velocity as originally found by Pasteur in yeast cells but in the organic flow of sugar carbons.

comment –  E-mail:

-About carcinogenesis- Electronegativity is a nucleus of an atom’s ability to attract and maintain a cloud of electrons. Copper atom electronegativity is higher than the Iron atom electronegativity. Atom with lower electronegativity (Iron),remove the atom with higher electronegativity (Copper) of combinations. This means that,in conditions of acidosis, we have cytochrome oxidase with iron (red, neoplastic), instead of cytochrome oxidase with copper (green, normal).I think this is the key to carcinogenesis. Siincerely, Dr. Viorel Bungau

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Scale‑Free Diagnosis of AMI from Clinical Laboratory Values

William P. Fisher, Jr., Larry H. Bernstein, Thomas A Naegele, Arden

Forrey, Asadullah Qamar, Joseph Babb, Eugene W. Rypka, Donna Yasick

Objective. Clinicians are often challenged with interpreting myriads of laboratory test results with few resources for knowing which values are most relevant, when any given value indicates a need for action, or how urgent the need for action is. The arrival of the electronic health record creates a context in which computational resources for meeting these challenges will be readily available. The purpose of this study was to evaluate the feasibility of employing probabilistic conjoint (Rasch) measurement models for creating the needed scale‑free standard measures and data quality standards.

Methods. Pathology data from 144 clients suspected of suffering myocardial infarctions were obtained. Thirty indicators were converted from their original values to ratings indicating a worsening of condition. These conversions took advantage of the fact that serial measurement of creatine kinase (CK; EC isoenzyme MB (CK‑MB) and lactic dehydrogenase (LD; EC isoenzyme 1 (LD‑1) in serum have characteristic evolutions in acute myocardial infarction (AMI). CK‑MB concentration begins to rise within 4 to 8 hours, peaks at 12 to 24 hours, and returns to normal within 48 to 72 hours. LD‑1 becomes elevated as early as 8 to 24 hours after infarction, and reaches a peak in 48 to 72 hours. However, the ratio of serum activity of LD‑1/total LD may be more definitive than LD‑1 activity itself. While these are most important in ECG negative AMI, they are not by themselves a “gold standard” for diagnosis.

The additional information and functionality required for such standards, including probabilistic estimates of scale parameters whose values do not depend on the calibrating sample and the capacity to deal with missing data, were sought by fitting the data to a Rasch partial credit model. This model estimates separate rating step values for each group of items sharing a common rating structure, en route to testing the hypothesis that the items work together to delineate a unidimensional measurement continuum defined by the repetition of a single unit quantity.

Results. Twenty of the 30 items were identified as delineating a unidimensional continuum.  Client measurement reliability was 0.90, and item calibration reliability was 0.96. Overall model fit is indicated by the client information‑ weighted mean square fit (infit) statistic (mean = .94, SD = .34) and  outlier‑ sensitive mean square fit (outfit) statistic (mean = 1.02, SD = .72), and the item infit (mean = .99, SD = .41) and outfit (mean = 1.04, SD = .72). The data‑to‑ model global fit is also indicated by the chi‑square of 3094.5, with 164 maximum independent parameters, 2766 maximum degrees of  freedom, and a probability (statistical significance) of less than .01 that this ora greater chi‑square would be observed with perfect data‑model fit.

Discussion. The analysis identified the 20 values most relevant to the diagnosis of AMI; these data may also support the construction of a unidimensional measure of AMI severity. If the construct supports both diagnostic and severity inferences, then the clinical action needed and its urgency will be indicated by the client’s measure. Similar analyses of data from other diagnostic groups will determine the extent to which lab value item relevance and hierarchies vary across diagnoses; such variation will be crucial to determining computer‑based decision support algorithms, which will match individual clients’ data with specific diagnostic profiles. Further analyses will also demonstrate the extent to which diagnosis is affected by missing data.


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