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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.

2.1.2.1 Cancer Cell Metabolism. Warburg and Beyond

Hsu PP, Sabatini DM
Cell, Sep 5, 2008; 134:703-707
http://dx.doi.org:/10.016/j.cell.2008.08.021

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?

2.1.2.2 Cancer metabolism. The Warburg effect today

Ferreira LMR
Exp Molec Pathol 2010; 89:372-383.
http://dx.doi.org/10.1016/j.yexmp.2010.08.006

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.

Introduction
Contestation to Warburg’s ideas
Glucose’s uptake and intracellular fates
Lactate production and induced acidosis
Hypoxia
Impairment of mitochondrial function
Tumour microenvironment
Proliferating versus cancer cells
More on cancer bioenergetics – integration of metabolism
Perspectives

2.1.2.3 New aspects of the Warburg effect in cancer cell biology

Bensinger SJ, Cristofk HR
Sem Cell Dev Biol 2012; 23:352-361
http://dx.doi.org:/10.1016/j.semcdb.2012.02.003

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.

Highlights

► 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.

2.1.2.4 Choosing between glycolysis and oxidative phosphorylation. A tumor’s dilemma

Jose C, Ballance N, Rossignal R
Biochim Biophys Acta 201; 1807(6): 552-561.
http://dx.doi.org/10.1016/j.bbabio.2010.10.012

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.

2.1.2.5 Mitohormesis

Yun J, Finkel T
Cell Metab May 2014; 19(5):757–766
http://dx.doi.org/10.1016/j.cmet.2014.01.011

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.

2.1.2.6 Mitochondrial function and energy metabolism in cancer cells. Past overview and future perspectives

Mayevsky A
Mitochondrion. 2009 Jun; 9(3):165-79
http://dx.doi.org:/10.1016/j.mito.2009.01.009

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)

2.1.2.7 Metabolic Reprogramming. Cancer Hallmark Even Warburg Did Not Anticipate

Ward PS, Thompson CB.
Cancer Cell 2012; 21(3):297-308
http://dx.doi.org/10.1016/j.ccr.2012.02.014

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.

2.1.2.8 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
http://dx.doi.org:/10.1016/j.molcel.2014.09.026

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.

2.1.2.9  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
http://dx.doi.org:/10.1016/j.cellsig.2014.11.004

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.

Conclusions

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
http://dx.doi.org/10.1016/j.cellsig.2014.11.004

References

[1] R.A. Cairns, I.S. Harris, T.W. Mak, Cancer 11 (2011) 85–95.
[2] O. Warburg, Science 123 (1956) 309–314.
[3] G.L. Semenza, D.Artemov, A.Bedi, …, J. Simons, P. Taghavi, H. Zhong, Novartis Found. Symp. 240 (2001) 251–260 (discussion 260–254).
[4] N.C. Denko, Cancer 8 (2008) 705–713.
[5] C. Chen, N. Pore, A. Behrooz, F. Ismail-Beigi, A. Maity, J. Biol. Chem. 276 (2001) 9519–9525.
[6] J.Lum, T.Bui, M.Gruber, J.D.Gordan, R.J.DeBerardinis,.. ,C.B. Thompson, Genes Dev. 21 (2007) 1037–1049.
[7] J.T. Chi, Z. Wang, D.S. Nuyten, E.H. Rodriguez, .., P.O. Brown, PLoS Med.
3 (2006) e47.
[8] G.L. Semenza, Cancer 3 (2003) 721–732.

2.1.2.10 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
http://dx.doi.org/10.1016/j.molcel.2014.09.025

Highlights

  • 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

Summary

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.

Read Full Post »


Warburg Effect Revisited – 2

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

Finding Dysregulation in the Cancer Cell

2.1.         Warburg Effect Revisited

One of the great observations of the 20th century was the behavior of cancer cells to proliferate and rely on anaerobic glycolysis for the source of energy.  This was a restatement of the Pasteur effect, described 60 years earlier by the great French scientist in yeast experiments.  The experiments with yeast were again reperformed by Jose EDS Roselino, a Brazilian biochemist, who established an explanation for it 50 years after Warburg.  It is quite amazing the mitochondria were not yet discovered at the time that Warburg carried out the single-cell thickness measurements in his respiratory apparatus. He concluded from the observation that the cancer cells grew in a media that became acidic from producing lactic acid, that the cells were dysfunctional in the utilization of oxygen, as nonmalignant cells efficiently utilized oxygen. He also related the metabolic events to observations made by Meyerhof.  The mitochondria and the citric acid cycle at this time had not yet been discovered, and the latter was, worked out by Hans Krebs and Albert Szent-Gyorgi, both of whom worked with him on mitochondrial metabolism.  The normal cell utilizes glucose efficiently and lipids as well, generating energy through oxidative phosphorylation, with the production of ATP in a manner previously described in these posts.  Greater clarity was achieved with the discovery of Coenzyme A, and finally the electron transport chain (ETC).  This requires that the pyruvate be directed into the tricarboxylic acid cycle and to go through a series of reactions producing succinate and finally malate.

The following great achievements were made with regard to elucidating these processes:

1922 Archibald Vivian Hill United Kingdom “for his discovery relating to the production of heat in the muscle[26]
Otto Fritz Meyerhof Germany “for his discovery of the fixed relationship between the consumption of oxygen and the metabolism of lactic acid in the muscle”[26]
1931 Otto Heinrich Warburg Germany “for his discovery of the nature and mode of action of the respiratory enzyme[34]
1937 Albert Szent-Györgyi von Nagyrapolt Hungary “for his discoveries in connection with the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid[40]
1953 Sir Hans Adolf Krebs United Kingdom “for his discovery of the citric acid cycle[53]
Fritz Albert Lipmann United States “for his discovery of co-enzyme A and its importance for intermediary metabolism”[53]
1955 Axel Hugo Theodor Theorell Sweden “for his discoveries concerning the nature and mode of action of oxidation enzymes”[55]
1978 Peter D. Mitchell United Kingdom “for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory[77]
1997 Paul D. Boyer United States “for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)”[96]
John E. Walker United Kingdom

 

 1967  Manfred Eigen   and the other half jointly to:

Ronald George Wreyford Norrish and Lord George Porter for their studies of extremely fast chemical reactions, effected by disturbing the equlibrium by means of very short pulses of energy.

1965   FRANÇOIS JACOB , ANDRÉ LWOFF And JACQUES MONOD for their discoveries concerning genetic control of enzyme and virus synthesis.

1964 KONRAD BLOCH And FEODOR LYNEN for their discoveries concerning the mechanism and regulation of the cholesterol and fatty acid metabolism.

If there is a more immediate need for energy (as in stressed muscular activity) with net oxygen insufficiency, the pyruvate is converted to lactic acid, with acidemia, and with much less ATP production, but the lactic academia and the energy deficit is subsequently compensated for.    The observation made by Jose EDS Rosalino was that yeast grown in a soil deficient in oxygen don’t put down roots.

^I. Topisirovic and N. Sonenberg

Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXVI

http://dx.doi.org:/10.1101/sqb.2011.76.010785 ”A prominent feature of cancer cells is the use of aerobic glycolysis under conditions in which oxygen levels are sufficient to support energy production in the mitochondria (Jones and Thompson 2009; Cairns et al. 2010). This phenomenon, named the “Warburg effect,” after its discoverer Otto Warburg, is thought to fuel the biosynthetic requirements of the neoplastic growth (Warburg 1956; Koppenol et al. 2011) and has recently been acknowledged as one of the hallmarks of cancer (Hanahan and Weinberg 2011). mRNA translation is the most energy-demanding process in the cell (Buttgereit and Brand 1995).

Again, the use of aerobic glycolysis expression has been twisted.”

To understand my critical observation consider this: Aerobic glycolysis is the carbon flow that goes from Glucose to CO2 and water (includes Krens cycle and respiratory chain for the restoration of NAD, FAD etc.

Anerobic glyclysis is the carbon flow that goes from glucose to lactate. It uses conversion of pyruvate to lactate to regenerate NAD.

“Pasteur effect” is an expression coined by Warburg, which refers to the reduction in the carbon flow from glucose when oxygen is offered to yeasts. The major reason for that is in general terms, derived from the fact that carbon flow is regulated by several cell requirements but mainly by the ATP needs of the cell. Therefore, as ATP is generated 10 more efficiently in aerobiosis than under anaerobiosis, less carbon flow is required under aerobiosis than under anaerobiosis to maintain ATP levels. Warburg, after searching for the same regulatory mechanism in normal and cancer cells for comparison found that transformed cell continued their large flow of glucose carbons to lactate despite the presence of oxygen.

So, it is wrong to describe that aerobic glycolysis continues in the presence of oxygen. It is what it is expected to occur. The wrong thing is that anaerobic glycolysis continues under aerobiosis.
^Aurelian Udristioiu (comment)
In cells, the immediate energy sources involve glucose oxidation. In anaerobic metabolism, the donor of the phosphate group is adenosine triphosphate (ATP), and the reaction is catalyzed via the hexokinase or glucokinase: Glucose +ATP-Mg²+ = Glucose-6-phosphate (ΔGo = – 3.4 kcal/mol with hexokinase as the co-enzyme for the reaction.).

In the following step, the conversion of G-6-phosphate into F-1-6-bisphosphate is mediated by the enzyme phosphofructokinase with the co-factor ATP-Mg²+. This reaction has a large negative free energy difference and is irreversible under normal cellular conditions. In the second step of glycolysis, phosphoenolpyruvic acid in the presence of Mg²+ and K+ is transformed into pyruvic acid. In cancer cells or in the absence of oxygen, the transformation of pyruvic acid into lactic acid alters the process of glycolysis.

The energetic sum of anaerobic glycolysis is ΔGo = -34.64 kcal/mol. However a glucose molecule contains 686kcal/mol and, the energy difference (654.51 kcal) allows the potential for un-controlled reactions during carcinogenesis. The transfer of electrons from NADPH in each place of the conserved unit of energy transmits conformational exchanges in the mitochondrial ATPase. The reaction ADP³+ P²¯ + H²à ATP + H2O is reversible. The terminal oxygen from ADP binds the P2¯ by forming an intermediate pentacovalent complex, resulting in the formation of ATP and H2O. This reaction requires Mg²+ and an ATP-synthetase, which is known as the H+-ATPase or the Fo-F1-ATPase complex. Intracellular calcium induces mitochondrial swelling and aging. [12].

The known marker of monitoring of treatment in cancer diseases, lactate dehydrogenase (LDH) is an enzyme that is localized to the cytosol of human cells and catalyzes the reversible reduction of pyruvate to lactate via using hydrogenated nicotinamide deaminase (NADH) as co-enzyme.

The causes of high LDH and high Mg levels in the serum include neoplastic states that promote the high production of intracellular LDH and the increased use of Mg²+ during molecular synthesis in processes pf carcinogenesis (Pyruvate acid>> LDH/NADH >>Lactate acid + NAD), [13].

The material we shall discuss explores in more detail the dysmetabolism that occurs in cancer cells.

Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?
https://pharmaceuticalintelligence.com/2014/06/21/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-century-view-2/

Warburg Effect Revisited
https://pharmaceuticalintelligence.com/2013/11/28/warburg-effect-revisited/

AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo
https://pharmaceuticalintelligence.com/2013/03/12/ampk-is-a-negative-regulator-of-the-warburg-effect-and-suppresses-tumor-growth-in-vivo/

AKT Signaling Variable Effects
https://pharmaceuticalintelligence.com/2013/03/04/akt-signaling-variable-effects/

Otto Warburg, A Giant of Modern Cellular Biology
https://pharmaceuticalintelligence.com/2012/11/02/otto-warburg-a-giant-of-modern-cellular-biology/

The Metabolic View of Epigenetic Expression
https://pharmaceuticalintelligence.com/2015/03/28/the-metabolic-view-of-epigenetic-expression/

Metabolomics Summary and Perspective
https://pharmaceuticalintelligence.com/2014/10/16/metabolomics-summary-and-perspective/

2.1.1       Cancer Metabolism

2.1.1.1  Oncometabolites: linking altered  metabolism with cancer

Ming Yang, Tomoyoshi Soga, and Patrick J. Pollard
J Clin Invest Sep 2013; 123(9):3652–3658
http://dx.doi.org:/10.1172/JCI67228

The discovery of cancer-associated mutations in genes encoding key metabolic enzymes has provided a direct link between altered metabolism and cancer. Advances in mass spectrometry and nuclear magnetic resonance technologies have facilitated high-resolution metabolite profiling of cells and tumors and identified the accumulation of metabolites associated with specific gene defects. Here we review the potential roles of such “oncometabolites” in tumor evolution and as clinical biomarkers for the detection of cancers characterized by metabolic dysregulation.

The emerging interest in metabolites whose abnormal accumulation causes both metabolic and nonmetabolic dysregulation and potential transformation to malignancy (herein termed “oncometabolites”) has been fueled by the identification of cancerassociated mutations in genes encoding enzymes with significant roles in cellular metabolism (1–5). Loss-of-function mutations in genes encoding the Krebs cycle enzymes fumarate hydratase (FH) and succinate dehydrogenase (SDH) cause the accumulation of fumarate and succinate, respectively (6), whereas gain-offunction isocitrate dehydrogenase (IDH) mutations increase levels of D–2-hydroxyglutarate (D-2HG) (7, 8). These metabolites have been implicated in the dysregulation of cellular processes including the competitive inhibition of α-ketoglutarate–dependent (α-KG–dependent) dioxygenase enzymes (also known as 2-oxoglutarate–dependent dioxgenases) and posttranslational modification of proteins (1, 4, 9–11). To date, several lines of biochemical and genetic evidence support roles for fumarate, succinate, and D-2HG in cellular transformation and oncogenesis (3, 12).

The Journal of Clinical Investigation   http://www.jci.org   Volume 123   Number 9   September 2013

ventional gene sequencing methods may lead to false positives due to genetic polymorphism and sequencing artifacts (98). In comparison, screening for elevated 2HG levels is a sensitive and specific approach to detect IDH mutations in tumors. Whereas patient sera/plasma can be assessed in the case of AML (7, 8, 21, 99), exciting advances with proton magnetic resonance spectroscopy (MRS) have been made in the noninvasive detection of 2HG in patients with gliomas (100–103). Using MRS sequence optimization and spectral fitting techniques, Maher and colleagues examined 30 patients with glioma and showed that the detection of 2HG correlated 100% with the presence of IDH1 or IDH2 mutations (102). Andronesi et al. further demonstrated that two-dimensional correlation spectroscopy could effectively distinguish 2HG from chemically similar metabolites present in the brain (103). Negative IHC staining for SDHB correlates with the presence of SDH mutations, whether in SDHB, SDHC, or SDHD (104). This finding is most likely explained by the fact that mutations in any of the four subunits of SDH can destabilize the entire enzyme complex. PGLs/PCCs associated with an SDHA mutation show negative staining for SDHA as well as SDHB (105). Therefore, IHC staining for SDHB is a useful diagnostic tool to triage patients for genetic testing of any SDH mutation, and subsequent staining for the other subunits may further narrow the selection of genes to be tested. In contrast, detection of FH protein is often evident in HLRCC tumors due to retention of the nonfunctional mutant allele (106). However, staining of cysts and tumors for 2SC immunoreactivity reveals a striking correlation between FH inactivation and the presence of 2SC-modified protein (2SCP), which is absent in non-HLRCC tumors and normal tissue controls (106). IHC staining for 2SCP thus provides a robust diagnostic biomarker for FH deficiency (107).

Therapeutic targeting Because D-2HG is a product of neomorphic enzyme activities, curtailing the D-2HG supply by specifically inhibiting the mutant IDH enzymes provides an elegant approach to target IDH-mutant cancers. Indeed, recent reports of small-molecule inhibitors against mutant forms of IDH1 and IDH2 demonstrated the feasibility of this method. An inhibitor against IDH2 R140Q was shown to reduce both intracellular and extracellular levels of D-2HG, suppress cell growth, and increase differentiation of primary human AML cells (108). Similarly, small-molecule inhibition of IDH1 R132H suppressed colony formation and increased tumor cell differentiation in a xenograft model for IDH1 R132H glioma (58). The inhibitors exhibited a cytostatic rather than cytotoxic effect, and therefore their therapeutic efficacy over longer time periods may need further assessment (109). Letouzé et al. showed that the DNA methytransferase inhibitor decitabine could repress the migration capacities of SDHB-mutant cells (40). However, for SDH- and FH-associated cancers, a synthetic lethality approach is worth exploring because of the pleiotrophic effects associated with succinate and fumarate accumulation.

Outlook The application of next-generation sequencing technologies in the field of cancer genomics has substantially increased our understanding of cancer biology. Detection of germline and somatic mutations in specific tumor types not only expands the current repertoire of driver mutations and downstream effectors in tumorigenesis, but also sheds light on how oncometabolites may exert their oncogenic roles. For example, the identification of mutually exclusive mutations in IDH1 and TET2 in AML led to the characterization of TET2 as a major pathological target of D-2HG (34, 110). Additionally, the discovery of somatic CUL3, SIRT1, and NRF2 mutations in sporadic PRCC2 converges with FH mutation in HLRCC, in which NRF2 activation is a consequence of fumarate-mediated succination of KEAP1, indicating the functional prominence of the NRF2 pathway in PRCC2 (73). In light of this, the identification of somatic mutations in genes encoding the chromatin-modifying enzymes histone H3K36 methyltransferase (SETD2), histone H3K4 demethylase JARID1C (KDM5C), histone H3K27 demethylase UTX (KDM6A), and the SWI/SNF chromatin remodelling complex gene PBRM1 in clear cell renal cell carcinoma (111–113) highlights the importance of epigenetic modulation in human cancer and raises the potential for systematic testing in other types of tumors such as those associated with FH mutations. Technological advances such as those in gas and liquidchromatography mass spectrometry (114, 115) and nuclear magnetic resonance imaging (102) have greatly improved the ability to measure low-molecular-weight metabolites in tumor samples with high resolution (116). Combined with metabolic flux analyses employing isotope tracers and mathematical modeling, modern-era metabolomic approaches can provide direct pathophysiological insights into tumor metabolism and serve as an excellent tool for biomarker discovery. Using a data-driven approach, Jain and colleagues constructed the metabolic profiles of 60 cancer cell lines and discovered glycine consumption as a key metabolic event in rapidly proliferating cancer cells (117), thus demonstrating the power of metabolomic analyses and the relevance to future cancer research and therapeutics.

Acknowledgments The Cancer Biology and Metabolism Group is funded by Cancer Research UK and the European Research Council under the European Community’s Seventh Framework Programme (FP7/20072013)/ERC grant agreement no. 310837 to Dr. Pollard. Professor Soga receives funding from a Grant-in-Aid for scientific research on Innovative Areas, Japan (no. 22134007), and the Yamagata Prefectural Government and City of Tsuruoka.

Address correspondence to: Patrick J. Pollard, Cancer Biology and Metabolism Group, Nuffield Department of Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom. Phone: 44.0.1865287780; Fax: 44.0.1865287787; E-mail:  patrick.pollard@well.ox.ac.uk.

  1. Yang M, Soga T, Pollard PJ, Adam J. The emerging role of fumarate as an oncometabolite. Front Oncol. 2012;2:85. 2. Ward PS, Thompson CB. Metabolic reprogramming: a cancer hallmark even warburg did not anticipate. Cancer Cell. 2012;21(3):297–308. 3. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009; 324(5930):1029–1033. 4. Thompson CB. Metabolic enzymes as oncogenes or tumor suppressors. N Engl J Med. 2009; 360(8):813–815. 5. Schulze A, Harris AL. How cancer metabolism is tuned for proliferation and vulnerable to disruption. Nature. 2012;491(7424):364–373.
  1. Pollard PJ, et al. Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations. Hum Mol Genet. 2005; 14(15):2231–2239. 7. Ward PS, et al. The common feature of leukemiaassociated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell. 2010; 17(3):225–234.

Because D-2HG is a product of neomorphic enzyme activities, curtailing the D-2HG supply by specifically inhibiting the mutant IDH enzymes provides an elegant approach to target IDH-mutant cancers. Indeed, recent reports of small-molecule inhibitors against mutant forms of IDH1 and IDH2 demonstrated the feasibility of this method. An inhibitor against IDH2 R140Q was shown to reduce both intracellular and extracellular levels of D-2HG, suppress cell growth, and increase differentiation of primary human AML cells (108). Similarly, small-molecule inhibition of IDH1 R132H suppressed colony formation and increased tumor cell differentiation in a xenograft model for IDH1 R132H glioma (58). The inhibitors exhibited a cytostatic rather than cytotoxic effect, and therefore their therapeutic efficacy over longer time periods may need further assessment (109). Letouzé et al. showed that the DNA methytransferase inhibitor decitabine could repress the migration capacities of SDHB-mutant cells (40). However, for SDH- and FH-associated cancers, a synthetic lethality approach is worth exploring because of the pleiotrophic effects associated with succinate and fumarate accumulation.

Technological advances such as those in gas and liquid chromatography mass spectrometry (114, 115) and nuclear magnetic resonance imaging (102) have greatly improved the ability to measure low-molecular-weight metabolites in tumor samples with high resolution (116). Combined with metabolic flux analyses employing isotope tracers and mathematical modeling, modern-era metabolomic approaches can provide direct pathophysiological insights into tumor metabolism and serve as an excellent tool for biomarker discovery. Using a data-driven approach, Jain and colleagues constructed the metabolic profiles of 60 cancer cell lines and discovered glycine consumption as a key metabolic event in rapidly proliferating cancer cells (117), thus demonstrating the power of metabolomic analyses and the relevance to future cancer research and therapeutics.

Figure 1 D-2HG produced by mutant IDH1/2 affects metabolism and epigenetics by modulating activities of α-KG–dependent oxygenases. Wild-type IDH1 and IDH2 catalyze the NADP+-dependent reversible conversion of isocitrate to α-KG, whereas cancer-associated gain-of-function mutations enable mutant IDH1/2 (mIDH1/2) to catalyze the oxidation of α-KG to D-2HG, using NADPH as a cofactor. Because D-2HG is structurally similar to α-KG, its accumulation can modulate the activities of α-KG–utilizing dioxygenases. Inhibition of 5mC hydroxylase TET2 and the KDMs results in increased CpG island methylation and increased histone methylation marks, respectively, thus blocking lineage-specific cell differentiation. Inhibition of collagen prolyl and lysyl hydroxylases (C-P4Hs and PLODs, respectively) leads to impaired collagen maturation and disrupted basement membrane formation. D-2HG can also stimulate the activities of HIF PHDs, leading to enhanced HIF degradation and a diminished HIF response, which are associated with increased soft agar growth of human astrocytes and growth factor independence of leukemic cells. Together these processes exert pleiotrophic effects on cell signaling and gene expression that probably contribute to the malignancy of IDH1/2-mutant cells.
Figure 2 Candidate oncogenic mechanisms of succinate and fumarate accumulation. SDH and FH are Krebs cycle enzymes and tumor suppressors. Loss-of-function mutations in SDH and FH result in abnormal accumulation of Krebs cycle metabolites succinate (Succ) and fumarate (Fum), respectively, both of which can inhibit the activities of α-KG–dependent oxygenases. Inhibition of HIF PHDs leads to activation of HIF-mediated pseudohypoxic response, whereas inhibition of KDMs and TET family of 5mC hydroxylases causes epigenetic alterations. Fumarate is electrophilic and can also irreversibly modify cysteine residues in proteins by succination. Succination of KEAP1 in FH deficiency results in the constitutive activation of the antioxidant defense pathway mediated by NRF2, conferring a reductive milieu that promotes cell proliferation. Succination of the Krebs cycle enzyme Aco2 impairs aconitase activity in Fh1-deficient MEFs. Fumarate accumulation may also affect cytosolic pathways by inhibiting the reactions involved in the biosynthesis of arginine and purine. AcCoA, acetyl CoA; Mal, malate; OAA, oxaloacetate; Succ-CA, succinyl CoA.

2.1.1.2. Emerging concepts: linking hypoxic signaling and cancer metabolism.

Lyssiotis CA, Vander-Heiden MG, Muñoz-Pinedo C, Emerling BM.
Cell Death Dis. 2012 May 3; 3:e303
http://dx.doi.org:/10.1038/cddis.2012.41

The Joint Keystone Symposia on Cancer and Metabolism and Advances in Hypoxic Signaling: From Bench to Bedside were held in Banff, Alberta, Canada from 12 to 17 February 2012. Drs. Reuben Shaw and David Sabatini organized the Cancer and Metabolism section, and Drs. Volker Haase, Cormac Taylor, Johanna Myllyharju and Paul Schumacker organized the Advances in Hypoxic Signaling section. Accumulating data illustrate that both hypoxia and rewired metabolism influence cancer biology. Indeed, these phenomena are tightly coupled, and a joint meeting was held to foster interdisciplinary interactions and enhance our understanding of these two processes in neoplastic disease. In this report, we highlight the major themes of the conference paying particular attention to areas of intersection between hypoxia and metabolism in cancer.

One opening keynote address was delivered by Craig Thompson (Memorial Sloan-Kettering, USA), in which he provided a comprehensive perspective on the current thinking around how altered metabolism supports cancer cell growth and survival, and discussed areas likely to be important for future discovery. In particular, Thompson highlighted the essential roles of glucose and glutamine in cell growth, how glucose- and glutamine-consuming processes are rewired in cancer and how this rewiring facilitates anabolic metabolism. These topics were at the core of many of the metabolism presentations that described in detail how some metabolic alterations contribute to the properties of transformed cells.

The other keynote address was delivered by Peter Ratcliffe (University of Oxford, UK), in which he provided a historical perspective on the progress of how signaling events sense oxygen. Mammals have evolved multiple acute and long-term adaptive responses to low oxygen levels (hypoxia). This response prevents a disparity in ATP utilization and production that would otherwise result in a bioenergetic collapse when oxygen level is low. Multiple effectors have been proposed to mediate the response to hypoxia including prolyl hydroxylases, AMPK, NADPH oxidases and the mitochondrial complex III. Currently, however, the precise mechanism by which oxygen is sensed in various physiological contexts remains unknown. Indeed, this was an active point of debate, with Peter Ratcliffe favoring the prolyl hydroxylase PHD2 as the primary cellular oxygen sensor.

Anabolic glucose metabolism and the Warburg effect

Nearly a century ago, Warburg noted that cancer tissues take up glucose in excess than most normal tissues and secrete much of the carbon as lactate. Recently, headway has been made toward determining how the enhanced glucose conversion to lactate occurs and contributes to cell proliferation and survival. Heather Christofk (University of California, Los Angeles, USA) and John Cleveland (the Scripps Research Institute, USA) described a role for the lactate/pyruvate transporter MCT-1 in carbon secretion, and suggested that blocking lactate or pyruvate transport may be a strategy to target glucose metabolism in cancer cells. Kun-Liang Guan (University of California, San Diego, USA) described a novel feedback loop to control glucose metabolism in highly glycolytic cells. Specifically, he discussed how glucose-derived acetyl-CoA can be used as a substrate to modify two enzymes involved in glucose metabolism, pyruvate kinase M2 (PKM2) and phosphoenolpyruvate carboxylase (PEPCK). In both cases, acetylation leads to protein degradation and decreased glycolysis and gluconeogenesis, respectively. Data presented from Matthew Vander Heiden’s laboratory (Koch Institute/MIT, USA) illustrated that loss of pyruvate kinase activity can accelerate tumor growth, suggesting that the regulation of glycolysis may be more complex than previously appreciated. Almut Schulze (London Research Institute, UK) discussed a novel regulatory role for phosphofructokinase in controlling glucose metabolism and Jeffrey Rathmell (Duke University, USA) discussed parallels between glucose metabolism in cancer cells and lymphocytes that suggest many of these phenotypes could be a feature of rapidly dividing cells.

Glutamine addiction

Cancer cells also consume glutamine to support proliferation and survival. Alfredo Csibi (Harvard Medical School, USA) described how mTORC1 promotes glutamine utilization by indirectly regulating the activity of glutamate dehydrogenase. This work united two major themes at the meeting, mTOR signaling and glutamine metabolism, highlighting the interconnectedness of signal transduction and metabolic regulation. Richard Cerione (Cornell University, USA) described a small molecule inhibitor of glutaminase that can be used to target glutamine-addicted cancer cells. Christian Metallo (University of California, San Diego, USA), Andrew Mullen (University of Texas Southwestern Medical School, USA) and Patrick Ward (Memorial Sloan-Kettering, USA) presented data demonstrating that the carbon skeleton of glutamine can be incorporated into newly synthesized lipids. This contribution of glutamine to lipid synthesis was most pronounced in hypoxia or when the mitochondrial electron transport chain was compromised.

Signal transduction and metabolism

The protein kinases AMPK and mTOR can function as sensors of metabolic impairment, whose activation by energy stress controls multiple cellular functions. Grahame Hardie (University of Dundee, UK) and Reuben Shaw (Salk Institute, USA) highlighted novel roles for AMPK, including inhibition of viral replication, and the control of histone acetylation via phosphorylation of class IIa HDACs, respectively. Brandon Faubert (McGill University, USA) reported on an AMPK-dependent effect on glucose metabolism in unstressed cells. Brendan Manning (Harvard Medical School, USA) found that chronic activation of mTOR in the mouse liver, due to genetic ablation of this complex, promotes the development of liver cancer. Kevin Williams (University of California, Los Angeles, USA) discussed how growth signaling can control both lipid and glucose metabolism by impinging on SREBP-1, a transcription factor downstream of mTOR. AMPK-independent control of mTOR was addressed by John Blenis (Harvard Medical School, USA), who discussed the possible role of mTOR stabilizing proteins as mediators of mTOR inactivation upon energetic stress. David Sabatini (Whitehead Institute/MIT, USA) discussed several aspects of amino-acid sensing by Rag GTPases and showed that constitutive activation of the Rag GTPases leads to metabolic defects in mice.

One of the outcomes of AMPK activation and mTOR inhibition is autophagy, which can provide amino acids and fatty acids to nutrient-deprived cells. Ana Maria Cuervo (Albert Einstein College of Medicine, USA) and Eileen White (Rutgers University, USA) illuminated the role of chaperone-mediated autophagy (CMA) and macroautophagy, respectively, in tumor survival. White described a role for macroautophagy in the regulation of mitochondrial fitness, maintenance of TCA cycle and tumorigenesis induced by oncogenic Ras. Cuervo described how CMA is consistently elevated in tumor cells, and how its inactivation leads to metabolic impairment via p53-mediated downregulation of glycolytic enzymes.

Oncogene-specific changes to metabolism

Lewis Cantley (Harvard Medical School, USA) described a metabolic role for oncogenic Kras in the rewiring of glucose metabolism in pancreatic cancer. Specifically, Myc-mediated transcription (downstream of MEK-ERK signaling) both enhances glucose uptake and diverts glucose carbon into the nonoxidative pentose phosphate pathway to facilitate nucleotide biosynthesis. Alejandro Sweet-Cordero (Stanford University, USA) described how oncogenic Kras increases glycolysis and represses mitochondrial respiration (via decreased pyruvate dehydrogenase phosphatase 1 (PDP1) expression) in colon cancer. While these studies indicate that hyperstimulation of the Erk pathway suppresses PDH flux through suppression of PDP1, Joan Brugge (Harvard Medical School, USA) described studies showing that reduction of Erk signaling in normal epithelial cells also causes suppression of PDH flux, in this case through loss of repression of PDK4. The seemingly contradictory nature of these results highlighted an important theme emphasized throughout the week-long conference—that cellular context has an important role in shaping how oncogenic mutations or pathway activation rewires metabolism.

Targeting cancer metabolism

There was extensive discussion around targeting metabolism for cancer therapy. Metformin and phenformin, which act in part by mitochondrial complex I inhibition, can activate AMPK and influence cancer cell metabolism. Kevin Struhl (Harvard Medical School, USA) described how metformin can selectively target cancer stem cells, whereas Jessica Howell (Harvard Medical School, USA) described how the therapeutic activity of metformin relies on both AMPK and mTOR signaling to mediate its effect. Similarly, David Shackelford (University of California, Los Angeles, USA) demonstrated efficacy for phenformin in LKB1-deficient mouse models.

Several presentations, including those by Taru Muranen (Harvard Medical School, USA), Karen Vousden and Eyal Gottlieb (both from the Beatson Institute for Cancer Research, UK), provided insight into genetic control mechanisms that cancer cells use to promote survival under conditions of increased biosynthesis. As an example, Vousden illustrated how p53 loss can make cancer cells more dependent on exogenous serine. Several additional presentations, including those by Gottlieb, Richard Possemato (Whitehead Institute/MIT, USA), Michael Pollak (McGill University, USA) and Kevin Marks (Agios Pharmaceuticals, USA), also included data highlighting the important role of serine biosynthesis and metabolism in cancer growth. Collectively, these data highlight a metabolic addiction that may be therapeutically exploitable. Similarly, Cristina Muñoz-Pinedo (Institut d’Investigació Biomèdica, Spain) described how mimicking glucose deprivation with 2-deoxyglucose can cause programmed cell death and may be an effective cancer treatment.

Regulation of hypoxic responses

Peter Carmeliet (University of Leuven, Belgium) highlighted the mechanisms of resistance against VEGF-targeted therapies. Roland Wenger (University of Zurich, Switzerland) discussed the oxygen-responsive transcriptional networks and, in particular, the difference between the transcription factors HIF-1α and HIF-2α. Importantly, he demonstrated a rapid role for HIF-1α, and a later and more persistent response for HIF-2α. These results were central to a recurrent theme calling for the distinction of HIF-1α and HIF-2α target genes and how these responses mediate divergent hypoxic adaptations.

Advances in hypoxic signaling

Brooke Emerling (Harvard Medical School, USA) introduced CUB domain-containing protein 1 (CDCP1) and showed persuasive data on CDCP1 being a HIF-2α target gene involved in cell migration and metastasis, and suggested CDCP1 regulation as an attractive therapeutic target. Johannes Schodel (University of Oxford, UK) described an elegant HIF-ChIP-Seq methodology to define direct transcriptional targets of HIF in renal cancer.

Randall Johnson (University of Cambridge, UK) emphasized that loss of HIF-1α results in decreased lung metastasis. Lorenz Poellinger (Karolinska Institutet, Sweden) focused on how hypoxia can alter the epigenetic landscape of cells, and furthermore, how the disruption of the histone demethylase JMJD1A and/or the H3K9 methyltransferase G9a has opposing effects on tumor growth and HIF target gene expression.

Paul Schumacker (Northwestern University, USA) further emphasized the importance of mitochondrial ROS signaling under hypoxic conditions showing that ROS could be detected in the inter-membrane space of the mitochondria before activating signaling cascades in the cytosol. He also presented evidence for mitochondria as a site of oxygen sensing in diverse cell types. Similarly, Margaret Ashcroft (University College London, UK) argued for a critical role of mitochondria in hypoxic signaling. She presented on a family of mitochondrial proteins (CHCHD4) that influence hypoxic signaling and tumorigenesis and suggested that CHCHD4 is important for HIF and tumor progression.

2.1.1.3  Glutaminolysis: supplying carbon or nitrogen or both for cancer cells?

Dang CV
Cell Cycle. 2010 Oct 1; 9(19):3884-6

A cancer cell comprising largely of carbon, hydrogen, oxygen, phosphorus, nitrogen and sulfur requires not only glucose, which is avidly transported and converted to lactate by aerobic glycolysis or the Warburg effect, but also glutamine as a major substrate. Glutamine and essential amino acids, such as methionine, provide energy through the TCA cycle as well as nitrogen, sulfur and carbon skeletons for growing and proliferating cancer cells. The interplay between utilization of glutamine and glucose is likely to depend on the genetic make-up of a cancer cell. While the MYC oncogene induces both aerobic glycolysis and glutaminolysis, activated β-catenin induces glutamine synthesis in hepatocellular carcinoma. Cancer cells that have elevated glutamine synthetase can use glutamate and ammonia to synthesize glutamine and are hence not addicted to glutamine. As such, cancer cells have many degrees of freedom for re-programming cell metabolism, which with better understanding will result in novel therapeutic approaches.

Figure 1. Glutamine, glucose and glutamate are imported into the cytoplasm of a cell. Glucose is depicted to be converted primarily (large powder blue arrow) to lactate via aerobic glycolysis or the Warburg effect or channeled into the mitochondrion as pyruvate and converted to acetyl-CoA for oxidation. Glutamine is shown imported and used for different processes including glutaminolysis, which involves the conversion of glutamine to glutamate and ammonia by glutaminase (GLS). Glutamate is further oxidized via the TCA cycle to produce ATP and contribute anabolic carbon skeletons. Some cells can import glutamate and use ammonia to generate glutamine through glutamine synthetase (GLUL); glutamine could then be used for different purposes including glutathione synthesis (not shown).

The liver is organized into lobules, which have zones of cells around the perivenous region enriched with glutamine synthetase, which detoxifies ammonia by converting it to glutamine through the amination of glutamate (Fig. 1). As such, liver cancers vary in the degree of glutamine synthetase expression depending on the extent of anaplasia or de-differentiation. Highly undifferentiated liver cancers tend to be more glycolytic than those that retain some of the differentiated characteristics of liver cells. Furthermore, glutamine synthetase (considered as a direct target of activated β-catenin, which also induces ornithine aminotransferase and glutamate transporters) expression in liver cancers has been directly linked to β-catenin activation or mutations.  Hence, the work by Meng et al. illustrates, first and foremost, the metabolic heterogeneity amongst cancer cell lines, such that the ability to utilize ammonia instead of glutamine by Hep3B cells depends on the expression of glutamine synthetase. The Hep3B cells are capable of producing glutamine from glutamate and ammonia, as suggested by the observation that a glutamine-independent derivative of Hep3B has high expression of glutamine synthetase. In this regard, Hep3B could utilize glutamate directly for the production of α-ketoglutarate or to generate glutamine for protein synthesis or other metabolic processes, such as to import essential amino acids.  In contrast to Hep3B, other cell lines in the Meng et al. study were not demonstrated to be glutamine independent and thus become ammonia auxotrophs. Hence, the mode of glutamine or glucose utilization is dependent on the metabolic profile of cancer cells.
The roles of glutamine in different cancer cell lines are likely to be different depending on their genetic and epigenetic composition. In fact, well-documented isotopic labeling studies have demonstrated a role for glutamine to provide anapleurotic carbons in certain cancer and mammalian cell types. But these roles of glutaminolysis, whether providing nitrogen or anabolic carbons, should not be generalized as mutually exclusive features of all cancer cells. From these considerations, it is surmised that the expression of glutamine synthetase in different cancers will determine the extent by which these cancers are addicted to exogenous glutamine.

2.1.1.4  The Warburg effect and mitochondrial stability in cancer cells

Gogvadze V, Zhivotovsky B, Orrenius S.
Mol Aspects Med. 2010 Feb; 31(1):60-74
http://dx.doi.org:/10.1016/j.mam.2009.12.004

The last decade has witnessed a renaissance of Otto Warburg’s fundamental hypothesis, which he put forward more than 80 years ago, that mitochondrial malfunction and subsequent stimulation of cellular glucose utilization lead to the development of cancer. Since most tumor cells demonstrate a remarkable resistance to drugs that kill non-malignant cells, the question has arisen whether such resistance might be a consequence of the abnormalities in tumor mitochondria predicted by Warburg. The present review discusses potential mechanisms underlying the upregulation of glycolysis and silencing of mitochondrial activity in cancer cells, and how pharmaceutical intervention in cellular energy metabolism might make tumor cells more susceptible to anti-cancer treatment.

mitochondrial stabilization gr1

mitochondrial stabilization gr1

http://ars.els-cdn.com/content/image/1-s2.0-S0098299709000934-gr1.sml

Fig. 1. (1) Oligomerization of Bax is mediated by the truncated form of the BH3-only, pro-apoptotic protein Bid (tBid); (2) Bcl-2, Bcl-XL, Mcl-1, and Bcl-w, interact with the pro-apoptotic proteins, Bax and Bak, to prevent their oligomerization; (3) The anti-apoptotic protein Bcl-XL prevents tBid-induced closure of VDAC and apoptosis by maintaining VDAC in open configuration allowing ADT/ATP exchange and normal mitochondrial functioning; (4) MPT pore is a multimeric complex, composed of VDAC located in the OMM, ANT, an integral protein of the IMM, and a matrix protein, CyPD; (5) Interaction with VDAC allows hexokinase to use exclusively intramitochondrial ATP to phosphorylate glucose, thereby maintaining high rate of glycolysis.

mitochodrial stabilization gr2

mitochodrial stabilization gr2

http://ars.els-cdn.com/content/image/1-s2.0-S0098299709000934-gr2.sml

Fig. 2. Different sites of therapeutic intervention in cancer cell metabolism. (1) The non-metabolizable analog of glucose, 2-deoxyglucose, decreases ATP level in the cell; (2) 3-bromopyruvate suppresses the activity of hexokinase, and respiration in isolated mitochondria; (3) Phloretin a glucose transporter inhibitor, decreases ATP level in the cell and markedly enhances the anti-cancer effect of daunorubicin; (4) Dichloroacetate (DCA) shifts metabolism from glycolysistoglucoseoxidation;(5)Apoptolidin,aninhibitorofmitochondrialATPsynthase,inducescelldeathindifferentmalignantcelllineswhenapplied together with the LDH inhibitor oxamate (6).

Warburg Symposium

https://youtu.be/LpE6w6J3jU0

2.1.1.5 Oxidative phosphorylation in cancer cells

Giancarlo Solaini Gianluca SgarbiAlessandra Baracca

BB Acta – Bioenergetics 2011 Jun; 1807(6): 534–542
http://dx.doi.org/10.1016/j.bbabio.2010.09.003

Research Highlights

►Mitochondrial hallmarks of tumor cells.►Complex I of the respiratory chain is reduced in many cancer cells.►Oligomers of F1F0ATPase are reduced in cancer cells.►Mitochondrial membranes are critical to the life or death of cancer cells.

Evidence suggests that mitochondrial metabolism may play a key role in controlling cancer cells life and proliferation. Recent evidence also indicates how the altered contribution of these organelles to metabolism and the resistance of cancer mitochondria against apoptosis-associated permeabilization are closely related. The hallmarks of cancer growth, increased glycolysis and lactate production in tumours, have raised attention due to recent observations suggesting a wide spectrum of oxidative phosphorylation deficit and decreased availability of ATP associated with malignancies and tumour cell expansion. More specifically, alteration in signal transduction pathways directly affects mitochondrial proteins playing critical roles in controlling the membrane potential as UCP2 and components of both MPTP and oxphos complexes, or in controlling cells life and death as the Bcl-2 proteins family. Moreover, since mitochondrial bioenergetics and dynamics, are also involved in processes of cells life and death, proper regulation of these mitochondrial functions is crucial for tumours to grow. Therefore a better understanding of the key pathophysiological differences between mitochondria in cancer cells and in their non-cancer surrounding tissue is crucial to the finding of tools interfering with these peculiar tumour mitochondrial functions and will disclose novel approaches for the prevention and treatment of malignant diseases. Here, we review the peculiarity of tumour mitochondrial bioenergetics and the mode it is linked to the cell metabolism, providing a short overview of the evidence accumulated so far, but highlighting the more recent advances. This article is part of a Special Issue entitled: Bioenergetics of Cancer.

Mitochondria are essential organelles and key integrators of metabolism, but they also play vital roles in cell death and cell signaling pathways critically influencing cell fate decisions [1][2] and [3]. Mammalian mitochondria contain their own DNA (mtDNA), which encodes 13 polypeptides of oxidative phosphorylation complexes, 12S and 16S rRNAs, and 22 tRNAs required for mitochondrial function [4]. In order to synthesize ATP through oxidative phosphorylation (oxphos), mitochondria consume most of the cellular oxygen and produce the majority of reactive oxygen species (ROS) as by-products [5]. ROS have been implicated in the etiology of carcinogenesis via oxidative damage to cell macromolecules and through modulation of mitogenic signaling pathways [6][7] and [8]. In addition, a number of mitochondrial dysfunctions of genetic origin are implicated in a range of age-related diseases, including tumours [9]. How mitochondrial functions are associated with cancer is a crucial and complex issue in biomedicine that is still unravelled [10] and [11], but it warrants an extraordinary importance since mitochondria play a major role not only as energy suppliers and ROS “regulators”, but also because of their control on cellular life and death. This is of particular relevance since tumour cells can acquire resistance to apoptosis by a number of mechanisms, including mitochondrial dysfunction, the expression of anti-apoptotic proteins or by the down-regulation or mutation of pro-apoptotic proteins [12].

Cancer cells must adapt their metabolism to produce all molecules and energy required to promote tumor growth and to possibly modify their environment to survive. These metabolic peculiarities of cancer cells are recognized to be the outcome of mutations in oncogenes and tumor suppressor genes which regulate cellular metabolism. Mutations in genes including P53, RAS, c-MYC, phosphoinosine 3-phosphate kinase (PI3K), and mTOR can directly or through signaling pathways affect metabolic pathways in cancer cells as discussed in several recent reviews [13][14][15][16] and [17]. Cancer cells harboring the genetic mutations are also able to thrive in adverse environments such as hypoxia inducing adaptive metabolic alterations which include glycolysis up-regulation and angiogenesis factor release [18] and [19]. In response to hypoxia, hypoxia-induced factor 1 (HIF-1) [20], a transcription factor, is up-regulated, which enhances expression of glycolytic enzymes and concurrently it down regulates mitochondrial respiration through up-regulation of pyruvate dehydrogenase kinase 1 (PDK1) (see recent reviews [21] and [22]). However, several tumours have been reported to display high HIF-1 activity even in normoxic condition, now referred to as pseudohypoxia [23][24] and [25]. In addition, not only solid tumours present a changed metabolism with respect to matched normal tissues, hematological cell malignancies also are characterized by peculiar metabolisms, in which changes of mitochondrial functions are significant [26],[27] and [28], therefore indicating a pivotal role of mitochondria in tumours independently from oxygen availability.

Collectively, actual data show a great heterogeneity of metabolism changes in cancer cells, therefore comprehensive cellular and molecular basis for the association of mitochondrial bioenergetics with tumours is still undefined, despite the numerous studies carried out. This review briefly revisits the data which are accumulating to account for this association and highlights the more recent advances, particularly focusing on the metabolic and structural changes of mitochondria.

Mitochondria-related metabolic changes of cancer cells

Accumulating evidence indicate that many cancer cells have an higher glucose consumption under normoxic conditions with respect to normal differentiated cells, the so-called “aerobic glycolysis” (Warburg effect), a phenomenon that is currently exploited to detect and diagnose staging of solid and even hematological malignancies [27]. Since the initial publication by Otto Warburg over half a century ago [29], an enormous amount of studies on many different tumours have been carried out to explain the molecular basis of the Warburg effect. Although the regulatory mechanisms underlying aerobic and glycolytic pathways of energy production are complex, making the prediction of specific cellular responses rather difficult, the actual data seem to support the view that in order to favour the production of biomass, proliferating cells are commonly prone to satisfy the energy requirement utilizing substrates other than the complete oxidation of glucose (to CO2 and H2O). More precisely, only part (40 to 75%, according to [30]) of the cells need of ATP is obtained through the scarcely efficient catabolism of glucose to pyruvate/lactate in the cytoplasm and the rest of the ATP need is synthesized in the mitochondria through both the tricarboxylic acid (TCA) cycle (one ATP produced each acetyl moiety oxidized) and the associated oxidative phosphorylation that regenerates nicotinamide- and flavin-dinucleotides in their oxidized state(NAD+ and FAD). This might be due to the substrate availability as it was shown in HeLa cells, where replacing glucose with galactose/glutamine in the culture medium induced increased expression of oxphos proteins, suggesting an enhanced energy production from glutamine [31]. As a conclusion the authors proposed that energy substrate can modulate mitochondrial oxidative capacity in cancer cells. A direct evidence of this phenomenon was provided a few years later in glioblastoma cells, in which it was demonstrated that the TCA cycle flux is significantly sustained by anaplerotic alfa-ketoglutarate produced from glutamine and by acetyl moieties derived from the pyruvate dehydrogenase reaction where pyruvate may have an origin other than glucose [32]. The above changes are the result of genetic alteration and environmental conditions that induce many cancer cells to change their metabolism in order to synthesize molecules necessary to survive, grow and proliferate, including ribose and NADPH to synthesize nucleotides, and glycerol-3 phosphate to produce phospholipids. The synthesis of the latter molecules requires major amount of acetyl moieties that are derived from beta-oxidation of fatty acids and/or from cytosolic citrate (citrate lyase reaction) and/or from the pyruvate dehydrogenase reaction. Given the important requirement for NADPH in macromolecular synthesis and redox control, NADPH production in cancer cells besides being produced through the phosphate pentose shunt, may be significantly sustained by cytosolic isocitrate dehydrogenases and by the malic enzyme (see Ref. [33] for a recent review). Therefore, many cancer cells tend to have reduced oxphos in the mitochondria due to either or both reduced flux within the tricarboxylic acid cycle and/or respiration (Fig. 1). The latter being also caused by reduced oxygen availability, a typical condition of solid tumors, that will be discussed below.

Schematic illustration of mitochondrial metabolism and metabolic reprogramming in tumours gr1

Schematic illustration of mitochondrial metabolism and metabolic reprogramming in tumours gr1

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Fig. 1. Schematic illustration of mitochondrial metabolism and metabolic reprogramming in tumours. In normal cells (A), glucose is phosphorylated by HK-I, then the major part is degraded via glycolysis to pyruvate, which prevalently enters the mitochondria, it is decarboxylated and oxidized by PDH to acetyl-coenzyme A, which enters the TCA cycle where the two carbons are completely oxidized to CO2 whereas hydrogen atoms reduce NAD+ and FAD, which feed the respiratory chain (turquoise). Minor part of glycolytic G-6P is diverted to produce ribose 5-phosphate (R-5P) and NADPH, that will be used to synthesize nucleotides, whereas triose phosphates in minimal part will be used to synthesize lipids and phospholipids with the contribution of NADPH and acetyl-coenzyme A. Amino acids, including glutamine (Gln) will follow the physiological turnover of the proteins, in minimal part will be used to synthesize the nucleotides bases, and the excess after deamination will be used to produce energy. In the mitochondria inner membranes are located the respiratory chain complexes and the ATP synthase (turquoise), which phosphorylates ADP releasing ATP, that in turn is carried to the cytosol by ANT (green) in exchange for ADP. About 1–2% O2 uptaken by the mitochondria is reduced to superoxide anion radical and ROS. In cancer cells (B), where anabolism is enhanced, glucose is mostly phosphorylated by HK-II (red), which is up-regulated and has an easy access to ATP being more strictly bound to the mitochondria. Its product, G-6P, is only in part oxidized to pyruvate. This, in turn, is mostly reduced to lactate being both LDH and PDH kinase up-regulated. A significant part of G-6P is used to synthesize nucleotides that also require amino acids and glutamine. Citrate in part is diverted from the TCA cycle to the cytosol, where it is a substrate of citrate lyase, which supplies acetyl-coenzyme A for lipid and phospholipid synthesis that also requires NADPH. As indicated, ROS levels in many cancer cells increase.

Of particular relevance in the study of the metabolic changes occurring in cancer cells, is the role of hexokinase II. This enzyme is greatly up-regulated in many tumours being its gene promoter sensitive to typical tumour markers such as HIF-1 and P53 [30]. It plays a pivotal role in both the bioenergetic metabolism and the biosynthesis of required molecules for cancer cells proliferation. Hexokinase II phosphorylates glucose using ATP synthesized by the mitochondrial oxphos and it releases the product ADP in close proximity of the adenine nucleotide translocator (ANT) to favour ATP re-synthesis within the matrix (Fig. 1). Obviously, the expression level, the location, the substrate affinity, and the kinetics of the enzyme are crucial to the balancing of the glucose fate, to either allowing intermediates of the glucose oxidation pathway towards required metabolites for tumour growth or coupling cytoplasmic glycolysis with further oxidation of pyruvate through the TCA cycle, that is strictly linked to oxphos. This might be possible if the mitochondrial-bound hexokinase activity is reduced and/or if it limits ADP availability to the mitochondrial matrix, to inhibit the TCA cycle and oxphos. However, the mechanism is still elusive, although it has been shown that elevated oncogene kinase signaling favours the binding of the enzyme to the voltage-dependent anion channel (VDAC) by AKT-dependent phosphorylation [34] (Fig. 2). VDAC is a protein complex of the outer mitochondrial membrane which is in close proximity of ANT that exchanges ADP for ATP through the inner mitochondrial membrane [35]. However, the enzyme may also be detached from the mitochondrial membrane, to be redistributed to the cytosol, through the catalytic action of sirtuin-3 that deacylates cyclophilin D, a protein of the inner mitochondrial membrane required for binding hexokinase II to VDAC (Fig. 2[36]. Removing hexokinase from the mitochondrial membrane has also another important consequence in cancer cells: whatever mechanism its removal activates, apoptosis is induced [37] and [38]. These observations indicate hexokinase II as an important tool used by cancer cells to survive and proliferate under even adverse conditions, including hypoxia, but it may result an interesting target to hit in order to induce cells cytotoxicity. Indeed, a stable RNA interference of hexokinase II gene showed enhanced apoptosis indices and inhibited growth of human colon cancer cells; in accordance in vivo experiments indicated a decreased tumour growth [39].

Schematic illustration of the main mitochondrial changes frequently occurring in cancer cells gr2

Schematic illustration of the main mitochondrial changes frequently occurring in cancer cells gr2

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Fig. 2. Schematic illustration of the main mitochondrial changes frequently occurring in cancer cells. The reprogramming of mitochondrial metabolism in many cancer cells comprises reduced pyruvate oxidation by PDH followed by the TCA cycle, increased anaplerotic feeding of the same cycle, mostly from Gln, whose entry in the mitochondrial matrix is facilitated by UCP2 up-regulation. This increases also the free fatty acids uptake by mitochondria, therefore β-oxidation is pushed to produce acetyl-coenzyme A, whose oxidation contributes to ATP production. In cancer cells many signals can converge on the mitochondrion to regulate the mitochondrial membrane permeability, which may respond by elevating the MPTP (PTP) threshold, with consequent enhancement of apoptosis resistance. ROS belong to this class of molecules since it can enhance Bcl2 and may induce DNA mutations. Dotted lines indicate regulation; solid lines indicate reaction(s).

Respiratory chain complexes and ATP synthase

Beyond transcriptional control of metabolic enzyme expression by oncogenes and tumour suppressors, it is becoming evident that environmental conditions affect the mitochondrial energy metabolism, and many studies in the last decade indicate that mitochondrial dysfunction is one of the more recurrent features of cancer cells, as reported at microscopic, molecular, biochemical, and genetic level [7], [40] and [41]. Although cancer cells under several conditions, including hypoxia, oncogene activation, and mDNA mutation, may substantially differ in their ability to use oxygen, only few reports have been able to identify a strict association between metabolic changes and mitochondrial complexes composition and activity. In renal oncocytomas [42] and in lung epidermoid carcinoma [43], the NADH dehydrogenase activity and protein content of Complex I were found to be strongly depressed; subsequently, in a thyroid oncocytoma cell line [44] a similar decrease of Complex I activity was ascribed to a specific mutation in the ND1 gene of mitochondrial DNA. However, among the respiratory chain complexes, significant decrease of the only Complex I content and activity was found in K-ras transformed cells in our laboratory [45], and could not be ascribed to mtDNA mutations, but rather, based on microarray analysis of oxphos genes, we proposed that a combination of genetic (low transcription of some genes) and biochemical events (assembly factors deficiency, disorganization of structured supercomplexes, and ROS-induced structural damage) might cause the Complex I defects.

In some hereditary tumours (renal cell carcinomas) a correlation has been identified between mitochondrial dysfunctions and content of oxphos complexes [46]. For instance, the low content of ATP synthase, often observed in clear cell type renal cell carcinomas and in chromophilic tumours, seems to indicate that the mitochondria are in an inefficient structural and functional state [46]. However, it cannot be excluded that, in some cases, the structural alteration of ATP synthase may offer a functional advantage to cells exhibiting a deficient respiratory chain for instance to preserve the transmembrane electrical potential (Δψm) [47]. It is likely that low levels of ATP synthases may play a significant role in cancer cell metabolism since it has been reported that in tumours from many different tissues, carcinogenesis specifically affects the expression of F1-ATPase β subunit, suggesting alterations in the mechanisms that control mitochondrial differentiation (see for a detailed review [48]). What it seems intriguing is the overexpression of the inhibitor protein, IF1, reported in hepatocellular carcinomas [49] and [50] and in Yoshida sarcoma [51]. Normally, this protein binds to the F1 domain of the ATP synthase inhibiting its activity [52], and it is believed to limit the ATP hydrolysis occurring in the mitochondria of hypoxic cells, avoiding ATP depletion and maintaining Δψm to a level capable to avoid the induction of cell death [5]. But why is its expression in cancer cells enhanced in front of a reduced F1-ATPase β subunit?

The first possibility is that IF1 has a function similar to that in normal cells, simply avoiding excessive ATP hydrolysis therefore limiting Δψm enhancement, but in cancer cells this is unlikely due to both the reduced level of ATP synthase [46] and the high affinity of IF1 for the enzyme. A second possibility might be that cancer cells need strongly reduced oxphos to adapt their metabolism and acquire a selective growth advantage under adverse environmental conditions such as hypoxia, as it has been experimentally shown [53]. Finally, IF1 might contribute to the saving of the inner mitochondrial membrane structure since it has been reported its capability to stabilize oligomers of ATP synthase, which in turn can determine cristae shapes [54]. In this regard, recent experimental evidence has shed some light on a critical role of mitochondrial morphology in the control of important mitochondrial functions including apoptosis [55] and oxidative phosphorylation [56]. In particular, dysregulated mitochondrial fusion and fission events can now be regarded as playing a role in cancer onset and progression [57]. Accordingly, mitochondria-shaping proteins seem to be an appealing target to modulate the mitochondrial phase of apoptosis in cancer cells. In fact, several cancer tissues: breast, head-and-neck, liver, ovarian, pancreatic, prostate, renal, skin, and testis, showed a pattern suggestive of enlarged mitochondria resulting from atypical fusion [58].

Mitochondrial membrane potential in cancer cells

Critical mitochondrial functions, including ATP synthesis, ion homeostasis, metabolites transport, ROS production, and cell death are highly dependent on the electrochemical transmembrane potential, a physico-chemical parameter consisting of two components, the major of which being the transmembrane electrical potential (Δψm) (see for a recent review [59]). In normal cells, under normoxic conditions, Δψm is build up by the respiratory chain and is mainly used to drive ATP synthesis, whereas in anoxia or severe hypoxia it is generated by the hydrolytic activity of the ATP synthase complex and by the electrogenic transport of ATP in exchange for ADP from the cytosol to the matrix, operated by the adenine nucleotide translocator [17]. Dissipation of the mitochondrial membrane potential (proton leak) causes uncoupling of the respiratory chain electron transport from ADP phosphorylation by the ATP synthase complex. Proton leak functions as a regulator of mitochondrial ROS production and its modulation by uncoupling proteins may be involved in pathophysiology, including tumours. In addition, Δψm plays a role in the control of the mitochondrial permeability transition pore (MPTP), that might be critical in determining reduced sensitivity to stress stimuli that were described in neoplastic transformation [60], implying that dysregulation of pore opening might be a strategy used by tumour cells to escape death. Indeed, it has recently been reported that ERK is constitutively activated in the mitochondria of several cancer cell types, where it inhibits glycogen synthase kinase-3-dependent phosphorylation of CyP-D and renders these cells more refractory to pore opening and to the ensuing cell death [61].

It is worth mentioning a second protein of the inner mitochondrial membrane, the uncoupling protein, UCP2 (Fig. 2), which contributes to regulate Δψm. Indeed, recent observations evidenced its overexpression in various chemoresistent cancer cell lines and in primary human colon cancer. This overexpression was associated with an increased apoptotic threshold [62]. Moreover, UCP2 has been reported to be involved in metabolic reprogramming of cells, and appeared necessary for efficient oxidation of glutamine [63]. On the whole, these results led to hypothesize an important role of the uncoupling protein in the molecular mechanism at the basis of the Warburg effect, that suppose a reduced Δψm-dependent entry of pyruvate into the mitochondria accompanied by enhanced fatty acid oxidation and high oxygen consumption (see for a review [64]). However, in breast cancer Sastre-Serra et al. [65] suggested that estrogens by down-regulating UCPs, increase mitochondrial Δψm, that in turn enhances ROS production, therefore increasing tumorigenicity. While the two above points of view concur to support increased tumorigenicity, the mechanisms at the basis of the phenomenon appear on the opposite of the other. Therefore, although promising for the multiplicity of metabolic effects in which UCPs play a role (see for a recent review [66]), at present it seems that much more work is needed to clarify how UCPs are related to cancer.

A novel intriguing hypothesis has recently been put forward regarding effectors of mitochondrial function in tumours. Wegrzyn J et al. [67] demonstrated the location of the transcription factor STAT3 within the mitochondria and its capability to modulate respiration by regulating the activity of Complexes I and II, and Gough et al. [68] reported that human ras oncoproteins depend on mitochondrial STAT3 for full transforming potential, and that cancer cells expressing STAT3 have increased both Δψm and lactate dehydrogenase level, typical hallmarks of malignant transformation (Fig. 2). A similar increase of Δψm was recently demonstrated in K-ras transformed fibroblasts [45]. In this study, the increased Δψm was somehow unexpected since the cells had shown a substantial decrease of NADH-linked substrate respiration rate due to a compatible reduced Complex I activity with respect to normal fibroblasts. The authors associated the reduced activity of the enzyme to its peculiar low level in the extract of the cells that was confirmed by oxphos nuclear gene expression analysis. This significant and peculiar reduction of Complex I activity relative to other respiratory chain complexes, is recurrent in a number of cancer cells of different origin [42][44][45] and [69]. Significantly, all those studies evidenced an overproduction of ROS in cancer cells, which was consistent with the mechanisms proposed by Lenaz et al. [70] who suggested that whatever factor (i.e. genetic or environmental) initiate the pathway, if Complex I is altered, it does not associate with Complex III in supercomplexes, consequently it does not channel correctly electrons from NADH through coenzyme Q to Complex III redox centres, determining ROS overproduction. This, in turn, enhances respiratory chain complexes alteration resulting in further ROS production, thus establishing a vicious cycle of oxidative stress and energy depletion, which can contribute to further damaging cells pathways and structures with consequent tumour progression and metastasis [69].

Hypoxia and oxidative phosphorylation in cancer cells

Tumour cells experience an extensive heterogeneity of oxygen levels, from normoxia (around 2–4% oxygen tension), through hypoxia, to anoxia (< 0.1% oxygen tension). The growth of tumours beyond a critical mass > 1–2 mm3 is dependent on adequate blood supply to receive nutrients and oxygen by diffusion [88]. Cells adjacent to capillaries were found to exhibit a mean oxygen concentration of 2%, therefore, beyond this distance, hypoxia occurs: indeed, cells located at 200 μm displayed a mean oxygen concentration of 0.2%, which is a condition of severe hypoxia [89]. Oxygen shortage results in hypoxia-dependent inhibition of mitochondrial activity, mostly mediated by the hypoxia-inducible factor 1 (HIF-1)[90] and [91]. More precisely, hypoxia affects structure, dynamics, and function of the mitochondria, and in particular it has a significant inhibitory effect on the oxidative phosphorylation machinery, which is the main energy supplier of cells (see Ref. [22] for a recent review). The activation of HIF-1 occurs in the cytoplasmic region of the cell, but the contribution of mitochondria is critical being both cells oxygen sensors and suppliers of effectors of HIF-1α prolyl hydroxylase like α-ketoglutarate and probably ROS, that inhibit HIF-1α removal [92]. As reported above, mitochondria can also promote HIF-1α stabilization if the TCA flux is severely inhibited with release of intermediate molecules like succinate and fumarate into the cytosol. On the other hand, HIF-1 can modulate mitochondrial functions through different mechanisms, that besides metabolic reprogramming [7][22][93] and [94], include alteration of mitochondrial structure and dynamics[58], induction of microRNA-210 that decreases the cytochrome c oxidase (COX) activity by inhibiting the gene expression of the assembly protein COX10 [95], that also increases ROS generation. Moreover, these stress conditions could induce the anti-apoptotic protein Bcl-2, which has also been reported to regulate COX activity and mitochondrial respiration [96] conferring resistance to cells death in tumours (Fig. 2). This effect might be further enhanced upon severe hypoxia conditions, since COX is also inhibited by NO, the product of activated nitric oxide synthases [97].

The reduced respiration rate occurring in hypoxia favours the release of ROS also by Complex III, which contribute to HIF stabilization and induction of Bcl-2 [98]. In addition, hypoxia reduces oxphos by inhibiting the ATP synthase complex through its natural protein inhibitor IF1 (discussed in a previous section), which contributes to the enhancement of the “aerobic glycolysis”, all signatures of cancer transformation.

The observations reported to date indicate that cancer cells exhibit large varieties of metabolic changes which are associated with alterations in the mitochondrial structure, dynamics and function, and with tumour growth and survival. On one hand, mitochondria can regulate tumour growth through modulation of the TCA cycle and oxidative phosphorylation. The altered TCA cycle provides intermediates for both macromolecular biosynthesis and regulation of transcription factors such as HIF, and it allows cytosolic reductive power enhancement. Oxphos provides significant amounts of ATP which varies among tumour types. On the other hand, mitochondria are crucial in controlling redox homeostasis in the cell, inducing them to be either resistant or sensitive to apoptosis. All these reasons locate mitochondria at central stage to understanding the molecular basis of tumour growth and to seeking for novel therapeutical approaches.

Due to the complexity and variability of mitochondrial roles in cancer, careful evaluation of mitochondrial function in each cancer type is crucial. Deeper and more integrated knowledge of mitochondrial mechanisms and cancer-specific mitochondrial modulating means are expected for reducing tumorigenicity and/or improving anticancer drugs efficacy at the mitochondrial level. Although the great variability of biochemical changes found in tumour mitochondria, some highlighted peculiarities such as reduced TCA cycle flux, reduced oxphos rate, and reduced Complex I activity with respect to tissue specific normal counterparts are more frequent. In addition, deeper examination of supramolecular organization of the complexes in the inner mitochondrial membrane has to be considered in relation to oxphos dysfunction.

2.1.1.6  Oxidation–reduction states of NADH in vivo: From animals to clinical use

Mayevsky A, Chance B.
Mitochondrion. 2007 Sep; 7(5):330-9
http://dx.doi.org:/10.1016/j.mito.2007.05.001

Mitochondrial dysfunction is part of many pathological states in patients, such as sepsis or stroke. Presently, the monitoring of mitochondrial function in patients is extremely rare, even though NADH redox state is routinely measured in experimental animals. In this article, we describe the scientific backgrounds and practical use of mitochondrial NADH fluorescence measurement that was applied to patients in the past few years. In addition to NADH, we optically measured the microcirculatory blood flow and volume, as well as HbO(2) oxygenation, from the same tissue area. The four detected parameters provide real time data on tissue viability, which is critical for patients monitoring.

(very important article)

2.1.1.7  Mitochondria in cancer. Not just innocent bystanders

Frezza C, and Gottlieb E
Sem Cancer Biol 2009; 19: 4-11
http://dx.doi.org:/10.1016/j.semcancer.2008.11.008

The first half of the 20th century produced substantial breakthroughs in bioenergetics and mitochondria research. During that time, Otto Warburg observed abnormally high glycolysis and lactate production in oxygenated cancer cells, leading him to suggest that defects in mitochondrial functions are at the heart of malignant cell transformation. Warburg’s hypothesis profoundly influenced the present perception of cancer metabolism, positioning what is termed aerobic glycolysis in the mainstream of clinical oncology. While some of his ideas stood the test of time, they also frequently generated misconceptions regarding the biochemical mechanisms of cell transformation. This review examines experimental evidence which supports or refutes the Warburg effect and discusses the possible advantages conferred on cancer cells by ‘metabolic transformation’.

Fig.1. Mitochondria as a crossroad for catabolic and anabolic pathways in normal and cancer cells. Glucose and glutamine are important carbon sources which are metabolized in cells for the generation of energy and anabolic precursors. The pathways discussed in the text are illustrated and colour coded: red, glycolysis; white, TCA cycle; pink, non-essential amino acids synthesis; orange, pentose phosphate pathway and nucleotide synthesis; green, fatty acid and lipid synthesis; blue, pyruvate oxidation in the mitochondria; brown, glutaminolysis; black, malic enzyme reaction. Solid arrows indicate a single step reaction;dashed-dotted arrows indicate transport across membranes and dotted arrows indicate multi-step reactions. Abbreviations: HK, hexokinase; AcCoA, acetyl co-enzyme A; OAA, oxaloacetate; αKG, α-ketoglutarate.

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Fig. 2. Mitochondria as a target for multiple metabolic transformation events. Principal metabolic perturbations of cancer cells are induced by genetic reprogramming and environmental changes. The activation of Akt and MYC oncogenes and the loss of p53 tumor suppressor gene are among the most frequent events in cancer. Furthermore, all solid tumors are exposed to oxidative stress and hypoxia hence to HIF activation.These frequent changes in cancer cells trigger a dramatic metabolic shift from oxidative phosphorylation to glycolysis. In addition, direct genetic lesions of mtDNA or of nuclear encoded mitochondrial enzyme (SDH or FH) can directly abrogate oxidative phosphorylation in cancer. 3- D structures of the respiratory complexes in the scheme were retrieved from Protein DataBank (PDB:www.rcsb.org) except for complex I which was retrieved from [87]. PDB codes are as follow: SDH (II), 1 LOV; complex III (III), 1BGY; COX (IV), 1OCC; ATP synthase (V), 1QO1.

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Fig. 3. The physiological roles of SDH in the TCA cycle and the ETC and its potential roles in cancer. (A) Ribbon diagram of SDH structure (PBD code: 1LOV). The catalytic subunits: the flavoprotein (SDHA) and the iron-sulphur protein (SDHB) are depicted in red and yellow, respectively, and the membrane anchors and ubiquinone binding proteins SDHC and SDHD are depicted in cyan and green, respectively. (B) Other than being a TCA enzyme, SDH is an additional entry point to the ETC (most electrons are donated from NADH to complex I—not shown in this diagram). The electron flow in and out of complex II and III is depicted by the yellow arrows. During succinate oxidation to fumarate by SDHA, a two-electron reduction of FAD to FADH2 occurs. Electrons are transferred through their on–Sulphur centres on SDHB to ubiquinone (Q) bound to SDHC and SDHD in the inner mitochondrial membrane (IMM), reducing it to ubiquinol (QH2). Ubiquinol transfers its electrons through complex III, in a mechanism named the Q cycle, to cytochrome c (PDB: 1CXA). Electrons then flow from cytochrome c to COX where the final four-electron reduction of molecular oxygen to water occurs (not shown in this diagram). Complex III is the best characterized site of ROS production in the ETC, where a single electron reduction of oxygen to superoxide can occur (red arrow). It was proposed that obstructing electron flow within complex II might support a single electron reduction of oxygen at the FAD site (red arrow). Superoxide is dismutated to hydrogen peroxide which can then leave the mitochondria and inhibit PHD in the cytosol, leading to HIF[1] stabilization. Succinate or fumarate, which accumulate in SDH- or FH-deficient tumors, can also leave the mitochondria and inhibit PHD activity in the cytosol. The red dotted line represents the outer mitochondrial membrane (OMM).

2.1.1.8  Mitochondria in cancer cells: what is so special about them?

Gogvadze V, Orrenius S, Zhivotovsky B.
Trends Cell Biol. 2008 Apr; 18(4):165-73
http://dx.doi.org:/10.1016/j.tcb.2008.01.006

The past decade has revealed a new role for the mitochondria in cell metabolism–regulation of cell death pathways. Considering that most tumor cells are resistant to apoptosis, one might question whether such resistance is related to the particular properties of mitochondria in cancer cells that are distinct from those of mitochondria in non-malignant cells. This scenario was originally suggested by Otto Warburg, who put forward the hypothesis that a decrease in mitochondrial energy metabolism might lead to development of cancer. This review is devoted to the analysis of mitochondrial function in cancer cells, including the mechanisms underlying the upregulation of glycolysis, and how intervention with cellular bioenergetic pathways might make tumor cells more susceptible to anticancer treatment and induction of apoptosis.

Glucose utilization pathway

Glucose utilization pathway

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Figure 1. Glucose utilization pathway. When glucose enters the cell, it is phosphorylated by hexokinase to glucose-6-phosphate, which is further metabolized by glycolysis to pyruvate. Under aerobic conditions, most of the pyruvate in non-malignant cells enters the mitochondria, with only a small amount being metabolized to lactic acid. In mitochondria, pyruvate dehydrogenase (PDH) converts pyruvate into acetyl-CoA, which feeds into the Krebs cycle. Oxidation of Krebs cycle substrates by the mitochondrial respiratory chain builds up the mitochondrial membrane potential (Dc) – the driving force for ATP synthesis. By contrast, in tumor cells, the oxidative (mitochondrial) pathway of glucose utilization is suppressed, and most of the pyruvate is converted into lactate. Thus, the fate of pyruvate is determined by the relative activities of two key enzymes – lactate dehydrogenase and pyruvate dehydrogenase.

Mechanisms of mitochondrial silencing in tumors

Mechanisms of mitochondrial silencing in tumors

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Figure 2. Mechanisms of mitochondrial silencing in tumors. The activity of PDH is regulated by pyruvate dehydrogenase kinase 1 (PDK1), the enzyme that phosphorylates and inactivates pyruvate dehydrogenase. HIF-1 inactivates PDH through PDK1 induction, resulting in suppression of the Krebs cycle and mitochondrial respiration. In addition, HIF-1 stimulates expression of the lactate dehydrogenase A gene, facilitating conversion of pyruvate into lactate by lactate dehydrogenase (LDH). Mutation of p53 can suppress the mitochondrial respiratory activity through downregulation of the Synthesis of Cytochrome c Oxidase 2 (SCO2) gene, the product of which is required for the assembly of cytochrome c oxidase (COX) of the mitochondrial respiratory chain. Thus, mutation of p53 can suppress mitochondrial respiration and shift cellular energy metabolism towards glycolysis.

Production of ROS by mitochondria

In any cell, the majority of ROS are by-products of mitochondrial respiration. Approximately 2% of the molecular oxygen consumed during respiration is converted into the superoxide anion radical, the precursor of most ROS. Normally, a four-electron reduction of O2, resulting in the production of two molecules of water, is catalyzed by complex IV (COX) of the mitochondrial respiratory chain. However, the electron transport chain contains several redox centers (e.g. in complex I and III) that can leak electrons to molecular oxygen, serving as the primary source of superoxide production in most tissues. The one-electron reduction of oxygen is thermodynamically favorable for most mitochondrial oxidoreductases. Superoxide-producing sites and enzymes were recently analyzed in detail in a comprehensive review [87]. ROS, if not detoxified, oxidize cellular proteins, lipids, and nucleic acids and, by doing so, cause cell dysfunction or death. A cascade of water and lipid soluble antioxidants and antioxidant enzymes suppresses the harmful ROS activity. An imbalance that favors the production of ROS over antioxidant defenses, defined as oxidative stress, is implicated in a wide variety of pathologies, including malignant diseases. It should be mentioned that mitochondria are not only a major source of ROS but also a sensitive target for the damaging effects of oxygen radicals. ROS produced by mitochondria can oxidize proteins and induce lipid peroxidation, compromising the barrier properties of biological membranes. One of the targets of ROS is mitochondrial DNA (mtDNA), which encodes several proteins essential for the function of the mitochondrial respiratory chain and, hence, for ATP synthesis by oxidative phosphorylation. mtDNA, therefore, represents a crucial cellular target for oxidative damage, which might lead to lethal cell injury through the loss of electron transport and ATP generation. mtDNA is especially susceptible to attack by ROS, owing to its close proximity to the electron transport chain, the major locus for free-radical production, and the lack of protective histones. For example, mitochondrially generated ROS can trigger the formation of 8-hydroxydeoxyguanosine as a result of oxidative DNA damage; the level of oxidatively modified bases in mtDNA is 10- to 20-fold higher than that in nuclear DNA. Oxidative damage induced by ROS is probably a major source of mitochondrial genomic instability leading to respiratory dysfunction.

Figure 3. Stabilization of mitochondria against OMM permeabilization in tumor cells. OMM permeabilization is a key event in apoptotic cell death. (a) During apoptosis, tBid-mediated oligomerization of Bax causes OMM permeabilization and release of cytochrome c (red circles). (b) Bcl-2 protein binds Bax and prevents its oligomerization. A shift in the balance between pro- apoptotic and antiapoptotic proteins in cancer cells, in favor of the latter, reduces the availability of Bax and prevents OMM permeabilization. (c) Upregulation of hexokinase in tumors and its binding to VDAC in the OMM not only facilitates glucose phosphorylation using mitochondrially generated ATP but keeps VDAC in the open state, preventing its interaction with tBid (de).

http://www.cell.com/cms/attachment/591821/4554543/gr4.sml

Figure 4. Shifting metabolism from glycolysis to glucose oxidation. Utilization of pyruvate is controlled by the relative activities of two enzymes, PDH and LDH. In cancer cells, PDH activity is suppressed by PDH kinase-mediated phosphorylation, and, therefore, instead of entering the Krebs cycle, pyruvate is converted into lactate. Several attempts have been made to redirect pyruvate towards oxidation in the mitochondria. Thus, inhibition of PDK1 by dichloroacetate might stimulate the activity of PDH and, hence, direct pyruvate to the mitochondria. A similar effect can be achieved by inhibition of LDH by oxamate. Overall, suppression of PDK1 and LDH activities will stimulate mitochondrial ATP production and might be lethal to tumor cells, even if these inhibitors are used at non-toxic doses. In addition, stimulation of mitochondrial function, for example though overexpression of mitochondrial frataxin, a protein associated with Friedreich ataxia, was shown to stimulate oxidative metabolism and inhibited growth in several cancer cell lines [86].
2.1.1.9  Glucose avidity of carcinomas

Ortega AD1, Sánchez-Aragó M, Giner-Sánchez D, Sánchez-Cenizo L, et al.
Cancer Letters 276 (2009) 125–135
http://dx.doi.org:/10.1016/j.canlet.2008.08.007

The cancer cell phenotype has been summarized in six hallmarks [D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (1) (2000) 57-70]. Following the conceptual trait established in that review towards the comprehension of cancer, herein we summarize the basis of an underlying principle that is fulfilled by cancer cells and tumors: its avidity for glucose. Our purpose is to push forward that the metabolic reprogramming that operates in the cancer cell represents a seventh hallmark of the phenotype that offers a vast array of possibilities for the future treatment of the disease. We summarize the metabolic pathways that extract matter and energy from glucose, paying special attention to the concerted regulation of these pathways by the ATP mass-action ratio. The molecular and functional evidences that support the high glucose uptake and the “abnormal” aerobic glycolysis of the carcinomas are detailed discussing also the role that some oncogenes and tumor suppressors have in these pathways. We overview past and present evidences that sustain that mitochondria of the cancer cell are impaired, supporting the original Warburg’s formulation that ascribed the high glucose uptake of cancer cells to a defective mitochondria. A simple proteomic approach designed to assess the metabolic phenotype of cancer, i.e., its bioenergetic signature, molecularly and functionally supports Warburg’s hypothesis. Furthermore, we discuss the clinical utility that the bioenergetic signature might provide. Glycolysis is presented as the “selfish” pathway used for cellular proliferation, providing both the metabolic precursors and the energy required for biosynthetic purposes, in the context of a plethora of substrates. The glucose avidity of carcinomas is thus presented as the result of both the installment of glycolysis for cellular proliferation and of the impairment of mitochondrial activity in the cancer cell. At the end, the repression of mitochondrial activity affords the cancer cell with a cell-death resistant phenotype making them prone to malignant growth.

Fig. 1. Pathways of glucose metabolism. The model shows some of the relevant aspects of the metabolism of glucose. After entering the cell by specific transporters, glucose can be (i) catabolized by the pentose phosphate pathway (PPP) to obtain reducing power in the form of NADPH, (ii) used for the synthesis of carbohydrates or (iii) utilized by glycolysis to generate pyruvate and other metabolic intermediates that could be used in different anabolic processes (blue rectangles). In the cytoplasm, the generated pyruvate can be reduced to lactate and further exported from the cell or oxidized in the mitochondria by pyruvate dehydrogenase to generate acetyl-CoA, which is condensed with oxaloacetate in the tricarboxylic acid cycle (TCA cycle). The operation of the TCA cycle completes the oxidation of mitochondrial pyruvate. Different pathways that drain intermediates of the TCA cycle (oxaloacetate, succinyl-CoA, a-ketoglutarate and citrate) for biosynthetic purposes (blue rectangles) are represented. The transfer of electrons obtained in biological oxidations (NADH/FADH2) to molecular oxygen by respiratory complexes of the inner mitochondrial membrane (in green) is depicted by yellow lines. The utilization of the proton gradient generated by respiration for the synthesis of ATP by the H+-ATP synthase (in orange) in oxidative phosphorylation (OXPHOS) is also indicated. The incorporation of glutamine carbon skeletons into the TCA cycle is shown. The utilization of NADPH in anabolic pathways is also indicated.

Fig. 3. Fluxes of matter and energy in differentiated, proliferating and cancer cells. In differentiated cells, the flux of glycolysis is low because the requirement for precursors for anabolic purposes is low and there is a high energy yield by the oxidation of pyruvate in mitochondrial oxidative phosphorylation (OXPHOS). In this situation, mitochondrial activity produces large amounts of ROS that are normally quenched by the cellular antioxidant defense. In proliferating and cancer cells, there is a high demand of glucose to provide metabolic precursors for the biosynthesis of the macromolecules of daughter cells and because most of the energy required for anabolic purposes derives from non-efficient non-respiratory modes (glycolysis, pentose phosphate pathway) of energy generation. Limiting mitochondrial activity in these situations ensures less ROS production and their further downstream consequences. In addition, cancer cells have less overall mitochondrial complement or activity than normal cells by repressing the biogenesis of mitochondria.

Fig. 2. Genetic alterations underlying the glycolytic phenotype of cancer cells. The diagram represents the impact of gain-of-function mutations in oncogenes (ovals) and loss-of-function mutations in tumor suppressors (rectangles) in glycolysis and in the mitochondrial utilization of pyruvate in cancer cells. Hypoxia (low O2) induces the stabilization of HIF-1, which promotes transcriptional activation of the glucose transporter, glycolytic genes and PDK1. The expression of PDK1 results in the inactivation of pyruvate dehydrogenase and thus in a decreased oxidation of pyruvate in the TCA cycle concurrent to its enhanced cytoplasmic reduction to lactate by lactate dehydrogenase (LDHA). In addition, HIF1a reciprocally regulates the expression of two isoforms of the cytochrome c oxidase complex. The oncogen myc also supports an enhanced glycolytic pathway by transcriptional activation of glycolytic genes. High levels of c-myc could also promote the production of reactive oxygen species (ROS) that could damage nuclear (nDNA) and mitochondrial (mtDNA). The loss-of-function of the tumor suppressor p53 promotes an enhanced glycolytic phenotype by the repression of TIGAR expression. Likewise, loss-of-function of p53 diminished the expression of SCO2, a gene required for the appropriate assembly of cytochrome c oxidase, and thus limits the activity of mitochondria in the cancer cell.
Discussion:

Jose E S Roselino

  1. Warburg Effect revisited
    It is very interesting the series of commentaries following Warburg Effect revisited. However, it comes as no surprise that almost all of them have small or greater emphasis in the molecular biology (changes in gene expression) events of the metabolic regulation involved.
    I would like to comment on some aspects: 1- Warburg did the initial experiments following Pasteur line of reasoning that aimed at carbon flow through the cell (yeast in his case) instead of describing anything inside the cell. It is worth to recall that for the sake of his study, Pasteur considered anything inside the cell under the domain of divine forces. He, at least in defence of his work, entirely made outside the cell, considered that inside the cells was beyond human capability of understanding – He has followed vitalism as his line of reasoning in defence of his work – Interestingly, the same scientist that has ruled out spontaneous generation when Pasteurization was started. Therefore, Pasteur measured everything outside the cell (mainly sugar, ethanol – the equivalent of our lactic acid end product of anaerobic metabolism) and found that as soon as yeasts were placed in the presence of oxygen, sugar was consumed at low speed in comparison with the speed measured in anaerobiosis and ethanol was also produced at reduced speed. This is an indication of a fast biological regulatory mechanism that obviously, do not require changes in gene expression. As previously said, Warburg work translated for republishing in the Journal Biological Chemistry mentioned “grana” for mitochondria calling attention on an “inside-the-cell” component. It seems that, there is not a unique, single site of metabolism, where the Pasteur Effect – Warburg Effect seems to be elicited by the shift from anaerobiosis to aerobiosis or vice versa.
    In order to find a core for the mechanism the best approach seems to take into account one of the most important contributions of one of the greatest north-American biochemists, Briton Chance. He has made it with his polarographic method of following continuously the oxygen consumption of the cell´s mitochondria.
    Mitochondria burn organic carbon molecules under a very stringent control mechanism of oxidative-phosphorylation ATP production. Measured in the form of changes in the speed of oxygen consumption over time as Respiratory Control Ratio (RCR). When no ATP is required by the cell, oxygen consumption goes at low speed (basal or state II or IV). When ADP is offered to the mitochondria as an indication that ATP synthesis is necessary, oxygen consumption is activated in state III respiration. Low respiration means low burning activity of organic (carbon) molecules what in this case, means indirectly low glucose consumption. While high respiration is the converse – greater glucose consumption.
    Aerobic metabolism of glucose to carbonic acid and water provides a change in free energy enough for 38 molecules of ATP (the real production is +/- 32 ATP in aerobic condition) while glucose to lactic acid metabolism in anaerobiosis leads to 2 ATP production after discounting the other 2 required at initial stages of glucose metabolism.
    The low ATP yield in anaerobiosis explains the fast glucose metabolism in anaerobiosis while the control by RCR in mitochondria explains the reduction in glucose metabolism under aerobiosis as long as the ATP requirements of the cell remains the same – This is what it is assumed to happen in quiescent cells. Not necessarily in fast growing cells as cancer cells are. However, this will not be discussed here. In my first experiments in the early seventies, with M. Rouxii a dimorphic mold-yeast biological system the environmental change (aerobic – anaerobic) led to morphogenetic change presented as morphogenetic expression of the Pasteur Effect. In this case, the enzyme that replaces mitochondria in ATP production (Pyruvate Kinase) converting phosphoenolpyruvate into pyruvate together with ADP into ATP, shows changes that can be interpreted as change in gene expression together with new self-assembly of enzyme subunits. (Dimer AA – yeast in anaerobic growth or sporangiospores- converted into dimer AB in aerobic mold). In Leloir opinions at that time, PK I (AA) was only highly glycosylated, while PK II (AB) was less glycosylated without changes in gene expression.

    In case you read comments posted, you will see that the reference to aerobic glycolysis, continues to be made together with, new deranged forms of reasoning as is indicated by referring to: Mitochondrial role in ion homeostasis…
    Homeostasis is a regulation of something, ions, molecules, pH etc. that is kept outside the cell, therefore any role for mitochondria on it is only made indirectly, by its ATP production.
    However, mitochondria has a role together with other cell components in the regulation of for instance, intracellular Ca levels (Something that is not a homeostatic regulation). This is a very important point for the following reason: Homeostasis is maintained as a composite result of several differentiated cellular, tissue and organ functions. Differentiated function is something clearly missing in cancer cells. The best form to refer to the mitochondrial function regarding ions is to indicate a mitochondrial role in ion fluxes.
    In short, to indicate how an environmental event or better saying condition could favour genetic changes instead of being caused by genetic changes is to follow the same line of reasoning that is followed in understanding the role of cardioplegia. To stop heart beating is adequate for heart surgery it is also adequate for heart cells by sparing the ATP use during surgery and therefore, offering better recovery condition to the heart afterwards.
    In the case, here considered, even assuming that the genome is not made more unstable during hypoxic condition it is quite possible to understand that sharing ATP with both differentiated cell function and replication may led quality control of DNA in short supply of much needed ATP and this led to maintenance of mutations as well as less organized genome.

    • Thank you. I enjoy reading your comments. They are very instructive. I don’t really think that I comprehend the use of the term “epigenetics” and longer. In fact, it was never clear to me when I first heard it used some years ago.

      The term may have been closely wedded to the classic hypothesis of a unidirectional DNA–> RNA–> protein model that really has lost explanatory validity for the regulated cell in its environment. The chromatin has an influence, and protein-protein interactions are everywhere. As you point out, these are adjusting to a fast changing substrate milieu, and the genome is not involved. But in addition, the proteins may well have a role in suppression or activation of signaling pathways, and thereby, may well have an effect on gene expression. I don’t have any idea about how it would work, but mutations would appear to follow the metabolic condition of the cell over time. It would appear to be – genomic modification.

  2. In aerobic glucose metabolism, the oxidation of citric acid requires ADP and Mg²+, which will increase the speed of the reaction: Iso-citric acid + NADP (NAD) — isocitrate dehydrogenase (IDH) = alpha-ketoglutaric acid. In the Krebs cycle (the citric cycle), IDH1 and IDH2 are NADP+-dependent enzymes that normally catalyze the inter-conversion of D-isocitrate and alpha-ketoglutarate (α-KG). The IDH1 and IDH2 genes are mutated in > 75% of different malignant diseases. Two distinct alterations are caused by tumor-derived mutations in IDH1 or IDH2: the loss of normal catalytic activity in the production of α-ketoglutarate (α-KG) and the gain of catalytic activity to produce 2-hydroxyglutarate (2-HG), [22].
    This product is a competitive inhibitor of multiple α-KG-dependent dioxygenases, including demethylases, prolyl-4-hydroxylase and the TET enzymes family (Ten-Eleven Translocation-2), resulting in genome-wide alternations in histones and DNA methylation. [23]
    IDH1 and IDH2 mutations have been observed in myeloid malignancies, including de novo and secondary AML (15%–30%), and in pre-leukemic clone malignancies, including myelodysplastic syndrome and myeloproliferative neoplasm (85% of the chronic phase and 20% of transformed cases in acute leukemia), [24].
    Normally, cells in the body communicate via intra-cytoplasmic channels and maintain the energetic potential across cell membranes, which is 1-2.5 µmol of ATP in the form of ATP-ADP/ATP-ADP-IMP. These normal energetic values occur during normal cell division. If the intra-cellular and extra-cellular levels of Mg2+ are high, the extra-cellular charges of the cells will not be uniformly distributed.
    This change in distribution induces a high net positive charge for the cell and induces a loss of contact inhibition via the electromagnetic induction of oscillation [28, 29, 30]. Thereafter, malignant cells become invasive and metastasize.
    ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
    -22. Hartmann C, Meyer J, Balss J. Capper D, et al. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta Neuropathol 2009; 118: 464-474.

    23. Raymakers R.A, Langemeijer S.M., Kuiper R.P, Berends M, et al. Acquired mutations in TET2 are common in myelodysplastic syndromes. Nat. Genet 2009; 41; 838–849.

    24 Wagner K, Damm F, Gohring G., Gorlich K et al. Impact of IDH1 R132 mutations and an IDH1 single nucleotide polymorphism in cytogenetically normal acute myeloid leukemia: SNP rs11554137 is an adverse prognostic factor. J. Clin. Oncol.2010; 28: 2356–2364.
    Plant Molecular Biology 1989; 1: 271–303.

    29. Chien MM, Zahradka CE, Newel MC, Fred JW. Fas induced in B cells apoptosis require an increase in free cytosolic magnesium as in early event. J Biol Chem.1999; 274: 7059-7066.

    30. Milionis H J, Bourantas C L, Siamopoulos C K, Elisaf MS. Acid bases and electrolytes abnormalities in Acute Leukemia. Am J Hematol 1999; (62): 201-207.

    31. Thomas N Seyfried; Laura M Shelton.Cancer as a Metabolic Disease. Nutr Metab 2010; 7: 7

    – Aurelian Udristioiu, M.D,
    – Lab Director, EuSpLM,
    – City Targu Jiu, Romania
    AACC, National Academy of Biochemical Chemistry (NACB) Member, Washington D.C, USA.

 

 

 

 

 

 

 

 

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Summary, Metabolic Pathways

Author: Larry H. Bernstein, MD, FCAP 

 

This portion of a series of chapters on metabolism, proteomics and metabolomics dealt mainly with carbohydrate metabolism. Amino acids and lipids are presented more fully in the chapters that follow. There are features on the

  • functioning of enzymes and proteins,
  • on sequential changes in a chain reaction, and
  • on conformational changes that we shall also cover.

These are critical to developing a more complete understanding of life processes.

I needed to lay out the scope of metabolic reactions and pathways, and their complementary changes. These may not appear to be adaptive, if the circumstances and the duration is not clear. The metabolic pathways map in total
is in interaction with environmental conditions – light, heat, external nutrients and minerals, and toxins – all of which give direction and strength to these reactions. A developing goal is to discover how views introduced by molecular biology and genomics don’t clarify functional cellular dynamics that are not related to the classical view.  The work is vast.

Carbohydrate metabolism denotes the various biochemical processes responsible for the formation, breakdown and interconversion of carbohydrates in living organisms. The most important carbohydrate is glucose, a simple sugar (monosaccharide) that is metabolized by nearly all known organisms. Glucose and other carbohydrates are part of a wide variety of metabolic pathways across species: plants synthesize carbohydrates from carbon dioxide and water by photosynthesis storing the absorbed energy internally, often in the form of starch or lipids. Plant components are consumed by animals and fungi, and used as fuel for cellular respiration. Oxidation of one gram of carbohydrate yields approximately 4 kcal of energy and from lipids about 9 kcal. Energy obtained from metabolism (e.g. oxidation of glucose) is usually stored temporarily within cells in the form of ATP. Organisms capable of aerobic respiration metabolize glucose and oxygen to release energy with carbon dioxide and water as byproducts.

Carbohydrates are used for short-term fuel, and even though they are simpler to metabolize than fats, they don’t produce as equivalent energy yield measured by ATP.  In animals, the concentration of glucose in the blood is linked to the pancreatic endocrine hormone, insulin. . In most organisms, excess carbohydrates are regularly catabolized to form acetyl-CoA, which is a feed stock for the fatty acid synthesis pathway; fatty acids, triglycerides, and other lipids are commonly used for long-term energy storage. The hydrophobic character of lipids makes them a much more compact form of energy storage than hydrophilic carbohydrates.

Glucose is metabolized obtaining ATP and pyruvate by way of first splitting a six-carbon into two three carbon chains, which are converted to lactic acid from pyruvate in the lactic dehydrogenase reaction. The reverse conversion is by a separate unidirectional reaction back to pyruvate after moving through pyruvate dehydrogenase complex.

Pyruvate dehydrogenase complex (PDC) is a complex of three enzymes that convert pyruvate into acetyl-CoA by a process called pyruvate decarboxylation. Acetyl-CoA may then be used in the citric acid cycle to carry out cellular respiration, and this complex links the glycolysis metabolic pathway to the citric acid cycle. This multi-enzyme complex is related structurally and functionally to the oxoglutarate dehydrogenase and branched-chain oxo-acid dehydrogenase multi-enzyme complexes. In eukaryotic cells the reaction occurs inside the mitochondria, after transport of the substrate, pyruvate, from the cytosol. The transport of pyruvate into the mitochondria is via a transport protein and is active, consuming energy. On entry to the mitochondria pyruvate decarboxylation occurs, producing acetyl CoA. This irreversible reaction traps the acetyl CoA within the mitochondria. Pyruvate dehydrogenase deficiency from mutations in any of the enzymes or cofactors results in lactic acidosis.

PDH-rxns The acetyl group is transferred to coenzyme A

PDH-rxns The acetyl group is transferred to coenzyme A

http://guweb2.gonzaga.edu/faculty/cronk/biochem/images/PDH-rxns.gif

Typically, a breakdown of one molecule of glucose by aerobic respiration (i.e. involving both glycolysis and Kreb’s cycle) is about 33-35 ATP. This is categorized as:

Glycogenolysis – the breakdown of glycogen into glucose, which provides a glucose supply for glucose-dependent tissues.

Glycogenolysis in liver provides circulating glucose short term.

Glycogenolysis in muscle is obligatory for muscle contraction.

Pyruvate from glycolysis enters the Krebs cycle, also known as the citric acid cycle, in aerobic organisms.

Anaerobic breakdown by glycolysis – yielding 8-10 ATP

Aerobic respiration by Kreb’s cycle – yielding 25 ATP

The pentose phosphate pathway (shunt) converts hexoses into pentoses and regenerates NADPH. NADPH is an essential antioxidant in cells which prevents oxidative damage and acts as precursor for production of many biomolecules.

Glycogenesis – the conversion of excess glucose into glycogen as a cellular storage mechanism; achieving low osmotic pressure.

Gluconeogenesis – de novo synthesis of glucose molecules from simple organic compounds. An example in humans is the conversion of a few amino acids in cellular protein to glucose.

Metabolic use of glucose is highly important as an energy source for muscle cells and in the brain, and red blood cells.

The hormone insulin is the primary glucose regulatory signal in animals. It mainly promotes glucose uptake by the cells, and it causes the liver to store excess glucose as glycogen. Its absence

  1. turns off glucose uptake,
  2. reverses electrolyte adjustments,
  3. begins glycogen breakdown and glucose release into the circulation by some cells,
  4. begins lipid release from lipid storage cells, etc.

The level of circulatory glucose (known informally as “blood sugar”) is the most important signal to the insulin-producing cells.

  • insulin is made by beta cells in the pancreas,
  • fat is stored n adipose tissue cells, and
  • glycogen is both stored and released as needed by liver cells.
  • no glucose is released to the blood from internal glycogen stores from muscle cells.

The hormone glucagon, on the other hand, opposes that of insulin, forcing the conversion of glycogen in liver cells to glucose, and then release into the blood. Growth hormone, cortisol, and certain catecholamines (such as epinepherine) have glucoregulatory actions similar to glucagon.  These hormones are referred to as stress hormones because they are released under the influence of catabolic proinflammatory (stress) cytokines – interleukin-1 (IL1) and tumor necrosis factor α (TNFα).

Net Yield of GlycolysisThe preparatory phase consumes 2 ATP

The pay-off phase produces 4 ATP.

The gross yield of glycolysis is therefore

4 ATP – 2 ATP = 2 ATP

The pay-off phase also produces 2 molecules of NADH + H+ which can be further converted to a total of 5 molecules of ATP* by the electron transport chain (ETC) during oxidative phosphorylation.

Thus the net yield during glycolysis is 7 molecules of ATP*
This is calculated assuming one NADH molecule gives 2.5 molecules of ATP during oxidative phosphorylation.

Cellular respiration involves 3 stages for the breakdown of glucose – glycolysis, Kreb’s cycle and the electron transport system. Kreb’s cycle produces about 60-70% of ATP for release of energy in the body. It directly or indirectly connects with all the other individual pathways in the body.

The Kreb’s Cycle occurs in two stages:

  1. Conversion of Pyruvate to Acetyl CoA
  2. Acetyl CoA Enters the Kreb’s Cycle

Each pyruvate in the presence of pyruvate dehydrogenase (PDH) complex in the mitochondria gets converted to acetyl CoA which in turn enters the Kreb’s cycle. This reaction is called as oxidative  decarboxylation as the carboxyl group is removed from the pyruvate molecule in the form of CO2 thus yielding 2-carbon acetyl group which along with the coenzyme A forms acetyl CoA.

The PDH requires the sequential action of five co-factors or co-enzymes for the combined action of dehydrogenation and decarboxylation to take place. These five are TPP (thiamine phosphate), FAD (flavin adenine dinucleotide), NAD (nicotinamide adenine dinucleotide), coenzyme A (denoted as CoA-SH at times to depict role of -SH group) and lipoamide.

Acetyl CoA condenses with oxaloacetate (4C) to form a citrate (6C) by transferring its acetyl group in the presence of enzyme citrate synthase. The CoA liberated in this reaction is ready to participate in the oxidative decarboxylation of another molecule of pyruvate by PDH complex.

Isocitrate undergoes oxidative decarboxylation by the enzyme isocitrate dehydrogenase to form oxalosuccinate (intermediate- not shown) which in turn forms α-ketoglutarate (also known as oxoglutarate) which is a five carbon compound. CO2 and NADH are released in this step. α-ketoglutarate (5C) undergoes oxidative decarboxylation once again to form succinyl CoA (4C) catalysed by the enzyme α-ketoglutarate dehydrogenase complex.

Succinyl CoA is then converted to succinate by succinate thiokinase or succinyl coA synthetase in a reversible manner. This reaction involves an intermediate step in which the enzyme gets phosphorylated and then the phosphoryl group which has a high group transfer potential is transferred to GDP to form GTP.

Succinate then gets oxidised reversibly to fumarate by succinate dehydrogenase. The enzyme contains iron-sulfur clusters and covalently bound FAD which when undergoes electron exchange in the mitochondria causes the production of FADH2.

Fumarate is then by the enzyme fumarase converted to malate by hydration(addition of H2O) in a reversible manner.

Malate is then reversibly converted to oxaloacetate by malate dehydrogenase which is NAD linked and thus produces NADH.

The oxaloacetate produced is now ready to be utilized in the next cycle by the citrate synthase reaction and thus the equilibrium of the cycle shifts to the right.

The NADH formed in the cytosol can yield variable amounts of ATP depending on the shuttle system utilized to transport them into the mitochondrial matrix. This NADH, formed in the cytosol, is impermeable to the mitochondrial inner-membrane where oxidative phosphorylation takes place. Thus to carry this NADH to the mitochondrial matrix there are special shuttle systems in the body. The most active shuttle is the malate-aspartate shuttle via which 2.5 molecules of ATP are generated for 1 NADH molecule. This shuttle is mainly used by the heart, liver and kidneys. The brain and skeletal muscles use the other shuttle known as glycerol 3-phosphate shuttle which synthesizes 1.5 molecules of ATP for 1 NADH.

Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose Phosphate Pathway. This enzyme is regulated by availability of the substrate NADP+. As NADPH is utilized in reductive synthetic pathways, the increasing concentration of NADP+ stimulates the Pentose Phosphate Pathway, to replenish NADPH. The importance of this pathway can easily be underestimated.  The main source for energy in respiration was considered to be tied to the high energy phosphate bond in phosphorylation and utilizes NADPH, converting it to NADP+. The pentose phosphate shunt is essential for the generation of nucleic acids, in regeneration of red cells and lens – requiring NADPH.

NAD+ serves as electron acceptor in catabolic pathways in which metabolites are oxidized. The resultant NADH is reoxidized by the respiratory chain, producing ATP.

The pyridine nucleotide transhydrogenase reaction concerns the energy-dependent reduction of TPN by DPNH. In 1959, Klingenberg and Slenczka made the important observation that incubation of isolated liver mitochondria with DPN-specific substrates or succinate in the absence of phosphate acceptor resulted in a rapid and almost complete reduction of  the intramitochondrial TPN. These and related findings led Klingenberg and co-workers (1-3) to postulate the occurrence of a ATP-controlled transhydrogenase reaction catalyzing the reduction of TPN by DPNH.  (The role of transhydrogenase in the energy-linked reduction of TPN.  Fritz Hommes, Ronald W. Estabrook, The Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden. Biochemical and Biophysical Research Communications 11, (1), 2 Apr 1963, Pp 1–6. http://dx.doi.org:/10.1016/0006-291X(63)90017-2/).

Further studies observed the coupling of TPN-specific dehydrogenases with the transhydrogenase and observing the reduction of large amounts of diphosphopyridine nucleotide (DPN) in the presence of catalytic amounts of triphosphopyridine nucleotide (TPN). The studies showed the direct interaction between TPNHz and DPN, in the presence of transhydrogenase to yield products having the properties of TPN and DPNHZ. The reaction involves a transfer of electrons (or hydrogen) rather than a phosphate. (Pyridine Nucleotide Transhydrogenase  II. Direct Evidence for and Mechanism of the Transhydrogenase Reaction* by  Nathan 0. Kaplan, Sidney P. Colowick, And Elizabeth F. Neufeld. (From The Mccollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland) J. Biol. Chem. 1952, 195:107-119.) http://www.JBC.org/Content/195/1/107.Citation
Notation: TPN, NADP; DPN, NAD+; reduced pyridine nucleotides: TPNH (NADPH2), DPNH (NADH).

Note: In this discussion there is a detailed presentation of the activity of lactic acid conversion in the mitochondria by way of PDH. In a later section there is mention of the bidirectional reaction of lactate dehydrogenase.  However, the forward reaction is dominant (pyruvate to lactate) and is described. This is not related to the kinetics of the LD reaction with respect to the defining characteristic – Km.

Biochemical Education Jan 1977; 5(1):15. Kinetics of Lactate Dehydrogenase: A Textbook Problem.
K.L. MANCHESTER. Department of Biochemistry, University of Witwatersrand, Johannesburg South Africa.

One presupposes that determined Km values are meaningful under intracellular conditions. In relation to teaching it is a simple experiment for students to determine for themselves the Km towards pyruvate of LDH in a post-mitochondrial supernatant of rat heart and thigh muscle. The difference in Km may be a factor of 3 or 4-fold.It is pertinent then to ask what is the range of suhstrate concentrations over which a difference in Km may be expected to lead to significant differences in activity and how these concentrations compare with pyruvate concentrations in the cell. The evidence of Vesell and co-workers that inhibition by pyruvate is more readily seen at low than at high enzyme concentration is important in emphasizing that under intracellular conditions enzyme concentrations may be relatively large in relation to the substrate available. This will be particularly so in relation to [NADH] which in the cytoplasm is likely to be in the ~M range.

A final point concerns the kinetic parameters for LDH quoted by Bergmeyer for lactate estimations a pH of 9 is recommended and the Km towards lactate at that pH is likely to be appreciably different from the quoted values at pH 7 — Though still at pH 9 showing a substantially lower value for lactate with the heart preparationhttp://onlinelibrary.wiley.com/doi/10.1016/0307-4412%2877%2990013-9/pdf

Several investigators have established that epidermis converts most of the glucose it uses to lactic acid even in the presence of oxygen. This is in contrast to most tissues where lactic acid production is used for energy production only when oxygen is not available. This large amount of lactic acid being continually produced within the epidermal cell must be excreted by the cell and then carried away by the blood stream to other tissues where the lactate can be utilized. The LDH reaction with pyruvate and NADH is reversible although at physiological pH the equilibrium position for the reaction lies very far to the right, i.e., in favor of lactate production. The speed of this reaction depends not only on the amount of enzyme present but also on the concentrations of the substances involved on both sides of the equation. The net direction in which the reaction will proceed depends solely on the relative concentrations of the substances on each side of the equation.
In vivo there is net conversion of pyruvate (formed from glucose) to lactate. Measurements of the speed of lactate production by sheets of epidermis floating on a medium containing glucose indicate a rate of lactate production of approximately 0.7 rn/sm/
mm/mg of fresh epidermis.Slice incubation experiments are presumably much closer to the actual in vivo conditions than
the homogenate experiments. The discrepancy between the
two indicates that in vivo conditions are far from optimal for the conversion of pyruvate to lactate. Only 1/100th of the maximal activity of the enzyme present is being achieved. The concentrations of the various substances involved are not
optimal in vivo since pyruvate and NADH concentrations are
lower than lactate and NAD concentrations and this might explain the in vivo inhibition of LDH activity. (Lactate Production And Lactate Dehydrogenase In The Human Epidermis*. KM. Halprin, A Ohkawara. J Invest Dermat 1966; 47(3): 222-6.)
http://www.nature.com/jid/journal/v47/n3/pdf/jid1966133a.pdf

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Epilogue: Envisioning New Insights in Cancer Translational Biology

Author and Curator: Larry H Bernstein, MD, FCAP

 

The foregoing  summary leads to a beginning as it is a conclusion.  It concludes a body of work in the e-book series,

Series C: e-Books on Cancer & Oncology

Series C Content Consultant: Larry H. Bernstein, MD, FCAP

 

VOLUME ONE 

Cancer Biology and Genomics for Disease Diagnosis

2014

Stephen J. Williams, PhD, Senior Editor

sjwilliamspa@comcast.net

Tilda Barliya, PhD, Editor

tildabarliya@gmail.com

Ritu Saxena, PhD, Editor

ritu.uab@gmail.com

Leaders in Pharmaceutical Business Intelligence 

that has been presented by the cancer team of professional experts, e-Book concept was conceived by Aviva Lev-Ari, PhD, RN, e-Series Editor-in-Chief and Founder of Leaders in Pharmaceutical Business Intelligence 

and the Open Access Online Scientific Journal

http://pharmaceuticalintelligence.com

Stephen J. Williams, PhD, Senior Editor, and other notable contributors in  various aspects of cancer research in the emerging fields of targeted  pharmacology,  nanotechnology, cancer imaging, molecular pathology, transcriptional and regulatory ‘OMICS’, metabolism, medical and allied health related sciences, synthetic biology, pharmaceutical discovery, and translational medicine.

This  volume and its content have been conceived and organized to capture the organized events that emerge in embryological development, leading to the major organ systems that we recognize anatomically and physiologically as an integrated being.  We capture the dynamic interactions between the systems under stress  that are elicited by cytokine-driven hormonal responses, long thought to be circulatory and multisystem, that affect the major compartments of  fat and lean body mass, and are as much the drivers of metabolic pathway changes that emerge as epigenetics, without disregarding primary genetic diseases.

The greatest difficulty in organizing such a work is in whether it is to be merely a compilation of cancer expression organized by organ systems, or whether it is to capture developing concepts of underlying stem cell expressed changes that were once referred to as “dedifferentiation”.  In proceeding through the stages of neoplastic transformation, there occur adaptive local changes in cellular utilization of anabolic and catabolic pathways, and a retention or partial retention of functional specificities.

This  effectively results in the same cancer types not all fitting into the same “shoe”. There is a sequential loss of identity associated with cell migration, cell-cell interactions with underlying stroma, and metastasis., but cells may still retain identifying “signatures” in microRNA combinatorial patterns.  The story is still incomplete, with gaps in our knowledge that challenge the imagination.

What we have laid out is a map with substructural ordered concepts forming subsets within the structural maps.  There are the traditional energy pathways with terms aerobic and anaerobic glycolysis, gluconeogenesis, triose phosphate branch chains, pentose shunt, and TCA cycle vs the Lynen cycle, the Cori cycle, glycogenolysis, lipid peroxidation, oxidative stress, autosomy and mitosomy, and genetic transcription, cell degradation and repair, muscle contraction, nerve transmission, and their involved anatomic structures (cytoskeleton, cytoplasm, mitochondria, liposomes and phagosomes, contractile apparatus, synapse.

Then there is beneath this macro-domain the order of signaling pathways that regulate these domains and through mechanisms of cellular regulatory control have pleiotropic inhibitory or activation effects, that are driven by extracellular and intracellular energy modulating conditions through three recognized structures: the mitochondrial inner membrane, the intercellular matrix, and the ion-channels.

What remains to be done?

  1. There is still to be elucidated the differences in patterns within cancer types the distinct phenotypic and genotypic features  that mitigate anaplastic behavior. One leg of this problem lies in the density of mitochondria, that varies between organ types, but might vary also within cell type of a common function.  Another leg of this problem has also appeared to lie in the cell death mechanism that relates to the proeosomal activity acting on both the ribosome and mitochondrion in a coordinated manner.  This is an unsolved mystery of molecular biology.

 

  1. Then there is a need to elucidate the major differences between tumors of endocrine, sexual, and structural organs, which are distinguished by primarily a synthetic or primarily a catabolic function, and organs that are neither primarily one or the other.  For example, tumors of the thyroid and paratnhyroids, islet cells of pancreas, adrenal cortex, and pituitary glands have the longest 5 year survivals.  They and the sexual organs are in the visceral compartment.  The rest of the visceral compartment would be the liver, pancreas, salivary glands, gastrointestinal tract, and lungs (which are embryologically an outpouching of the gastrointestinal tract), kidneys and lower urinary tract.  Cancers of these organs have a much less favorable survival (brain, breast and prostate, lymphatic, blood forming organ, skin).  The case  is intermediate for breast and prostate between the endocrine organs and GI tract, based on natural history, irrespective of the available treatments.  Just consider the dilemma over what we do about screening for prostate cancer in men over the age of 60 years age who have a 70 percent incident silent carcinoma of the prostate that could be associated with unrelated cause of death.  The very rapid turnover of the gastric and colonic GI epithelium, and of the  subepithelial  B cell mucosal lymphocytic structures  is associated  with a greater aggressiveness of the tumor.

 

  1. However, we  have to reconsider the observation by NO Kaplan than the synthetic and catabolic functions are highlighted by differences in the expressions of the balance of  the two major pyridine nucleotides – DPN (NAD) and TPN (NADP) – which also might be related to the density of mitochondria  which is associated with both NADP and synthetic activity, and  with efficient aerobic function.  These are in an equilibrium through the “transhydrogenase reaction” co-discovered by Kaplan, in Fritz Lipmann’s laboratory. There does  arise a conundrum involving the regulation of mitochondria in these high turnover epithelial tissues  that rely on aerobic energy, and generate ATP through TPN linked activity, when they undergo carcinogenesis. The cells  replicate and they become utilizers of glycolysis, while at the same time, the cell death pathway is quiescent. The result becomes the introduction of peripheral muscle and liver synthesized protein cannabolization (cancer cachexia) to provide glucose  from proteolytic amino acid sources.

 

  1. There is also the structural compartment of the lean body mass. This is the heart, skeletal  structures (includes smooth muscle of GI tract, uterus, urinary bladder, brain, bone, bone marrow).  The contractile component is associated with sarcomas.  What is most striking is that the heart, skeletal muscle, and inflammatory cells are highly catabolic, not anabolic.  NO Kaplan referred tp them as DPN (NAD) tissues. This compartment requires high oxygen supply, and has a high mechanical function. But again, we return to the original observations of enrgy requirements at rest being different than at high demand.  At work, skeletal muscle generates lactic acid, but the heart can use lactic acid as fuel,.

 

  1. The liver is supplied by both the portal vein and the hepatic artery, so it is not prone to local ischemic injury (Zahn infarct). It is exceptional in that it carries out synthesis of all the circulating transport proteins, has a major function in lipid synthesis and in glycogenesis and glycogenolysis, with the added role of drug detoxification through the P450 system.  It is not only the largest organ (except for brain), but is highly active both anabolically and catabolically (by ubiquitilation).
  2. The expected cellular turnover rates for these tissues and their balance of catabolic and anabolic function would have to be taken into account to account for the occurrence and the activities of oncogenesis. This is by no means a static picture, but a dynamic organism constantly in flux imposed by internal and external challenges.  It is also important to note the the organs have a concentration of mitochondria, associated with energy synthetic and catabolic requirements provided by oxygen supply and the electron transport mechanism for oxidative phosphorylation.  For example, tissues that are primarily synthetic do not have intermitent states of resting and high demand, as seen in skeletal muscle, or perhaps myocardium (which is syncytial and uses lactic acid generated from skeletal muscle when there is high demand).
  3. The existence of  lncDNA has been discovered only as a result of the human genome project (HGP). This was previously known only as “dark DNA”.  It has become clear that lncDNA has an important role in cellular regulatory activities centered in the chromatin modeling.  Moreover, just as proteins exhibit functionality in their folding, related to tertiary structure and highly influenced by location of –S-S- bridges and amino acid residue distances (allosteric effects), there is a less studied effect as the chromatin becomes more compressed within the nucleus, that should have a bearing on cellular expression.

According to Jose Eduardo de Salles Roselino , when the Na/Glucose transport system (for a review Silvermann, M. in Annu. Rev. Biochem.60: 757-794(1991)) was  found in kidneys as well as in key absorptive cells of digestive tract, it should be stressed its functional relationship with “internal milieu” and real meaning, homeostasis. It is easy to understand how the major topic was presented as how to prevent diarrheal deaths in infants, while detected in early stages. However, from a biochemical point of view, as presented in Schrödinger´s What is life?, (biochemistry offering a molecular view for two legs of biology, physiology and genetics). Why should it be driven to the sole target of understanding genetics? Why the understanding of physiology in molecular terms should be so neglected?

From a biochemical point of view, here in a single protein. It is found the transport of the cation most directly related to water maintenance, the internal solvent that bath our cells and the hydrocarbon whose concentration is kept under homeostatic control on that solvent. Completely at variance with what is presented in microorganisms as previously mentioned in Moyed and Umbarger revision (Ann. Rev42: 444(1962)) that does not regulates the environment where they live and appears to influence it only as an incidental result of their metabolism.

In case any attempt is made in order to explain why the best leg that supports scientific reasoning from biology for medical purposes was led to atrophy, several possibilities can be raised. However, none of them could be placed strictly in scientific terms. Factors that bare little relationship with scientific progress in general terms must also be taken into account.

One simple possibility of explanation can be found in one review (G. Scatchard – Solutions of Electrolytes Ann. Rev. Physical Chemistry 14: 161-176 (1963)).  A simple reading of it and the sophisticated differences among researchers will discourage one hundred per cent of biologists to keep in touch with this line of research. Biochemists may keep on reading.  However, consider that first: Complexity is not amenable to reductionist vision in all cases. Second, as coupling between scalar flows such as chemical reactions and vector flows such as diffusion flows, heat flows, and electrical current can occur only in anisotropic system…let them with their problems of solvents, ions and etc. and let our biochemical reactions on another basket. At the interface, for instance, at membrane level, we will agree that ATP is converted to ADP because it is far from equilibrium and the continuous replenishment of ATP that maintain relatively constant ATP levels inside the cell and this requires some non-stationary flow.

Our major point must be to understand that our biological limits are far clearer present in our limited ability to regulate the information stored in the DNA than in the amount of information we have in the DNA as the master regulator of the cells.

The amazing revelation that Masahiro Chiga   (discovery of liver adenylate kinase  distinct from that of muscle) taught  me (LHB) is – draw 2 circles  that intersect, one of which represents what we know, the other – what we don’t know.  We don’t teach how much we don’t know!  Even today, as much as 40 years ago, there is a lot we need to get on top of this.

 

The observation is rather similar to the presentations I  (Jose Eduardo de Salles Rosalino) was previously allowed to make of the conformational energy as made by R Marcus in his Nobel lecture revised (J. of  Electroanalytical Chemistry 438:(1997) p251-259. His description of the energetic coordinates of a landscape of a chemical reaction is only a two-dimensional cut of what in fact is a volcano crater (in three dimensions) ( each one varie but the sum of the two is constant. Solvational+vibrational=100% in ordinate) nuclear coordinates in abcissa. In case we could represent it by research methods that allow us to discriminate in one by one degree of different pairs of energy, we would most likely have 360 other similar representations of the same phenomenon. The real representation would take into account all those 360 representation together. In case our methodology was not that fine, for instance it discriminate only differences of minimal 10 degrees in 360 possible, will have 36 partial representations of something that to be perfectly represented will require all 36 being taken together. Can you reconcile it with ATGC? Yet, when complete genome sequences were presented they were described as we will know everything about this living being. The most important problems in biology will be viewed by limited vision always and the awareness of this limited is something we should acknowledge and teach it. Therefore, our knowledge is made up of partial representations.

 

Even though we may have complete genome data for the most intricate biological problems, they are not so amenable to this level of reductionism. However, from general views of signals and symptoms we could get to the most detailed molecular view and in this case the genome provides an anchor. This is somehow, what Houssay was saying to me and to Leloir when he pointed out that only in very rare occasions biological phenomena could be described in three terms: Pacco, the dog and the anesthetic (previous e-mail). The non-coding region, to me will be important guiding places for protein interactions.

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A Synthesis of the Beauty and Complexity of How We View Cancer


A Synthesis of the Beauty and Complexity of How We View Cancer

Author: Larry H. Bernstein, MD, FCAP

Cancer Volume One – Summary

A Synthesis of the Beauty and Complexity of How We View Cancer

 

This document has covered a broad spectrum of the research, translational biology, diagnostics (both laboratory and imaging methodologies), and treatments for a variety of cancers, mainly by organs, and selectively by the most common cancers seen in human populations. A number of observations stand out on review of all the material presented. 1. The most common cancers affecting humans is spread worldwide, with some variation by region. 2. Cancers within geographic regions may be expressed differently in relationship to population migrations, the incidence of specific environmental pollutants, occurrence of insect transmitted and sexually transmitted diseases (HIV, HCV, HPV), and possibly according to age, or relationship to ultraviolet or high dose radiation exposure. 3. Cancers are expressed within generally recognized age timelines. For example, acute lymphocytic leukemia and neuroblastoma in children under 10 years age; malignant giant cell tumor and osteosarcoma in the third and fourth decade; prostate cancer and breast cancer over age 40, and are more aggressive at an earlier age, both having a strong sex hormone dependence. 4. There is dispute about the effectiveness of screening for cancer with respect to what age, excessive risk in treatment modality, and the duration of progression free survival. Despite the evidence of several years potential life extension, a long term survival of 10 years is not the expected outcome. However, the quality of life in the remaining years is a valid point in favor of progress. 5. There has been a significant reduction in toxicity of treatment, but attention has been focused on a patient-centric decision process. 6. There has been a dramatic improvement in surgical approaches, post-surgical surveillance, and in diagnosis by invasive and noninvasive methods, especially in the combination of needle biopsy and imaging techniques. 7. There is significant variation within cancer cell types with respect to disease-free survival.

The work presented has several main components: First, there is the biology and mechanisms involved in carcinogenesis related to (1) mutations; (2) carcinogenesis; (3) cell regulatory mechanisms; (4) cell signaling pathways; (5) apoptosis (6) ubitination (7) mitochondrial dysfunction; (8) cell-cell interactions; (9) cell migration; (10) metastasis. Then there are large portions covering (1) imaging; (2) specific targeted therapy; (3) nanotechology-based therapy; (4) specific organ-type cancers; (5) genomics-based testing; (6) circulating cancer cells; (7) miRNAs; (8) siRNAs; (9) cancer immunology and (10) immunotherapy.

Classically, we refer to cancer development in terms of the germ cell layers – ectoderm, mesoderm, and endoderm. These are formative in embryonic development. The most active development occurs during embryonic development, with a high growth rate of cells and also a high utilization of energy. The cells utilize oxidation for energy in this period characterized by movement of cells in differentiation and organogenesis. This was observed to be unlike the cell metabolism in carcinogenesis, which is characterized by impaired mitochondrial function and reliance on lactate production for energy – termed anaerobic glycolysis, as investigated by Meyerhof, Embden, Warburg, Szent-Gyorgy, H. Krebs, Theorell, AV Hill, B Chance, P Mitchell, P Boyer, F Lippman, and others.

In addition, the body economy has been divided into two major metabolic compartments: fat and lean body mass (LBM), which is further denoted as visceral and structural. This denotes the gut, kidneys, liver, lung, pancreas, sexual organs, endocrines, brain and fat cells in one compartment, and skeletal muscle, bone and cardiovascular in another. LBM is calculated as fat free mass. Further, brown fat is distinguished from white fat. But this was a first layer of construction of the human body. One peels away this layer to find a second layer. For example, the gut viscera have an inner (outer) epithelial layer, a muscularis, and a deep epithelium, which has circulation and fat. There is also an interstitium between the gut epithelium and muscularis. The lung has an epithelium exposed to the airspaces, then capillaries, and then epithelium, designed for exchange of O2 and CO2, the source of heat generation. The pancreas has an endocrine portion in the islets that are embedded in an exocrine secretory organ. The sexual organs have a combination of glandular structures embedded in a mesothelium.

The structural compartment is entirely accounted for by the force of contraction. If this is purely anatomical, that is not really the case when one goes into the functioning substructures of these tissues – cytoplasm, endoplasmic reticulum (ribosomal), mitochondria, liposomes, chromatin apparatus, cell membrane and vesicles. Within and between these structures are the working and interacting mechanisms of the cell in its unique role. What ties these together was first thought to be found in the dogma following the discovery of the genetic code in 1953 that begat DNA to RNA to protein.

This led to many other discoveries that made it clear that it was only a first approximation. It did not account for noncoding DNA, which became unmasked with the culmination of the Human Genome Project and concurrent advances in genomics (mtDNA, mtRNA, siRNA, exosomes, proteomics, synthetic biology, predictive analytics, and regulatory pathways directed by signaling molecules. Here is a list of signaling pathways: 1. JAK-STAT 2. GPCR 3. Endocrine 4. Cytochemical 5. RTK 6. P13K 7. NF-KB 8. MAPK 9. Ubiquitin 10. TGF-beta 11. Stem cell These signaling pathways have become the basis for the discovery of inhibitors of signaling pathways (suppressors), as well as activators, as these have been considered as specific targets for selective therapy. (.See Figure below) Of course, extensive examination of these pathways has required that all such findings are validated based on the STRENGTH of their effect on the target and in the impact of suppression.

inhibitors of signaling pathways-1

http://www.SelleckChem.com

 

Let us continue this discussion elucidating several major points.  While the early observations that drove the interest in biochemical behavior of cancer cells has been displaced, it has not faded from view.

Bioenergetics of Cancer cells

Michael J. Gonzalez (Bioenergetic_Theory_of_Carcinigenesis. http://www.academia.edu/2224071/ Bioenergetic_Theory_of_Carcinigenesis) maintains that the altered energy metabolism of tumor cells provides a viable target for a non-toxic chemotherapeutic approach.  An increased glucose consumption rate  has been observed in malignant cells. Warburg (NobelLaureate in medicine) postulated that the respiratory process of malignant cells was impaired in the malignant transformation. Szent-Györgyi (Nobel in medicine) also viewed cancer as originating from insufficient oxygen utilization. Oxygen inhibits anaerobic  metabolism (fermentation and lactic acid production). Interestingly, during cell differentiation (where cell energy level is high) there is an increased cellular production of oxidation products that appear to provide physiological stimulation for changes in gene expression that may lead to a terminal differentiated state. The failure to maintain high ATP production (high cell energy levels) may be a consequence of inactivation of key enzymes, especially those related to the Krebs cycle and the electron transport system. A distorted mitochondrial function (transmembrane potential) may result.  This  aspect could be suggestive of an important mitochondrial involvement in the carcinogenic process in addition to presenting it as a possible therapeutic target for cancer. Intermediate metabolic correction of the mitochondria is postulated as a possible non-toxic therapeutic approach for cancer.

Fermentation is the anaerobic metabolic breakdown of glucose without net oxidation. Fermentation does not release all the available energy of glucose or need oxygen as part of its biochemical reactions ;  it merely allows glycolysis  (a process that yields two ATP per mole of glucose) to continue by replenishing reduced coenzymes and yields lactate as its final product. The first step in aerobic and anaerobic energy producing pathways, it occurs in the cytoplasm of cells, not in specialized organelles, and is found in all living organisms.  Cancer cells have a fundamentally different energy metabolism compared to normal cells, that  are obligate aerobes (oxygen-requiring cells)  meeting their energy needs with oxidative metabolic processes., while cancer cells do not  require oxygen for their survival. This increase in glycolytic  flux is a metabolic strategy of tumor cells to ensure growth and    survival  in  environments  with  low   oxygen concentrations.

Radoslav Bozov has commented that the process of genomic evolution cannot be fully revealed through comparative genomicsHe states that DNA would be entropic- favorable stable state going towards absolute ZERO temp. Themodynamics measurement in subnano discrete space would go negative towards negativity. DNA is like a cold melting/growing crystal, quite stable as it appears not due to hydrogen bonding , but due to interference of C-N-O. That force is contradicted via proteins onto which we now know large amount of negative quantum redox state carbon attaches. The more locally one attempts to observe, the more hidden variables would emerge as a consequence of discrete energy spaces opposing continuity of matter/time. But stability emerges out of non-stable states, and never reaches absolute stability, for there would be neither feelings nor freedom.

Membrane potential(Vm)

Membrane potential (Vm), the voltage across the plasma membrane, arises because of the presence of differention channels/transporters with specific ion selectivity and permeability. Vm is a key biophysical signal in non-excitable cells, modulating important cellular activities, such as proliferation and differentiation. Therefore, the multiplicities of various ion channels/transporters expressed on different cells are finely tuned in order to regulate the Vm. (M Yang and WJ Brackenbury.

Membrane potential and cancer progression. Frontiers in Physiol.  2013(4); 185: 1.  http://dx.doi.org/10.3389/fphys.2013.00185)

It is well-established that cancer cells possess distinct bioelectrical properties. Notably, electrophysiological analyses in many cancer cell types have revealed a depolarized Vm that favors cell proliferation. Ion channels/transporters control cell volume and migration, and emerging data also suggest that the level of Vm has functional roles in cancer cell migration. In addition, yperpolarization is necessary for stem cell differentiation. For example, both osteogenesis and adipogenesis are hindered in human mesenchymal stem cells (hMSCs) under depolarizing conditions. Therefore, in the context of cancer, membrane depolarization might be important for the emergence and maintenance of cancer stem cells (CSCs), giving rise to sustained tumor growth. This review aims to provide a broad understanding of the Vm as a bioelectrical signal in cancer cells by examining several key types of ion channels that contribute to its regulation. The mechanisms by which Vm regulates cancer cell proliferation, migration, and differentiation will be discussed. In the long term, Vm might be avaluable clinical marker for tumor detection with prognostic value, and could even be artificially modified in order to inhibit tumor growth and metastasis.

Perspective beyond Cancer Genomics: Bioenergetics of Cancer Stem Cells

Hideshi Ishii, Yuichiro Doki, and Masaki Mori
Yonsei Med J 2010; 51(5):617-621.  http://dx.doi.org/10.3349/ymj.2010.51.5.617   pISSN: 0513-5796, eISSN: 1976-2437

Although the notion that cancer is a disease caused by genetic and epigenetic alterations is now widely accepted, perhaps more emphasis has been given to the fact that cancr is a genetic disease. It should be noted that in the post-genome sequencing project period of the 21st century, the underlined phenomenon nevertheless could not be discarded towards the complete control of cancer disaster as the whole strategy, and in depth investigation of the factors associated with tumorigenesis is required for achieving it. Otto Warburg has 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. Furthermore, 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.

Efficient execution of cell death in non-glycolytic cells requires the generation of ROS controlled by the activity of mitochondrial H+-ATP synthase.

Gema Santamaría1,#, Marta Martínez-Diez1,#, Isabel Fabregat2 and José M. Cuezva1,*
Carcinogenesis 2006 27(5):925-935      http://dx.doi.org/10.1093/carcin/bgi315

There is a large body of clinical data documenting that most human carcinomas contain reduced levels of the catalytic subunit of the mitochondrial H+-ATP synthase. In colon and lung cancer this alteration correlates with a poor patient prognosis. Furthermore, recent findings in colon cancer cells indicate that down-regulation of the H+-ATP synthase is linked to the resistance of the cells to chemotherapy. However, the mechanism by which the H+-ATP synthase participates in cancer progression is unknown. In this work, we show that inhibitors of the H+-ATP synthase delay

staurosporine-induced cell death in liver cells that are dependent on oxidative phosphorylation for energy provision whereas it has no effect on glycolytic cells. Efficient execution of cell death requires the generation of reactive oxygen species (ROS) controlled by the activity of the H+-ATP synthase in a process that is concurrent with the rapid disorganization of the cellular mitochondrial network. The generation of ROS after staurosporine treatment is highly dependent on the mitochondrial membrane potential and most likely caused by reverse electron flow to Complex I. The generated ROS promote the carbonylation and covalent modification of cellular and mitochondrial proteins. Inhibition of the activity of the H+-ATP synthase blunted ROS production, prevented the oxidation of cellular proteins and the modification of mitochondrial proteins, delaying the release of cyt c and the execution of cell death. The results in this work establish the down-regulation of the H+-ATP synthase, and thus of oxidative phosphorylation, as part of the molecular strategy adapted by cancer cells to avoid reactive oxygen species-mediated cell death. Furthermore, the results provide a mechanistic explanation to understand chemotherapeutic resistance of cancer cells that rely on glycolysis as main energy provision pathway.

see also –

The tumor suppressor function of mitochondria: Translation into the clinics

José M. CuezvaÁlvaro D. OrtegaImke Willers, et al.  
Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease  Dec 2009;  1792(12): 1145–1158  http://dx.doi.org/10.1016/j.bbadis.2009.01.006

Recently, the inevitable metabolic reprogramming experienced by cancer cells as a result of the onset of cellular proliferation has been added to the list of hallmarks of the cancer cell phenotype. Proliferation is bound to the synchronous fluctuation of cycles of an increased glycolysis concurrent with a restrained oxidative phosphorylation. Mitochondria are key players in the metabolic cycling experienced during proliferation because of their essential roles in the transduction of biological energy and in defining the life–death fate of the cell. These two activities are molecularly and functionally integrated and are both targets of commonly altered cancer genes. Moreover, energetic metabolism of the cancer cell also affords a target to develop new therapies because the activity of mitochondria has an unquestionable tumor suppressor function. In this review, we summarize most of these findings paying special attention to the opportunity that translation of energetic metabolism into the clinics could afford for the management of cancer patients. More specifically, we emphasize the role that mitochondrial β-F1-ATPase has as a marker for the prognosis of different cancer patients as well as in predicting the tumor response to therapy.

Self-Destructive Behavior in Cells May Hold Key to a Longer Life

Carl Zimmer, MY Times  October 5, 2009

In recent years, scientists have found evidence of autophagy in preventing a much wider range of diseases. Many disorders, like Alzheimer’s disease, are the result of certain kinds of proteins forming clumps. Lysosomes can devour these clumps before they cause damage, slowing the onset of diseases.

Lysosomes may also protect against cancer. As mitochondria get old, they cast off charged molecules that can wreak havoc in a cell and lead to potentially cancerous mutations. By gobbling up defective mitochondria, lysosomes may make cells less likely to damage their DNA. Many scientists suspect it is no coincidence that breast cancer cells are often missing autophagy-related genes. The genes may have been deleted by mistake as a breast cell divided. Unable to clear away defective mitochondria, the cell’s descendants become more vulnerable to mutations.

Unfortunately, as we get older, our cells lose their cannibalistic prowess. The decline of autophagy may be an important factor in the rise of cancer, Alzheimer’s disease and other disorders that become common in old age. Unable to clear away the cellular garbage, our bodies start to fail.

If this hypothesis turns out to be right, then it may be possible to slow the aging process by raising autophagy. It has long been known, for example, that animals that are put on a strict low-calorie diet can live much longer than animals that eat all they can. Recent research has shown that caloric restriction raises autophagy in animals and keeps it high. The animals seem to be responding to their low-calorie diet by feeding on their own cells, as they do during famines. In the process, their cells may also be clearing away more defective molecules, so that the animals age more slowly.

Some scientists are investigating how to manipulate autophagy directly. Dr. Cuervo and her colleagues, for example, have observed that in the livers of old mice, lysosomes produce fewer portals on their surface for taking in defective proteins. So they engineered mice to produce lysosomes with more portals. They found that the altered lysosomes of the old experimental mice could clear away more defective proteins. This change allowed the livers to work better.

 

Essentiality of pyruvate kinase, oxidation, and phosphorylation

We can move to the next level with greater clarity. Yu et al. reported an important relationship between Pyruvate kinase M2 (PKM2) and the Warburg effect of cancer cells ( M Yu, et al. PIM2 phosphorylates PKM2 and promotes Glycolysis in Cancer Cells. J Biol Chem (PMID: 24142698) http://dx.doi.org10.1074/jbc.M113.508226 ).  They found that PIM2 could directly phosphorylate PKM2 on the Thr454 residue, which resulted in an increase of PKM2 protein levels. PKM2 with a phosphorylation-defective mutation displayed a reduced effect on glycolysis compared to the wild-type, thereby co-activating HIF-1α and β-catenin, and enhanced mitochondria respiration and chemotherapeutic sensitivity of cancer cells. This indicated that PIM2-dependent phosphorylation of PKM2 is critical for regulating the Warburg effect in cancer, highlighting PIM2 as a potential therapeutic target.

In another study of the effect of 3 homoplastic mtDNA mutations on oxidative metabolism of osteosarcoma cells, there was a difference proportional to the magnitude of the defect. (Iommarini L, et al. Different mtDNA mutations modify tumor progression in dependence of the degree of respiratory complex I impairment. Hum Mol Genet. 2013 Nov 11. [Epub ahead of print]; PMID: 24163135 ).   Osteosarcoma cells carrying the most marked impairment of the gene encoding mitochondrial complex I  (CI) of oxidative phosphorylation displayed a reduced tumorigenic potential both in vitro and in vivo, when compared with cells with mild CI dysfunction. The severe CI dysfunction was an energetic defect associated with a compensatory increase in glycolytic metabolism and AMP-activated protein kinase activation.  The result suggested that mtDNA mutations may display diverse impact on tumorigenic potential depending on the type and severity of the resulting oxidative phosphorylation dysfunction. The modulation of tumor growth was independent from reactive oxygen species production but correlated with hypoxia-inducible factor 1α stabilization, indicating that structural and functional integrity of CI and oxidative phosphorylation are required for hypoxic adaptation and tumor progression.

An unrelated finding shares some agreement with what has been identified (Systematic isolation of context-dependent vulnerabilities in NSCLC. Cell, 24 Oct 2013; 155 (3): 552-566, http://dx.doi.org/10.1016/ j.cell.2013.09.041). They report  three distinct target/response-indicator pairings that are represented with significant frequencies (6%–16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature.   This is depicted in the Figure below.  The authors noted a frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets.

Print

 

 

The forging of a cancer-metabolism link and twists in the chain (Biome 19th April 2013)

Ten years ago, Grahame Hardie and Dario Alessi discovered that the elusive upstream kinase required for the activation of AMP-activated protein kinase (AMPK) by metabolic stress that the Hardie lab had been pursuing in their research on the metabolic regulator AMPK was the tumor suppressor, LKB1, that the neighbouring Alessi lab was working on at the time. This finding represented the first clear link between AMPK and cancer.

The resulting paper [1], published in 2003 in what was then Journal of Biology (now BMC Biology), was one [1] of three [2, 3] connecting these two kinases and that helped to swell of a surge of interest in the metabolism of tumor cells that was just beginning at about that time and is still growing. (LKB1 and AMPK and the cancer-metabolism link – ten years after.  D Grahame Hardie, and Dario R Alessi.  BMC Biology 2013, 11:36.   http://dx doi.org.10.1186/1741-7007-11-36.)

 

In September 2003, both groups published a joint paper [1] in Journal of Biology (now BMC Biology) that identified the long-sought and elusive upstream kinase acting on AMP-activated protein kinase (AMPK) as a complex containing LKB1, a known tumor suppressor. Similar findings were reported at about the same time by David Carling and Marian Carlson [2] and by Reuben Shaw and Lew Cantley [3]; at the time of writing these three papers have received between them a total of over 2,000 citations. These findings provided a direct link between a protein kinase, AMPK, which at the time was mainly associated with regulation of metabolism, and another protein kinase, LKB1, which was known from genetic studies to be a tumor suppressor. While the idea that cancer is in part a metabolic disorder (first suggested by Warburg in the 1920s [4]) is well recognized today [5], this was not the case in 2003, and our paper perhaps contributed towards its renaissance.

The distinctive metabolic feature of tumor cells that enables them to meet the demands of unrestrained growth is the switch from oxidative generation of ATP to aerobic glycolysis – a phenomenon now well known as the Warburg effect. Operating this switch is one of the central functions of the AMP-activated protein kinase (AMPK) that has long been the focus of research in the Hardie lab. AMPK is an energy sensor that is allosterically tuned by competitive binding of ATP, ADP and AMP to sites on its g regulatory subunit (its portrait here, with AMP bound at two sites, was kindly provided by Bing Xiao and Stephen Gamblin). When phosphorylated by LKB1, AMPK responds to depletion of ATP by turning off anabolic reactions required for growth, and turning on catabolic reactions and oxidative phosphorylation – the reverse of the Warburg effect. In this light, it is not surprising that LKB1  is inactivated in some proportion of many different types of tumors.

AMPK as an energy sensor and metabolic switch

AMPK was discovered as a protein kinase activity that phosphorylated and inactivated two key enzymes of fatty acid and sterol biosynthesis: acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). The ACC kinase activity was reported to be activated by 5’-AMP, and the HMGR kinase activity by reversible phosphorylation, but for many years the two activities were thought to be due to distinct enzymes. However, in 1987 the DGH laboratory showed that both were functions of a single protein kinase, which we renamed AMPK after its allosteric activator, 5’-AMP. It was subsequently found that AMPK regulated not only lipid biosynthesis, but also many other metabolic pathways, both by direct phosphorylation of metabolic enzymes, and through longer-term effects mediated by phosphorylation of transcription factors and co-activators. In general, AMPK switches off anabolic pathways that consume ATP and NADPH, while switching on catabolic pathways that generate ATP (Figure 1).

 

target proteins and metabolic pathways regulated by AMPK 1741-7007-11-36-1_1

 

Summary of a selection of target proteins and metabolic pathways regulated by AMPK. Anabolic pathways switched off by AMPK are shown in the top half of the ‘wheel’ and catabolic pathways switched on by AMPK in the bottom half. Where a protein target for AMPK responsible for the effect is known, it is shown in the inner wheel; a question mark indicates that it is not yet certain that the protein is directly phosphorylated. For original references see [54].

Key to acronyms: ACC1/ACC2, acetyl-CoA carboxylases-1/-2; HMGR, HMG-CoA reductase; SREBP1c, sterol response element binding protein-1c; CHREBP, carbohydrate response element binding protein; TIF-1A, transcription initiation factor-1A; mTORC1, mechanistic target-of-rapamycin complex-1; PFKFB2/3, 6-phosphofructo-2-kinase, cardiac and inducible isoforms; TBC1D1, TBC1 domain protein-1; SIRT1, sirtuin-1; PGC-1α, PPAR-γ coactivator-1α; ULK1, Unc51-like kinase-1.

Regulation of AMPK  1741-7007-11-36-3

 

Regulation of AMPK. AMPK can be activated by increases in cellular AMP:ATP or ADP:ATP ratio, or Ca2+ concentration. AMPK is activated >100-fold on conversion from a dephosphorylated form (AMPK) to a form phosphorylated at Thr172 (AMPK-P) catalyzed by at least two upstream kinases: LKB1, which appears to be constitutively active, and CaMKKβ, which is only active when intracellular Ca2+ increases. Increases in AMP or ADP activate AMPK by three mechanisms: (1) binding of AMP or ADP to AMPK, causing a conformational change that promotes phosphorylation by upstream kinases (usually this will be LKB1, unless [Ca2+] is elevated); (2) binding of AMP or ADP, causing a conformational change that inhibits dephosphorylation by protein phosphatases; (3) binding of AMP (and not ADP), causing allosteric activation of AMPK-P. All three effects are antagonized by ATP, allowing AMPK to act as an energy sensor.

AMPK and AMPK-related kinase (ARK) family  1741-7007-11-36-4

 

Members of the AMPK and AMPK-related kinase (ARK) family. All the kinases named in the figure are phosphorylated and activated by LKB1, although what regulates this phosphorylation is known only for AMPK. Alternative names are shown, where applicable.

AMPK-activating drugs metformin or phenformin might provide protection against cancer 1741-7007-11-36-5

 

 

Three possible mechanisms to explain how the AMPK-activating drugs metformin or phenformin might provide protection against cancer. (a) Metformin acts on the liver and other insulin target tissues by activating AMPK (and probably via other targets), normalizing blood glucose; this reduces insulin secretion from pancreatic β cells, reducing the growth-promoting effects of insulin (and high glucose) on tumor cells. Since metformin does not reduce glucose levels in normoglycemic individuals, this mechanism would only operate in insulin-resistant subjects. (b) Metformin or phenformin activates AMPK in pre-neoplastic cells, restraining their growth and proliferation and thus delaying the onset of tumorigenesis; this mechanism would only operate in cells where the LKB1-AMPK pathway was intact. (c) Metformin or phenformin inhibits mitochondrial ATP synthesis in tumor cells, promoting cell death. If the LKB1-AMPK pathway was down-regulated in the tumor cells, they would be more sensitive to cell death induced by the biguanides than surrounding normal cells.

Metformin and phenformin are biguanides that inhibit mitochondrial function and so deplete ATP by inhibiting its production . AMPK is activated by any metabolic stress that depletes ATP, either by inhibiting its production (as do hypoxia, glucose deprivation, and treatment with biguanides) or by accelerating its consumption (as does muscle contraction). By switching off anabolism and other ATP-consuming processes and switching on alternative ATP-producing catabolic pathways, AMPK acts to restore cellular energy homeostasis.

Findings that AMPK is activated in skeletal muscle during exercise and that it increases muscle glucose uptake and fatty acid oxidation led to the suggestion that AMPK-activating drugs might be useful for treating type 2 diabetes. Indeed, it turned out that AMPK is activated by metformin, a drug that had at that time been used to treat type 2 diabetes for over 40 years, and by phenformin , a closely related drug that had been withdrawn for treatment of diabetes due to side effects of lactic acidosis.

If only it were so simple. Effects of metformin on cancer in type 2 diabetics could be secondary to reduction in insulin levels, and although there is evidence for direct effects of AMPK activation on the development of tumors in mice, there is also recent evidence that tumors that become established without down-regulating LKB1 survive metformin better than those that have lost it – probably because metformin poisons the mitochondrial respiratory chain, depressing ATP levels, and cells in which AMPK can still be activated in response to the challenge do better than those in which it can’t.

In their review, Hardie and Alessi chart these  twists and turns, and point to the explosion of further possibilities opened up by the discovery, since their 2003 publication, of at least one other class of kinase upstream of AMPK (the CaM kinases), and at least a dozen other downstream targets of LKB1 (AMPK-related kinases, or ARKs) – not to mention the innumerable downstream targets of AMPK; all which make half their schematic illustrations look like hedgehogs.

Analysis of respiration  in human cancer

Bioenergetic profiling of cancer cells is of great potential because it can bring forward new and effective

Therapeutic  strategies along with early diagnosis. Metabolic Control Analysis (MCA) is a methodology that enables quantification of the flux control exerted by different enzymatic steps in a metabolic network thus assessing their contribution to the system‘s function.

(T Kaambre,V Chekulayev, I Shevchuk, et al. Metabolic control analysis of respiration  in human cancer tissue.  Frontiers Physiol 2013 (4); 151:  1. http://dx.doi.org/10.3389/fphys.2013.00151)

Our main goal is to demonstrate the applicability of MCA for in situ studies of energy

Metabolism in human breast and colorectal cancer cells as well as in normal tissues .We seek to determine the metabolic conditions leading to energy flux redirection in cancer cells. A main result obtained is that the adenine nucleotide translocator exhibits the highest control of respiration in human breast cancer thus becoming a prospective therapeutic target. Additionally, we present evidence suggesting the existence of mitochondrial respiratory supercomplexes that may represent a way by which cancer cells avoid apoptosis. The data obtained show that MCA applied in situ can be insightful in cancer cell energetic research.

Metabolic control analysis of respiration in human cancer tissue. fphys-04-00151-g001

Metabolic control analysis of respiration in human cancer tissue.

Representative traces of change in the rate of oxygen consumption by permeabilized human colorectal cancer (HCC) fibers after their titration with increasing concentrations of mersalyl, an inhibitor of inorganic phosphate carrier (panel A). The values of respiration rate obtained were plotted vs. mersalyl concentration (panel B) and from the plot the corresponding flux control coefficient was calculated. Bars are ±SEM.

Oncologic diseases such as breast and colorectal cancers are still one of the main causes of premature death. The low efficiency of contemporary medicine in the treatment of these malignancies is largely mediated by a poor understanding of the processes involved in metastatic dissemination of cancer cells as well as the unique energetic properties of mitochondria from tumors. Current knowledge supports the idea that human breast and colorectal cancer cells exhibit increased rates of glucose consumption displaying Warburg phenotype,i.e.,elevated glycolysis even in the presence of oxygen (Warburg and Dickens, 1930; Warburg, 1956 ;Izuishietal., 2012). Notwithstanding,  there are some evidences that in these malignancies mitochondrial oxidative phosphorylation (OXPHOS) is the main source of ATP rather than glycolysis. Cancer cells have been classified according to their pattern of metabolic remodeling depending of the relative balance between aerobic glycolysis and OXPHOS (Bellanceetal.,2012). The first type of tumor cells is highly glycolytic, the second OXPHOS deficient and the third type of tumors dislay enhanced OXPHOS. Recent studies strongly sug gest  that cancer cells can utilize lactate, free fatty acids, ketone bodies, butyrate and glutamine as key respiratory substrate selic iting metabolic remodeling of normal surrounding cells toward aerobic glycolysis—“reverse Warburg”effect (Whitaker-Menezes et al.,2011;Salem et al.,2012;Sotgia et al.,2012;Witkiewicz et al., 2012).

In normal cells,the OXPHOS system is usually closely linked to phosphotransfer systems, including various creatine kinase(CK) isotypes,which ensure a safe operation of energetics over a broad functional range of cellular activities (Dzejaand Terzic,2003).  However, our current knowledge about the function of CK/creatine (Cr) system in human breast and colorectal cancer is insufficient. In some malignancies, for example sarcomas the CK/Cr system was shown to be strongly downregulated (Beraetal.,2008;Patraetal.,2008).  Our previous studies showed  that the mitochondrial-bound CK (MtCK) activity was significantly decreased in HL-1 tumor cells (Mongeetal.,2009), as compared to normal parent cardiac cells where the OXPHOS is the main ATP source of and the CK system is a main energy carrier. In the present study,we estimated the role of MtCK in maintaining energy homeostasis in human colorectal cancer cells. Understanding the control and regulation of energy metabolism requires analytical tools that take into account  the existing interactions between individual network components and their impact on systemic network function. Metabolic Control Analysis(MCA) is a theoretical framework relating the properties of metabolic systems to the kinetic characteristics of their individual enzymatic components (Fell,2005). An experimental approach of MCA has been already successfully applied to the studies of OXPHOS in isolated mitochondria (Tageretal.,1983; Kunzetal.,1999; Rossignoletal.,2000)  and in skinned muscle fibers (Kuznetsovetal.,1997;Teppetal.,2010).

Metabolic control analysis of respiration in human cancer tissue

Values of basal (Vo) and maximal respiration rate (Vmax, in the presence of 2 mM ADP) and apparent Michaelis Menten constant (Km) for ADP in permeabilized human breast and colorectal cancer samples as well as health tissue. – See more at: http://journal.frontiersin.org/Journal/10.3389/fphys.2013.00151/full#sthash.VBXPdodj.dpuf

Role of Uncoupling Proteins in Cancer

Adamo Valle, Jordi Oliver and Pilar Roca *
Cancers 2010; 2: 567-591;   http://dx.doi.org/10.3390/cancers2020567

Since Otto Warburg discovered that most cancer cells predominantly produce energy by glycolysis rather than by oxidative phosphorylation in mitochondria, much interest has been focused on the alterations of these organelles in cancer cells. 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.

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. In this review we compile the latest evidence showing a link between uncoupling and the carcinogenic process, paying special attention to their involvement in cancer initiation, progression and drug chemoresistance.

The Warburg Effect

Uncoupling the Warburg effect from cancer

A Najafov and DR Alessi
Proc Nat Acad Sci                                      www.pnas.org/cgi/doi/10.1073/pnas.1014047107
A remarkable trademark of most tumors is their ability to break down glucose by glycolysis at a vastly higher rate than in normal tissues, even when oxygen is copious. This phenomenon, known as the Warburg effect, enables rapidly dividing tumor cells to generate essential biosynthetic building blocks such as nucleic acids, amino acids, and lipids from glycolytic intermediates to permit growth and duplication of cellular components during  division (1). An assumption dominating research in this area is that the Warburg effect is specific to cancer. Thus, much of the focus has been on uncovering mechanisms by which cancer-causing mutations influence metabolism to stimulate glycolysis.

This has lead to many exciting discoveries. For example, the p53 tumor suppressor can suppress glycolysis through its ability to control expression of key metabolic genes, such as phosphoglycerate mutase (2), synthesis of cytochrome C oxidase-2 (3), and TP53-induced glycolysis and apoptosis regulator (TIGAR) (4). Many cancer-causing mutations lead to activation of the Akt and mammalian target of rapamycin (mTOR) pathway that profoundly influences metabolism and expression of metabolic enzymes to promoteglycolysis (5).

Strikingly, all cancer cells but not nontransformed cells express a specific splice variant of pyruvate kinase, termed M2-PK, that is less active, leading to the build up of phosphoenolpyruvate (6). Recent work has revealed that reduced activity of M2-PK promotes a unique glycolytic pathway in which phosphoenolpyruvate is converted to pyruvate by a histidine-dependent phosphorylation of phosphoglycerate mutase, promoting assimilation of glycolytic products into biomass (7). However, despite these observations, one might imagine that the Warburg effect need not be specific for cancer and that any normal cell would need to stimulate glycolysis to generate sufficient biosynthetic materials to fuel expansion and division.

Recent work by Salvador Moncada’s group published in PNAS (8) and other recent work from the same group (9, 10) provides exciting evidence supporting the idea that the Warburg effect is also required for the proliferation of noncancer cells.

The key discovery was that the anaphase promoting complex/cyclosome-Cdh1(APC/C-Cdh1), a master regulator of the transition of G1 to S phase of the cell cycle, inhibits glycolysis in proliferating noncancer cells by mediating the degradation of two key metabolic enzymes, namely 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase isoform3 (PFKFB3) (9, 10) and glutaminase-(Fig. 1) (8).

Fig. 1. Mechanism by which APC_C-Cdh1 inhibits glycolysis and glutaminolysis to suppress cell proliferation

 

Fig.  Mechanism by which APC/C-Cdh1 inhibits glycolysis and glutaminolysis to suppress cell proliferation.

APC/C-Cdh1 E3 ligase recognizes KEN-box–containing metabolic enzymes, such as PFKFB3 and glutaminase-1 (GLS1), and ubiquitinates and targets them for proteasomal degradation. This inhibits glycolysis and glutaminolysis, leading to decrease in metabolites that can be assimilated into biomass, thereby suppressing proliferation.

PFKFB3 potently stimulates glycolysis by catalyzing the formation of fructose-2,6-bisphosphate, the allosteric activatorof 6-phosphofructo-1-kinase (11). Glutaminase-1 is the first enzyme in glutaminolysis, converting glutamine to lactate, yielding biosyntheticintermediates required for cell proliferation (12).

APC/C is a cell cycle-regulated E3 ubiquitin ligase that promotes ubiquitination of a distinct set of cell cycle proteins containing either a D-box (destruction box) or a KEN-box, named after the essential Lys-Glu-Asn motif required for APC recognition (13). Among its well-known substrates are crucial cell cycle proteins, such as cyclin B1, securin, and Plk1. By ubiquitinating and targeting its substrates to 26S proteasome-mediated degradation, APC/C regulates processes in late mitotic stage, exit  from mitosis, and several events in G1 (14). The Cdh1 subunit is the KENbox binding adaptor of the APC/C ligase and is essential for G1/S transition.

Importantly, APC/C-Cdh1 is inactivated at the initiation of the S-phase of the cell cycle when DNA and cellular organelles are replicated at the time of the greatest need for generation of biosynthetic materials. APC/C-Cdh1 is reactivated later at the mitosis/G1 phase of the cell cycle when there is a lower requirement for biomassgeneration.

Both PFKFB3 (9, 10) and glutaminase-1 (8) possess a KEN-box and are rapidly degraded in nonneoplastic lymphocytes during the cell cycle when APC/C-Cdh1 is active. Consistent with destruction being mediated by APC-C-Cdh1, ablation of the KEN-box prevents degradation of PFKFB3 (9, 10) and glutaminase-1 (8). Inhibiting the proteasomal-dependent degradation with the MG132 inhibitor

markedly increases levels of ubiquitinated PFKFB3 and glutaminase-1 (8). Moreover, overexpression of Cdh1 to activate APC/C-Cdh1 decreases levels of PFKFB3 as well as glutmaninase-1 and concomitantly inhibited glycolysis, as judged by decrease in lactate production. This effect is also observed when cells were treated with a glutaminase-1 inhibitor (6-diazo-5- oxo-L-norleucine) (8). The final evidence supporting the authors’ hypothesis is that proliferation and glycolysis is inhibited after shRNA-mediated silencing of either PFKFB3 or glutaminase-1 (8).

These results are interesting, because unlike most recent work in this area, Colombo et al. (8) link the Warburg effect to the machinery of the cell cycle that is present in all cells rather than to cancer driving mutations. Further work is required to properly define the overall importance of this pathway, which has thus far only been studied in a limited number of cells. It would also be of value to undertake a more detailed analysis of how the rate of glycolysis and other metabolic pathways vary during the cell cycle of normal and cancer cells…(see full 2 page article) at PNAS.

 

The Warburg Effect Suppresses Oxidative Stress Induced Apoptosis in a Yeast Model for Cancer

C Ruckenstuhl, S Buttner, D Carmona-Gutierre, et al.
PLoS ONE 2009; 4(2): e4592.  http://dx.doi.org/10.1371/journal.pone.0004592

Colonies of Saccharomyces cerevisiae, suitable for manipulation of mitochondrial respiration and shows mitochondria-mediated cell death, were used as a model. Repression of respiration as well as ROS-scavenging via glutathione inhibited apoptosis, conferred a survival advantage during seeding and early development of this fast proliferating solid cell population. In contrast, enhancement of respiration triggered cell death.

Conclusion/Significance: 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.

 

PIM2 phosphorylates PKM2 and promotes Glycolysis in Cancer Cells
Z Yu, L Huang, T Zhang, et al.
J Biol Chem 2013;                               http://dx.doi.org/10.1074/jbc.M113.508226

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.508226

Serine/threonine protein kinase PIM2, a known oncogene is a binding partner of pyruvate kinase M2 (PKM2), a key player in the Warburg effect of cancer cells.   PIM2 interacts with PKM2 and phosphorylates PKM2 on the Thr454 residue.

The phosphorylation of PKM2 increases glycolysis and proliferation in cancer cells.

The PIM2-dependent phosphoirylation of ZPKM2 is critical for regulating the Warburg effect in cancer.

 

Genome-Scale Metabolic Modeling Elucidates the Role of Proliferative Adaptation in Causing the Warburg Effect

Shlomi T, Benyamini T, Gottlieb E, Sharan R, Ruppin E
PLoS Comput Biol 2011; 7(3): e1002018.    http://dx.doi.org/10.1371/journal.pcbi.1002018
The Warburg effect – a classical hallmark of cancer metabolism – is a counter-intuitive phenomenon in which rapidly proliferating cancer cells resort to inefficient ATP production via glycolysis leading to lactate secretion, instead of relying primarily on more efficient energy production through mitochondrial oxidative phosphorylation, as most normal cells do.

The causes for the Warburg effect have remained a subject of considerable controversy since its discovery over 80 years ago, with several competing hypotheses. Here, utilizing a genome-scale human metabolic network model accounting for stoichiometric and enzyme solvent capacity considerations, we show that the Warburg effect is a direct consequence of the metabolic adaptation of cancer cells to increase biomass production rate. The analysis is shown to accurately capture a three phase metabolic behavior that is observed experimentally during oncogenic progression, as well as a prominent characteristic of cancer cells involving their preference for glutamine uptake over other amino acids.

 

The metabolic advantage of tumor cells

Maurice Israël and Laurent Schwartz

Additional article information

Abstract

1- Oncogenes express proteins of “Tyrosine kinase receptor pathways”, a receptor family including insulin or IGF-Growth Hormone receptors. Other oncogenes alter the PP2A phosphatase brake over these kinases.

2- Experiments on pancreatectomized animals; treated with pure insulin or total pancreatic extracts, showed that choline in the extract, preserved them from hepatomas.

Since choline is a methyle donor, and since methylation regulates PP2A, the choline protection may result from PP2A methylation, which then attenuates kinases.

3- Moreover, kinases activated by the boosted signaling pathway inactivate pyruvate kinase and pyruvate dehydrogenase. In addition, demethylated PP2A would no longer dephosphorylate these enzymes. A “bottleneck” between glycolysis and the oxidative-citrate cycle interrupts the glycolytic pyruvate supply now provided via proteolysis and alanine transamination. This pyruvate forms lactate (Warburg effect) and NAD+ for glycolysis. Lipolysis and fatty acids provide acetyl CoA; the citrate condensation increases, unusual oxaloacetate sources are available. ATP citrate lyase follows, supporting aberrant transaminations with glutaminolysis and tumor lipogenesis. Truncated urea cycles, increased polyamine synthesis, consume the methyl donor SAM favoring carcinogenesis.

4- The decrease of butyrate, a histone deacetylase inhibitor, elicits epigenic changes (PETEN, P53, IGFBP decrease; hexokinase, fetal-genes-M2, increase)

5- IGFBP stops binding the IGF – IGFR complex, it is perhaps no longer inherited by a single mitotic daughter cell; leading to two daughter cells with a mitotic capability.

6- An excess of IGF induces a decrease of the major histocompatibility complex MHC1, Natural killer lymphocytes should eliminate such cells that start the tumor, unless the fever prostaglandin PGE2 or inflammation, inhibit them…

Introduction

The metabolic network of biochemical pathways forms a system controlled by a few switches, changing the finality of this system. Specific substrates and hormones control such switches. If for example, glycemia is elevated, the pancreas releases insulin, activating anabolism and oxidative glycolysis, energy being required to form new substance or refill stores. If starvation decreases glycemia, glucagon and epinephrine activate gluconeogenesis and ketogenesis to form nutriments, mobilizing body stores. The different finalities of the system are or oriented by switches sensing the NADH/NAD+, the ATP/AMP, the cAMP/AMP ratios or the O2 supply… We will not describe here these metabolic finalities and their controls found in biochemistry books.

Many of the switches depend of the phosphorylation of key enzymes that are active or not. Evidently, there is some coordination closing or opening the different pathways. Take for example gluconeogenesis, the citrate condensation slows down, sparing OAA, which starts the gluconeogenic pathway. In parallel, one also has to close pyruvate kinase (PK); if not, phosphoenolpyruvate would give back pyruvate, interrupting the pathway. Hence, the properties of key enzymes acting like switches on the pathway specify the finality of the system. Our aim is to show that tumor cells invent a new specific finality, with mixed glycolysis and gluconeogenesis features. This very special metabolism gives to tumor cells a selective advantage over normal cells, helping the tumor to develop at the detriment of the rest of the body.

I Abnormal metabolism of tumors, a selective advantage

The initial observation of Warburg 1956 on tumor glycolysis with lactate production is still a crucial observation [1]. Two fundamental findings complete the metabolic picture: the discovery of the M2 pyruvate kinase (PK) typical of tumors [2] and the implication of tyrosine kinase signals and subsequent phosphorylations in the M2 PK blockade [35].

A typical feature of tumor cells is a glycolysis associated to an inhibition of apoptosis. Tumors over-express 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 [6,7]. 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 [8]. 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 bis phosphate; 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 phosphoenolpyruvate (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.

Well, tumors have a PK and a PDH 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.

We also know that starvation mobilizes lipid stores from adipocyte to form ketone bodies, they are like glucose, nutriments for cells. Growth hormone, cAMP, AMP, activate a lipase, which provides fatty acids; their β oxidation cuts them into acetyl CoA in mitochondria and in peroxisomes for very long fatty acids; forming ketone bodies. Normally, citrate synthase slows down, to spare acetyl CoA for the ketogenic route, and OAA for the gluconeogenic pathway. Like for starvation, tumors mobilize lipid stores. But here, citrate synthase activity is elevated, condensing acetyl CoA and OAA [1113]; citrate increases, ketone bodies decrease. Consequently, ketone bodies will stop stimulating Pcarb. In tumors, the OAA needed for citrate synthase will presumably come from PEP, via reversible PEP carboxykinase or other sources. The quiescent Pcarb will not process the pyruvate produced by alanine transamination after proteolysis, leaving even more pyruvate to lactate dehydrogenase, increasing the lactate released by the tumor, and the NAD+ required for 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 [14] 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; since PDH that might have provided acetyl CoA remains in tumors, like PK, in the inactive phosphorylated form. The blockade of PDH [15] was recently reversed by inhibiting its kinase [16,17].

The key question is then to find out why NADH, a natural citrate synthase inhibitor did not switch off the enzyme in tumor cells. Probably, the synthesis of NADH by the dehydrogenases of the Krebs cycle and malate/aspartate shuttle, was too low, or the oxidation of NADH via the respiratory electron transport chain and mitochondrial complex1 (NADH dehydrogenase) was abnormally elevated. Another important point concerns PDH and α ketoglutarate dehydrogenase that are homologous enzymes, they might be regulated in a concerted way; when PDH is off, α ketoglutarate dehydrogenase might be also be slowed. Moreover, this could be associated to an upstream inhibition of aconinase by NO, or more probably to a blockade of isocitrate dehydrogenase, which favors in tumor cells, the citrate efflux from mitochondria, and the ATP citrate lyase route.

Normally, an increase of NADH inhibits the citrate condensation, favoring the ketogenic route associated to gluconeogenesis, which turns off glycolysis. Apparently, this regulation does not occur in tumors, since citrate synthase remains active. Moreover, in tumor cells, the α ketoglutarate not processed by
α ketoglutarate dehydrogenase converts to glutamate, via glutamate dehydrogenase, in this direction the reaction forms NAD+, backing up the LDH production. Other sources of glutamate are glutaminolysis, which increases in tumors [2].

The Figure Figure11 shows how tumors bypass the PK and PDH bottlenecks and evidently, the increase of glucose influx above the bottleneck, favors the supply of substrates to the pentose shunt, as pentose is needed for synthesizing ribonucleotides, RNA and DNA. The Figure Figure11 represents the stop below the citrate condensation. Hence, citrate quits the mitochondria to give via ATP citrate lyase, acetyl CoA and OAA in the cytosol of tumor cells. Acetyl CoA supports the synthesis of fatty acids and the formation of triglycerides. The other product of the ATP citrate lyase reaction, OAA, drives the transaminase cascade (ALAT and GOT transaminases) in a direction that consumes GLU and glutamine and converts in fine alanine into pyruvate and lactate plus NAD+. This consumes protein body stores that provide amino acids and much alanine (like in starvation).

The Figure Figure11 indicates that malate dehydrogenase is a source of NAD+ converting OAA into malate, which backs-up LDH. Part of the malate converts to pyruvate (malic enzyme) and processed by LDH. Moreover, malate enters in mitochondria via the shuttle and gives back OAA to feed the citrate condensation. Glutamine will also provide amino groups for the “de novo” synthesis of purine and pyrimidine bases particularly needed by tumor cells. The Figure Figure11 indicates that ASP shuttled out of the mitochondrial, joins the ASP formed by cytosolic transaminases, to feed the synthesis of pyrimidine bases via ASP transcarbamylase, a process also enhanced in tumor cells. In tumors, this silences the argininosuccinate synthetase step of the urea cycle [1820].

This blockade also limits the supply of fumarate to the Krebs cycle. The latter, utilizes the α ketoglutarate provided by the transaminase reaction, since α ketoglutarate coming via aconitase slows down. Indeed, NO and peroxynitrite increase in tumors and probably block aconitase. The Figure Figure11 indicates the cleavage of arginine into urea and ornithine. In tumors, the ornithine production increases, following the polyamine pathway. Ornithine is decarboxylated into putrescine by ornithine decarboxylase, then it captures the backbone of S adenosyl methionine (SAM) to form polyamines spermine then spermidine, the enzyme controlling the process is SAM decarboxylase. The other reaction product, 5-methlthioribose is then decomposed into methylthioribose and adenine, providing purine bases to the tumor. We shall analyze below the role of SAM in the carcinogenic mechanism, its destruction aggravates the process.

metabolic pathways 1476-4598-10-70-1
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,

In summary, it is like 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! Tumor cell metabolism gives them a selective advantage over normal cells. However, one may attack some vulnerable points.

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 β-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, ATPcitrate 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”. (Black arrows = interrupted pathways).

 II Starters for cancer metabolic anomaly

1. Lessons from oncogenes

Following the discovery of Rous sarcoma virus transmitting cancer [21], we have to wait the work of Stehelin [22] to realize that this retrovirus only transmitted a gene captured from a previous host. When one finds that the transmitted gene encodes the Src tyrosine kinase, we are back again to the tyrosine kinase signals, similar to those activated by insulin or IGF, which control carbohydrate metabolism, anabolism and mitosis.

An up regulation of the gene product, now under viral control causes tumors. However, the captured viral oncogene (v-oncogene) derives from a normal host gene the proto-oncogene. The virus only perturbs the expression of a cellular gene the proto-oncogene. It may modify its expression, or its regulation, or transmit a mutated form of the proto-oncogene. Independently of any viral infection, a similar tumorigenic process takes place, if the proto-oncogene is translocated in another chromosome; and transcribed under the control of stronger promoters. In this case, the proto-oncogene becomes an oncogene of cellular origin (c-oncogene). The third mode for converting a prot-oncogene into an oncogene occurs if a retrovirus simply inserts its strong promoters in front of the proto-oncogene enhancing its expression.

It is impressive to find that retroviral oncogenes and cellular oncogenes disturb this major signaling pathway: the MAP kinases mitogenic pathways. At the ligand level we find tumors such Wilm’s kidney cancer, resulting from an increased expression of insulin like growth factor; we have also the erbB or V-int-2 oncogenes expressing respectively NGF and FGF growth factor receptors. The receptors for these ligands activate tyrosine kinase signals, similarly to insulin receptors. The Rous sarcoma virus transmits the src tyrosine kinase, which activates these signals, leading to a chicken leukemia. Similarly, in murine leukemia, a virus captures and retransmits the tyrosine kinase abl. Moreover, abl is also stimulated if translocated and expressed with the bcr gene of chromosome 22, as a fusion protein (Philadelphia chromosome). Further, ahead Ras exchanging protein for GTP/GDP, and then the Raf serine-threonine kinases proto-oncogenes are known targets for oncogenes. Finally, at the level of transcription factors activated by MAP kinases, one finds cjun, cfos or cmyc. An avian leucosis virus stimulates cmyc, by inserting its strong viral promoter. The retroviral attacks boost the mitogenic MAP kinases similarly to inflammatory cytokins, or to insulin signals, that control glucose transport and gycolysis.

In addition to the MAP kinase mitogenic pathway, tyrosine kinase receptors activate PI3 kinase pathways; PTEN phosphatase counteracts this effect, thus acting as a tumor suppressor. Recall that a DNA virus, the Epstein-Barr virus of infectious mononucleose, gives also the Burkitt lymphoma; the effect of the virus is to enhance PI3 kinase. Down stream, we find mTOR (the target of rapamycine, an immune-suppressor) mTOR, inhibits PP2A phosphatase, which is also a target for the simian SV40 and Polyoma viruses. Schematically, one may consider that the different steps of MAP kinase pathways are targets for retroviruses, while the different steps of PI3 kinase pathway are targets for DNA viruses. The viral-driven enhanced function of these pathways mimics the effects of their prolonged activation by their usual triggers, such as insulin or IGF; one then expects to find an associated increase of glycolysis. The 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 [35] 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.

2. The methylation hypothesis and the role of PP2A phosphatase

In a remarkable comment, Newberne [23] highlights interesting observations on the carcinogenicity of diethanolamine [24] showing that diethanolamine decreased choline derivatives and methyl donors in the liver, like does 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 [25]. 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 must now find how the lipotropic methyl donor controls the signaling pathway. 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 in works on Alzheimer’s disease [26]. Indeed, the catalytic C subunit of PP2A is associated to a structural subunit A. When C receives a methyle, the dimer recruits a regulatory subunit B. The trimer then targets specific proteins that are dephosphorylated [27].

In Alzheimer’s disease, the poor methylation of PP2A is associated to an increase of homocysteine in the blood [26]. The result of the PP2A methylation failure is a hyperphosphorylation of Tau protein and the formation of tangles in the brain. Tau protein is involved in tubulin polymerization, controlling axonal flow but also the mitotic spindle. It is thus possible that 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 [28].

3. Cellular distribution of PP2A

In fact, 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 [29]. The targeting of PP2A towards proteins of the signaling pathway depends of the assembly of the different holoenzymes. The carboxyl methylation of C-terminal leucine 309 of the catalytic C unit, permits to a dimeric form made of C and a structural unit A, to recruit one of the many regulatory units B, giving a great diversity of possible enzymes and effects. The different methylated ABC trimers would then find specific targets. It is consequently essential to have more information on methyl transferases and methyl esterases that control the assembly or disassembly of PP2A trimeric forms.

A specific carboxyl methyltransferase for PP2A [30] was purified and shown to be essential for normal progression through mitosis [31]. In addition, a specific methylesterase that demethylates PP2A has been purified [32]. Is seems that the methyl esterase cancels the action of PP2A, on signaling kinases that increase in glioma [33]. Evidently, the cellular localization of the methyl transferase (LCMT-1) and the phosphatase methyl esterase (PME-1) are crucial for controlling PP2A methylation and targeting. Apparently, LCMT-1 mainly localizes to the cytoplasm and not in the nucleus, where PME-1 is present, and the latter harbors a nuclear localization signal [34]. From these observations, one may suggest that PP2A gets its methyles in the cytoplasm and regulates the tyrosine kinase-signaling pathway, attenuating its effects.

A methylation deficit should then decrease the methylation of PP2A and boost the mitotic insulin signals as discussed above for choline deficiency, steatosis and hepatoma. At the nucleus, where PME-1 is present, it will remove the methyl, from PP2A, favoring the formation of dimeric AC species that have different targets, presumably proteins involved in the cell cycle. It is interesting to quote here the structural mechanism associated to the demethylation of PP2A. The crystal structures of PME-1 alone or in complex with PP2A dimeric core was reported [35] PME-1 binds directly to the active site of PP2A and this rearranges the catalytic triad of PME-1 into an active conformation that should demethylate PP2A, but this also seems to evict a manganese required for the phosphatase activity. Hence, demethylation and inactivation would take place in parallel, blocking mitotic actions.

However, another player is here involved, the so-called PTPA protein, which is a PP2A phosphatase activator. Apparently, this activator is a new type of cis/trans of prolyl isomerase, acting on Pro190 of the catalytic C unit isomerized in presence of Mg-ATP [36], which would then cancel the inactivation mediated by PME-1. Following the PTPA action, the demethylated phosphatase would become active again in the nucleus, and stimulate cell cycle proteins [37,38] inducing mitosis. Unfortunately, the ligand of this new prolyl isomerase is still unknown. Moreover, we have to consider that other enzymes such as cytochrome P450 have also demethylation properties.

In spite of deficient methylations and choline dehydrogenase pathway, tumor cells display an enhanced choline kinase activity, associated to a parallel synthesis of lecithin and triglycerides.

The hypothesis to consider is that triglycerides change the fate of methylated PP2A, by targeting it to the nucleus, there a methylesterase demethylates it; the phosphatase attacks new targets such as cell cycle proteins, inducing mitosis. Moreover, the phosphatase action on nuclear membrane proteins may render the nuclear membrane permeable to SAM the general methyl donor; promoters get methylated inducing epigenetic changes.

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.

Hypoxia is an essential issue to discuss

Many adequate “adult proteins” replace their fetal isoform: muscle proteins utrophine, switches to dystrophine; enzymes such as embryonic M2 PK [39] is replaced by M1. 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 deacetylase (HDAC) inhibition or of histone acetyltransferase (HAT) activation, and represents the main switch. Second, a more specific switch must be open, indicating the adult/fetal gene couple concerned, or more generally the isoform of a given gene that is more adapted to the specific situation. When the adult gene mutates, an unbound ligand may indeed indicate, directly or indirectly, the particular fetal copy gene to reactivate [40]. In anoxia, lactate is more difficult to release against its external gradient, leading to a cytosolic increase of up-stream glycolytic products, 3P glycerate or others. These products may then be a second signal controlling the specific switch for triggering the expression of fetal genes, such as fetal hemoglobin or the embryonic M2 PK; this takes place if histones (main switch) are in an acetylated form.

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 β 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, but since citrate synthase activity is elevated in tumors, ketone bodies do not form.

Compounds for correcting tumor metabolism

The figure figure1 indicates interrupted and enhanced metabolic pathways in tumor cells.

In table table1,1, the numbered pathways represent possible therapeutic targets; they cover several enzymes. When the activity of the pathway is increased, one may give inhibitors; when the activity of the pathway decreases, we propose possible activators

Table - metabolic  targets

Table 1 Mol Cancer. 2011; 10 70. Published online Jun 7, 2011. doi  10.1186_1476-4598-10-70

The origin of Cancers by means of metabolic selection

The disruption of cells by internal or external compounds, releases substrates stimulating the tyrosine kinase signals for anabolism proliferation and stem cell repair, like for most oncogenes. If such signals are not limited, there is a parallel blockade of key metabolic enzymes by activated kinases or inhibited phosphatases. The result is a metabolism typical of tumor cells, which gives them a selective advantage; stabilized by epigenetic changes. A proliferation process, in which the two daughter cells divide, increases the tumor mass at the detriment of the body. Inevitable mutations follow.

Maurice Israël, et al. Mol Cancer. 2011;10:70-70.
Transcriptomics and Regulatory Processes

What are lncRNAs?

It was traditionally thought that the transcriptome would be mostly comprised of mRNAs, however advances in high-throughput RNA sequencing technologies have revealed the complexity of our genome. Non-coding RNA is now known to make up the majority of transcribed RNAs and in addition to those that carry out well-known housekeeping functions (e.g. tRNA, rRNA etc), many different types of regulatory RNAs have been and continue to be discovered.

Long noncoding RNAs (lncRNAs) are a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins. Their expression is developmentally regulated and lncRNAs can be tissue- and cell-type specific. A significant proportion of lncRNAs are located exclusively in the nucleus. They are comprised of many types of transcripts that can structurally resemble mRNAs, and are sometimes transcribed as whole or partial antisense transcripts to coding genes. LncRNAs are thought to carry out important regulatory functions, adding yet another layer of complexity to our understanding of genomic regulation.

lncRNA-s   A summary of the various functions described for lncRNA

 

The evolution of genome-scale models of cancer metabolism
The importance of metabolism in cancer is becoming increasingly apparent with the identification of metabolic enzyme mutations and the growing awareness of the influence of metabolism on signaling, epigenetic markers, and transcription. However, the complexity of these processes has challenged our ability to make sense of the metabolic changes in cancer. Fortunately, constraint-based modeling, a systems biology approach, now enables one to study the entirety of cancer metabolism and simulate basic phenotypes. With the newness of this field, there has been a rapid evolution of both the scope of these models and their applications. (NE Lewis and AM.Abdel-Haleem. frontiers physiol  2013;4(237): 1   http://dx.doi.org/10.3389/fphys.2013.00237)

Here we review the various constraint-based models built for cancer metabolism and how their predictions are shedding new light on basic cancer phenotypes, elucidating pathway differences between tumors, and discovering putative anti-cancer targets. As the field continues to evolve, the scope of these genome-scale cancer models must expand beyond central metabolism to address questions related to the diverse processes contributing to tumor development and metastasis.

“One of the goals of cancer research is to ascertain the mechanisms of cancer.”These words, penned by Dulbecco (1986), began a treatise on how a mechanistic understanding of cancer requires a sequenced human genome. Now with the abundance of sequence data, we are finding diverse genetic changes among different cancers (Vogelstein et al.,2013). While we are cataloging these mutations, the associated mechanisms leading to phenotypic changes are often unclear since mutations occur in the context of complex biological networks. For example, mutations to isocitrate dehydrogenase lead to oncometabolite synthesis, which alters DNA methylation and ultimately changes gene expression and the balance of normal cell processes (Sasakietal.,2012). Furthermore, many different combinations of mutations can lead to cancer. Since the genetic heterogeneity between tumors can be large, the biomolecular mechanisms underlying tumor physiology can vary substantially.

This is apparent in metabolism, where tumors can differ in serine metabolism  dependence (Possematoetal., 2011) or TCA cycle function (Frezzaetal., 2011b). In addition, diverse mutations can alter NADPH synthesis by differentially regulat ing  signaling pathways, such as the AMPK pathway (Cairnsetal., 2011; Jeonetal., 2012). The challenges regarding complexity and heterogeneity in cancer metabolism are beginning to be addressed with the COnstraint-Based Reconstruction and Analysis (COBRA) approach (Hernández Patiñoetal., 2012; Sharma and König,  2013), an emerging field in systems biology.Specifically, it accounts for the complexity of the perturbed biochemical processes by using genome-scale metabolic network reconstructions (Duarteetal., 2007; Maetal., 2007;Thieleetal., 2013).

In a reconstruction, the stoichiometric chemical reactions in a cell are carefully annotated and stitched together into a large network, often containing thousands of reactions. Genes and enzymes associated with each reaction are also delineated. The networks are converted into computational models and analyzed using many algorithms (Lewisetal., 2012). COBRA approaches are also beginning to address heterogeneity in cancer by integrating experimental data with the reconstructions (Blazier and Papin, 2012; Hydukeetal., 2013)  to tailor the models to the unique gene expression profiles of general cancer tissue, and even individual cell lines and tumors. Here we describe the recent conceptual evolution that has occurred for constraint-based cancer modeling.

Targeting of  gene expression

Tumor Suppressor Genes and its Implications in Human Cancer

Gain-of-function mutations in oncogenes and loss-of-function mutations in tumor suppressor genes (TSG) lead to cancer. In most human cancers, these mutations occur in somatic tissues. However, hereditary forms of cancer exist for which individuals are heterozygous for a germline mutation in a TSG locus at birth. The second allele is frequently inactivated by gene deletion, point mutation, or promoter methylation in classical TSGs that meet Knudson’s two-hit hypothesis. Conversely, the second allele remains as wild-type, even in tumors in which the gene is haplo-insufficient for tumor suppression. (K Inoue, EA Fry and Pj Taneja. Recent Progress in Mouse Models for Tumor Suppressor Genes and its Implications in Human Cancer. Clinical Medicine Insights: Oncology2013:7 103–122). This article highlights the importance of PTEN, APC, and other tumor suppressors for counteracting aberrant PI3K, β-catenin, and other oncogenic signaling pathways. We discuss the use of gene-engineered mouse models (GEMM) of human cancer focusing on Pten and Apc knockout mice that recapitulate key genetic events involved in initiation and progression of human neoplasia.

Targeting cancer metabolism – aiming at a tumour’s sweet-spot
Neil P. Jones and Almut Schulze
Drug Discovery Today   January 2012

Targeting cancer metabolism has emerged as a hot topic for drug discovery. Most cancers have a high demand for metabolic inputs (i.e. glucose/glutamine), which aid proliferation and survival. Interest in targeting cancer metabolism has been renewed in recent years with the discovery that many cancer related (e.g. oncogenic and tumor suppressor) pathways have a profound effect on metabolism and that many tumors become dependent on specific metabolic processes. Considering the recent increase in our understanding of cancer metabolism and the increasing knowledge of the enzymes and pathways involved, the question arises: could metabolism be cancer’s Achilles heel?
During recent years, interest into the possible therapeutic benefit of targeting metabolic pathways in cancer has increased dramatically with academic and pharmaceutical groups actively pursuing this aspect of tumor physiology. Therefore, what has fuelled this revived interest in targeting cancer metabolism and what are the major advances and potential challenges faced in the race to develop new therapeutics in this area? This review will attempt to answer these questions and illustrate why we, and others, believe that targeting metabolism in cancer presents such a promising therapeutic rationale.

Oncogenes and cancer metabolism
Glycolysis  TCA cycle  Pentose phosphate pathway

 FIGURE 1

Schematic representation of the regulation of cancer metabolism pathways. Metabolic enzymes are regulated by signaling pathways involving oncogenes and tumor suppressors. Complex regulatory mechanisms, key pathway interactions and enzymes are shown along with key metabolic endpoints (shown in purple) necessary for proliferation and survival (biosynthetic intermediates and NADPH). Key oncogenic pathways are shown in green and key tumor suppressor pathways are shown in red. Mutant IDH (mIDH) pathway is listed but is only functional in cancers containing mIDH.

FIGURE 2

Schematic representation of key components of the pentose phosphate pathway (PPP). Key enzymes are shown in blue boxes and key intermediates in purple text/box outline. DNA damage can activate ATM which in turn activates G6PDH to upregulate nucleotide synthesis for DNA repair and NAPDH to combat reactive oxygen species. PPP is also regulated by the tumour suppressor p53. The PPP can function as two separate branches (oxidative and non-oxidative) or be coupled into a recycling pathway – the pentose phosphate shunt – for maximum NADPH production.

Serine biosynthesis

Another branch diverting from glycolysis recently implicated in cancer is the serine biosynthesis pathway which converts the glycolytic intermediate 3-phosphoglycerate into serine (Fig. 3). Serine is an amino acid and an important neurotransmitter but can also provide fuel for the synthesis of other amino acids and nucleotides. The serine biosynthesis pathway also provides another key metabolic intermediate, a-KG, from glutamate breakdown via the action of phosphoserine aminotransferase (PSAT1). This pathway couples glycolysis (via 3-phosphoglycerate) with glutaminolysis (via glutamate), thereby linking two metabolic pathways known to be activated in many cancers.

FIGURE 3

Schematic representation of the serine biosynthesis pathway. Synthesis of serine involves integration of metabolites from glycolysis and  glutaminolysis pathways  and generates a-ketoglutarate, a key biosynthetic intermediate, and serine. Serine has many essential uses in the cell including amino acid, phospholipid and nucleotide synthesis.

 

Silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1)

Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) contributes to silencing of tumor suppressorgenes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely,demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action.  (Mayada Achour, et al. Epigallocatechin-3-gallate up-regulates tumor suppressor gene expression via a reactive oxygen species-dependent down-regulation of UHRF1.  Biochemical and Biophysical Research Communications 430 (2013) 208–212.    http://dx.doi.org/10.1016/j.bbrc.2012.11.087)

Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16INK4A genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16INK4A  is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16INK4A and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16INK4A and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.

Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant

ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and –active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored.  (Citation: Richard M. Gallo, et al. (2013) Multiple Functional Motifs Are Required for the Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant. J Cancer Res Therap Oncol 1: 1-10)

Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavage by gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.

EGF Receptor

 Initiation of pancreatic ductal adenocarcinoma (PDA) is definitively linked to activating mutations in the KRAS oncogene. However, PDA mouse models show that mutant Kras expression early in development gives rise to a normal pancreas, with tumors forming only after a long latency or pancreatitis induction.

(CM Ardito,BM Gruner. ,EGF Receptor Is Required for KRAS-Induced Pancreatic Tumorigenesis.  http://dx.doi.org/10.1016/j.ccr.2012.07.024)

Here, we show that oncogenic KRAS upregulates endogenous EGFR expression and activation, the latter being dependent on the EGFR ligand sheddase, ADAM17. Genetic ablation or pharmacological inhibition of EGFR or ADAM17 effectively eliminates KRAS-driven tumorigenesis in vivo. Without EGFR activity, active RAS levels are not sufficient to induce robust MEK/ERK activity, a requirement for epithelial transformation

The almost universal lethality of PDA has led to the intense study of genetic mutations responsible for its formation and progression. The most common oncogenic mutations associated with all PDA stages are found in the KRAS gene, suggesting it as the primary initiator of pancreatic neoplasia. However, mutant Kras expression throughout the mouse pancreatic parenchyma shows that the oncogene remains largely indolent until secondary events, such as pancreatitis, unlock its transforming potential. We find KRAS requires an inside-outside-in signaling axis that involves ligand-dependent EGFR activation to initiate the signal transduction and cell biological changes that link PDA and pancreatitis. (Cancer Cell (2012); 22: 304–317).

HER4 (EGFR/ErbB, HER2/Neu, HER3)

 ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and –active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored.

ErbB4 Possesses Multiple Functional Motifs and Mutations Have Been Engineered to Target These Motifs.

The organization of ErbB4 is as indicated in this schematic. The extracellular ligand-binding motifs reside in the amino-terminal region upstream of amino acid residue 651. The singlepass transmembrane domain consists of amino acid residues 652-675. The cytoplasmic tyrosine kinase domain consists of amino acid residues 713-989. The majority of cytoplasmic sites of tyrosine phosphorylation reside in amino acid residues 990-1308, most notably Tyr1056. Additional putative functional motifs include a TACE cleavage site, a gamma-secretase cleavage site, two LXXLL (steroid hormone receptor binding) motifs, a BH3 domain, three WW domain binding motifs, and a PDZ domain binding motif. Mutations that disrupt these motifs are noted. Finally, note the two locations of alternative transcriptional splicing, resulting in a total of four different splicing isoforms.

 

 

 

Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavageby gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.

(Richard M. Gallo, et al. (2013) Multiple Functional Motifs Are Required for the Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant. J Cancer Res Therap Oncol 1: 1-10)

Resistance to Receptor Tyrosine Kinase Inhibition

Receptor tyrosine kinases (RTKs) are activated by somatic genetic alterations in a subset of cancers, and such cancers are often sensitive to specific inhibitors of the activated kinase. Two well-established examples of this paradigm include lung cancers with either EGFR mutations or ALK translocations. In these cancers, inhibition of the corresponding RTK leads to suppression of key downstream signaling pathways, such as the PI3K (phosphatidylinositol 3-kinase)/AKT and MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal–regulated kinase) pathways, resulting in cell growth arrest and death. Despite the initial clinical efficacy of ALK (anaplastic lymphoma kinase) and EGFR (epidermal growth factor receptor) inhibitors in these cancers, resistance invariably develops, typically within 1 to 2 years. (MJ Niederst and JA Engelman. Sci Signal, 24 Sep 2013; 6(294), p. re6 .  http://dx.doi.org/10.1126/scisignal.2004652)

Over the past several years, multiple molecular mechanisms of resistance have been identified, and some common themes have emerged. One is the development of resistance mutations in the drug target that prevent the drug from effectively inhibiting the respective RTK. A second is activation of alternative RTKs that maintain the signaling of key downstream pathways despite sustained inhibition of the original drug target. Indeed, several different RTKs have been implicated in promoting resistance to EGFR and ALK inhibitors in both laboratory studies and patient samples. In this mini-review, we summarize the concepts underlying RTK-mediated resistance, the specific examples known to date, and the challenges of applying this knowledge to develop improved therapeutic strategies to prevent or overcome resistance.

The TGF-β Pathway

Aberrations in the enzymes that modify ubiquitin moieties have been observed to cause a myriad of diseases, including cancer. Therefore a better understanding of these enzymes and their substrates will lead to the identification of prospective druggable targets. Here we discuss the role of ubiquitin modifying enzymes in the canonical TGF-β pathway highlighting the ubiquitin regulating enzymes, which may potentially be targeted by small molecule inhibitors. (Pieter Eichhorn. (DE) -Ubiquitination in The TGF-β Pathway. J Cancer Res Therap Oncol 2013; 1: 1-6).

TGF-β is a multifunctional cytokine that plays a key role in embryogenesis and adult tissue homoeostasis. TGF-β is secreted by a myriad of cell types triggering a varied array of cellular functions including apoptosis, proliferation, migration, endothelial and mesenchymal transition, and extracellular matrix production. Downstream TGFβ responses can also be modulated by other signalling pathways (i.e. PI3K, ERK, WNT, etc.) resulting in a complex web of TGF-β pathway activation or repression depending on the nature of the signal and cellular context. Apart from TGF-β mediated cell autonomous effects TGF-β can further play an important function in regulating tumour microenvironments effecting the interaction between stromal fibroblasts and tumour cells.
Due to the central role of TGF-β in cellular processes it is therefore unsurprising that loss of TGF-β pathway integrity is frequently observed in a variety of human diseases, including cancer. However, the TGF-β pathway plays a complex dual role in cancer. In normal epithelial cells and premalignant cells TGF-β acts a potent tumor suppressor eliciting a cytostatic response inhibiting tumor progression. Supporting this notion, inactivating mutations in members of the TGF-βpathway have been observed in a variety of cancers including pancreatic, colorectal, and head and neck cancer.

In contrast, during tumor progression the TGF-β antiproliferative function is lost, and in certain advanced cancers TGF-β becomes an oncogenic factor inducing cellular proliferation, invasion, angiogenesis, and immune suppression. As a consequence, the TGFβ pathway is currently considered a therapeutic target in advanced cancers and several anti- TGF-β agents in clinical trials have shown promising results. However, due to the complex dichotomous role of TGF-β in oncogenesis a detailed understanding of TGF-β biology is required in order to design successful therapeutic strategies to identify patient populations that will benefit most from these compounds.

G protein receptor

 G protein-coupled receptors (GPCRs) modulate a vast array of cellular processes. The current review gives an overview of the general characteristics of GPCRs and their role in physiological conditions. In addition, it describes the current knowledge of the physiological and pathophysiological functions of GPR55, an orphan GPCR, and how it can be exploited as a therapeutic target to combat various cancers.

(D Leyva-Illades, S DeMorrow . Orphan G protein receptor GPR55 as an emerging target in cancer therapy and management.  Cancer Management and Research 2013:5 147–155)

Signal transduction is essential for maintaining cellular homeostasis and to coordinate the activity of cells in all organisms. Proteins localized in the cell membrane serve as the interface between the outside and inside of the cell. G protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and are encoded by at least 800 genes in the human genome. GPCRs are also known as seven-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors. GPCRs can detect an expansive array of extracellular signals or ligands that include photons, ions, odors, pheromones, hormones, and neurotransmitters. Nonsensory GPCRs (excluding light, odor, and taste receptors) have been classified into four families: class A rhodopsin-like, class B secretin-like, class C metabotropic glutamate/pheromone, and frizzled receptors. They have a peculiar structure that has been highly conserved over the course of evolution and are made up of an amino acid chain, the N-terminal of which is localized outside of the cellular membrane and the C-terminal in the cytoplasm. The amino acid chain spans the cellular membrane seven times and has three intracellular and three extracellular loops.

GPCRs are called that because they exert their actions by associating with a family of heterotrimeric proteins (made up of α, β, and γ subunits) that are capable of binding and hydrolyzing guanosine triphosphate (GTP).To date, 16 different α subunits, five β subunits, and 11 γ subunits have been described in mammalian tissues. When activated, these receptors undergo conformational changes that are mechanically transduced to the G proteins, which then initiate a cycle of activation and inactivationassociated with the binding and hydrolysis of GTP. Activated G proteins can then positively or negatively modulate ion channels (mainly potassium and calcium) or the second messenger generating enzymes (ie, adenylate cyclase and phospholipase C [PLC]) that allow the signal to be propagated to the interior of the cell to ultimately affect cell function.

 Matrix Metalloproteinases

Degradation of extracellular matrix is crucial for malignant tumour growth, invasion, metastasis and angiogenesis. Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases collectively capable of degrading essentially all  components of the ECM. Elevated levels of distinct MMPs can be detected in tumour tissue or serumof patients with advanced cancer and their role as prognostic indicators in cancer is studied. In addition, therapeutic intervention of tumour growth and invasion based on inhibition of MMP activity is under intensive investigation and several MMP inhibitors are in clinical trials in cancer. In this review, we discuss the current view on the feasibility of MMPs as prognostic markers and as targets for therapeutic intervention in cancer.

(MATRIX METALLOPROTEINASES IN CANCER: PROGNOSTIC MARKERS AND THERAPEUTIC TARGETS.

Pia Vihinen and Veli-Matti Kahari.  Int. J. Cancer 2002;99: 157–166. http://dx.doi.org/10.1002/ijc.10329

Common properties of the MMPs include the requirement of zinc in their catalytic site for activity and their synthesis as inactive zymogens that generally need to be proteolytically cleaved to be active. Normally the MMPs are expressed only when and where needed for tissue remodeling accompanies various processes such as during embryonic development, wound healing, uterine and mammary involution, cartilage-to-bone transition during ossification, and trophoblast invasion into the endometrial stoma during placenta development. However, aberrant expression of various MMPs has been correlated with pathological conditions, such as periodontitis, rheumatoid arthritis, and tumor cell invasion and metastasis .

There are now over 20 members of the MMP family, and they can be subgrouped based on their structures. The minimal domain structure consists of a signal peptide, prodomain, and catalytic domain. The propeptide domain contains a conserved cysteine residue (the “cysteine switch”) that coordinates to the catalytic zinc to maintain inactivity. MMPs with only the minimal domain are referred to as matrilysins (MMP-7 and -26). The most common structures for secreted MMPs, including collagenases and stromelysins, have an additional hemopexin-like domain connected by a hinge region to the catalytic domain (MMP-1, -3, -8, -10, -12, -13, -19, and -20).

Terms: 1FN, fibronectin; 2M, 2-macroglobulin; 1PI, 1-proteinase inhibitor; COMP, cartilage oligomeric matrix protein; ND, not determined; TACE, TNF-converting enzyme; OP, osteopontin

FIGURE 1 – Structure of human matrix metalloproteinases

 

FIGURE 1 – Structure of human matrix metalloproteinases. The signal peptide directs the proenzyme for secretion. The propeptide contains a conserved sequence (PRCGxPD), in which the cysteine forms a covalent bond (cysteine switch), with the catalytic zinc (Zn2_) to maintain the latency of proMMPs. Catalytic domain contains the highly conserved zinc binding site (HExGHxxGxxHS) in which Zn2_is coordinated by 3 histidines. The proline-rich hinge region links the catalytic domain to the hemopexin domain, which determines the substrate specificity of specific MMPs. The hemopexin domain is absent in matrilysin (MMP-7) and matrilysin-2 (endometase, MMP-26). Gelatinases  A and B (MMP-2 and MMP-9, respectively) contain 3 repeats of the fibronectin-type II domain inserted in the catalytic domain. MT1-, MT2-, MT3- and MT5-MMP contain a transmembrane domain and MT4- and MT6-MMPs contain a glycosylphosphatidylinositol (GPI) anchor in the C-terminus of the molecule, which attach these MMPs to the cell surface. MT-MMPs, MMP-11, MMP-23 and MMP-28 contain a furin cleavage site (RxKR) between the propeptide and catalytic domain, making these proenzymes susceptible to activation by intracellular furin convertases. MMP-23 contains an N-terminal signal anchor, which anchors proMMP-23 to the Golgi complex and has a different C-terminal domain instead of hemopexin-like domain.

The physiologic expression of MMP-13 in vivo is limited to situations, such as fetal bone development and fetal wound repair, in which rapid remodeling of collagenous ECM is required. MMP-13 is expressed in pathologic conditions, such as arthritis, chronic dermal and intestinal ulcers, chronic periodontal inflammation and atherosclerotic plaques. The expression of MMP-13 is detected in vivo in invasive malignant tumours, breast carcinomas, squamous cell carcinomas (SCCs) of the head and neck and vulva, malignant melanomas, chondrosarcomas and urinary bladder carcinomas.

Table I. Human MMPS, their chromosomal localization, substrates, exogenous activators, and activating capacity1
Enzyme Chromosomal location Substrates Activated by Activator of
  • FN, fibronectin; 2M, 2-macroglobulin; 1PI, 1-proteinase inhibitor; COMP, cartilage oligomeric matrix protein; ND, not determined; TACE, TNF-converting enzyme; OP, osteopontin.

    …………..

Collagenases
 Collagenase-1 (MMP-1) 11q22.2-22.3 Collagen I, II, III, VII, VIII, X, aggregan, serpins, 2M MMP-3, -7, -10, plasmin kallikrein, chymase MMP-2
 Collagenase-2 (MMP-8) 11q22.2-22.3 Collagen I, II, III, aggregan, serpins, 2M MMP-3, -10, plasmin ND
 Collagenase-3 (MMP-13) 11q22.2-22.3 Collagen I, II, III, IV, IX, X, XIV, gelatin, FN, laminin, large tenascin aggrecan, fibrillin, osteonectin, serpins MMP-2, -3, -10, -14, -15, plasmin MMP-2, -9
Stromelysins
 Stromelysin-1 (MMP-3) 11q22.2-22.3 Collagen IV, V, IX, X, FN, elastin, gelatin, laminin, aggrecan, nidoge fibrillin*, osteonectin*, 1PI*, myelin basic protein*, OP, E-cadherin Plasmin, kallikrein, chymas tryptase MMP-1, -8, -9, -13
 Stromelysin-2 (MMP-10) 11q22.2-3 As MMP-3, except * Elastase, cathepsin G MMP-1, -7, -8, -9, -13
Stromelysin-like MMPs
 Stromelysin-3 (MMP-11) 22q11.2 Serine proteinase inhibitors, 1PI Furin ND
 Metalloelastase (MMP-12) 11q22.2-22.3 Collagen IV, gelatin, FN, laminin, vitronectin, elastin, fibrillin, 1-PI, myelin basic protein, apolipoprotein A ND ND
Matrilysins
 Matrilysin (MMP-7) 11q22.2-22.3 Elastin, FN, laminin, nidogen, collagen IV, tenascin, versican, 1PI, O E-cadherin, TNF- MMP-3, plasmin MMP-9
 Matrilysin-2 (MMP-26) 11q22.2 Gelatin, 1PI, synthetic MMP-substrates, TACE-substrate ND ND
Gelatinases
 Gelatinase A (MMP-2) 16q13 Gelatin, collagen I, IV, V, VII, X, FN, tenascin, fibrillin, osteonectin, Monocyte chemoattractant protein 3 MMP-1, -13, -14, -15, -16, -tryptase? MMP-9, -13
 Gelatinase B (MMP-9) 20q12-13 Gelatin, collagen IV, V, VII, XI, XIV, elastin, fibrillin, osteonectin 2 MMP-2, -3, 7, -13, plasmin, trypsin, chymotrypsin, cathepsin G ND
Membrane-type MMPs
 MT1-MMP (MMP-14) 14q12.2 Collagen I, II, III, gelatin, FN, laminin, vitronectin, aggrecan, tenasci nidogen, perlecan, fibrillin, 1PI, 2M, fibrin Plasmin, furin MMP-2, -13
 MT2-MMP (MMP-15) 16q12.2 FN, laminin, aggrecan, tenascin, nidogen, perlecan ND MMP-2, -13

 

MMP expression and activity are regulated at several levels. In most cases, MMPs are not synthesized until needed. Transcription can be induced by various signals including cytokines, growth factors, and mechanical stress. In certain cases, regulation of mRNA stability and translational efficiencyhave been reported. Because most MMPs are secreted as inactive zymogens, they need to be activated, usually by proteolytic cleavage of their NH2-terminal prodomains. Some MMPs are activated by other serine proteases such as plasmin and furin, whereas some of the MMPs can activate other members of their family. The most well characterized is the activation of pro-MMP-2 by MT1-MMP.

A number of MMPs have been strongly implicated in multiple stages of cancer progression including the acquisition of invasive and metastatic properties. Thus, efforts have been made for the past 20 years to develop MMPIs that can be used to halt the spread of cancer, which is what ultimately kills the person. However, initial clinical trials using first generation MMPIs proved to be disappointing . In the ensuing years, much has been learned about the roles of specific MMPs in the different processes of carcinogenesis and more specific MMPIs are being developed and brought to clinical trials.

However, the dosing and scheduling for optimal efficacy is not the same as required for conventional cytotoxic drugs because the MMPIs do not directly kill cancer cells, but instead target such processes as angiogenesis (the development of new blood vessels), invasion, and metastatic spread. (Matrix Metalloproteinases, Angiogenesis, and Cancer. Joyce E. Rundhaug.  Commentary re: A. C. Lockhart et al., Reduction of Wound Angiogenesis in Patients Treated with BMS-275291, a Broad Spectrum Matrix Metalloproteinase Inhibitor. Clin. Cancer Res., 2003; 9551–554).

 Role of p38 MAP Kinase Signal Transduction in Solid Tumors

HK Koul, M Pal, and S Koul. Genes & Cancer  2013 ; 4(9-10) 342–359.  http://dx.doi.org/10.1177/ 1947601913507951

Mitogen-activated protein kinases (MAPKs) mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the main subgroups, the p38 MAP kinases, has been implicated in a wide range of complex biologic processes, such as cell proliferation, cell differentiation, cell death, cell migration, and invasion. Dysregulation of p38 MAPK levels in patients are associated with advanced stages and short survival in cancer patients (e.g., prostate, breast, bladder, liver, and lung cancer). p38 MAPK plays a dual role as a regulator of cell death, and it can either mediate cell survival or cell death depending not only on the type of stimulus but also in a cell type specific manner. In addition to modulating cell survival, an essential role of p38 MAPK in modulation of cell migration and invasion offers a distinct opportunity to target this pathway with respect to tumor metastasis. The specific function of p38 MAPK appears to depend not only on the cell type but also on the stimuli and/or the isoform that is activated.

Mitogen-activated protein kinase (MAPK) signal transduction pathways are evolutionarily conserved among eukaryotes and have been implicated to play key roles in a number of biological processes, including cell growth, differentiation, apoptosis, inflammation, and responses to environmental stresses.

They are typically organized in 3-tiered architecture consisting of a MAPK, a MAPK activator (MAPK kinase), and a MAPKK activator (MAPKK kinase). The MAPK pathways can be regulated at multiple levels as well as via multiple mechanisms, of which the regulation of mitogen-activated protein kinase kinase kinase (MAPKKK/MAP3K) has been proved to be the most challenging due to the great diversity and versatility between different modules at this level. The complex array of growth factors and other ligands that can initiate intracellular cell signaling requires a very high level of coordination among the different proteins involved.

GTP cyclohydrolase (GCH1)

GTP cyclohydrolase (GCH1) is the key-enzyme to produce the essential enzyme cofactor, tetrahydrobiopterin. The byproduct, neopterin is increased in advanced human cancer and used as cancer-biomarker, suggesting that pathologically increased GCH1 activity may promote tumor growth.

(G Picker, Hee-Young Lim, et al. Inhibition of GTP cyclohydrolase attenuates tumor growth by reducing angiogenesis and M2-like polarization of tumor associated macrophages. Int. J. Cancer 2003; 132: 591–604 (2013)  http://dx.doi.org/10.1002/ijc.27706 )

We found that inhibition or silencing of GCH1 reduced tumor cell proliferation and survival and the tube formation of human umbilical vein endothelial cells, which upon hypoxia increased GCH1 and

endothelial NOS expression, the latter prevented by inhibition of GCH1. In nude mice xenografted with HT29-Luc colon cancer cells GCH1 inhibition reduced tumor growth and angiogenesis, determined by in vivo luciferase and near-infrared imaging of newly formed blood vessels. The treatment with the GCH1 inhibitor shifted the phenotype of tumor associated macrophages from the proangiogenic M2 towards M1, accompanied with a shift of plasma chemokine profiles towards tumor-attacking chemokines including CXCL10 and RANTES. GCH1 expression was increased in mouse AOM/DSS-induced colon tumors and in high grade human colon and skin cancer and oppositely, the growth of GCH1-deficient HT29-Luc tumor cells in mice was strongly reduced. The data suggest that GCH1 inhibition reduces tumor growth by (i) direct killing of tumor cells, (ii) by inhibiting angiogenesis, and (iii) by enhancing the antitumoral immune response.

The Role of Stroma in Tumour-Host Co-Existence

Molnár et al.,  The Role of Stroma in Tumour-Host Co-Existence: Some Perspectives in Stroma-Targeted Therapy of Cancer   Biochem Pharmacol 2013, 2:1    http://dx.doi.org/10.4172/2167-0501.1000107

 Cancer grows at the expense of the host as a parasite or superparasite following the second law of thermodynamics (conservation of energy). When the cancer cell progresses via replication to the special state called “spheroid”, a new phase begins with its intimate interaction and development of responses from the stroma which together assist in the formation of a full blown cancer. Among the processes involved are the development of blood vessels and lymphatic channels which are essential for maintenance and further growth of the cancer mass. In this way the condition of “parasitism” is completed with simultaneous suppression of the immune response of the host to the histo-incompatability of the tumor mass. Stroma/parenchyma promotes cancer invasion by feeding cancer cells and inducing immune tolerance. The dynamic changes in composition of stroma and biological consequences as feeder of cancer cells and immune tolerance can give a perspective for rational drug design in anti-stromal therapy. There are differences between normal and cancer cells at subcellular level such as compartmentalzation and structure of cytoskeleton and energy distribution (that is low generally, but locally high in normal cells). In cancer cannibalism of normal cells, the growing cancer mass is a factor for progression and invasion.

Cancer cells have been shown to kill normal cells and the products of cell death used for progression of growth of the cancer cell. Serum and growth factors produced by tumor stroma also provide the needed nutrients and conditions for further tumor growth. Cancer cannot feed off other cancer cells and therefore grow poorly. Probably, although not yet proven, the inability of cancer to “parasitise” other cancer cell types is probably due to some kind of competition or interference. The tumor is in charge of its own development due to its induction proteinases, lipid mobilization factors and angiogenetic factors as well as its ability to negate immune responses of the host response to what is in essence a foreign body.

In our review co-existence of normal and cancer cells in tumor with the growth promoting factors, and the immune tolerance mediating factors produced in the stromal and cancer cells/tissues will be discussed with perspective of stroma targeted therapy.

The clinical significance of cell cannibalism is well defined and described in a large number of publications. The direction of process of cancer development is defined as the tumor invades the normal tissue which never occurs in the reverse direction. This suggests that the cancer cell strives to achieve the lowest energy level possible. Therefore the first of the development of a full blown cancer can be considered as the 2nd Thermodynamic principle  that explains, describes and drives the invading cancer into normal surrounding tissue.

From the normal living state, under particular conditions such as hypoxia, where ATP synthesis is decreased resulting in a switch to glycolytic pathways, cancer cells are selected from a fraction of the population [4]. Energetically, in the presence of electron transfer, by using high energy from respiration, the proliferating state is more stable than resting cells where a higher degree of protein stabilization occurs such as that needed for maintainance of the cytoskeleton of the cell. It was proposed that tumor-promotion might be controlled or modulated by small electronic currents originating from reactive oxygen species and transported through the cytoskeletal microfilament network of the cancer cell.

Aerobic glycolysis is the main energy producing process in cancer cells. Among many other aspects, recently the mitochondria have also been regarded as potential targets in the therapy of cancer. Several small molecules have been tested to restore their dysfunctional functions either by direct or indirect effects. Because of poorly functioning mitochondria, the electron transfer component of the respiration cycle is inefficient; therefore, cancer cells have smaller Gibbs energy than healthy cells. This means, that these cancer cells exists in a metastable state and are not able maintain normal cell structure.

Therefore, the cytoskeleton system is collapsed and dielectric bilayers are formed as a lower grade of cellular structure with decreased electron conductivity. Consequently, to halt cancer growth, one has to evaluate the process of cancer cell development in situ, where the primary tumor is growing as well as that of the metastatic cell that is invading surrounding or distal tissues. This affords one to suggest that the stroma is formed first during long term repeated oxidative stress, a process that is initially accompanied with inflammation due to an active immune response to the histoincompatability antigens present on the surface of the cancer cell. If the cancer cell evades the activity of killer T cells (Treg cells) by either secreting agents that reduce the response of the Treg cells or the immune system for whatever reason is ineffective (immunosuppressed states such as HIV/AIDS, pregnancy, transplantation  therapy, etc.), the formed cancer cells have the opportunity to initiate tumor development. Because of the limited capacity of its electron transfer cycle, cancer cells are essentially starving cells that require glycolytically useful substrates. These substrates are obtained from the killing of normal cells by agents secreted by the cancer cell and the products yielded from dead normal cells “eaten” (phagocytosed) by the starving cancer cell which is digested by the cancer cells lysosomal system. This autophagic process of cannibalism keeps the cancer cell alive and thriving and is known as cytophagy, i.e., cannibalism of normal cells. This type of autophagocytosis  results in a parasitic co-existence of tumor cells with normal cells and will determine the main pathway of interaction between the growing cancer tissue (tumor) and normal tissue where the cancer tissue gradually destroys normal tissues. This process obeys the second law of thermodynamics-conservation of energy within a defined system.

Treatments for Cancer

 Bosutinib: a SRC–ABL tyrosine kinase inhibitor for treatment of chronic myeloid leukemia. 

FE Rassi, HJ Khoury. Pharmacogenomics and Personalized Medicine  2013:6 57–62.

Bosutinib is one of five tyrosine kinase inhibitors commercially available in the United States for the treatment of chronic myeloid leukemia. This review of bosutinib summarizes the mode of action, pharmacokinetics, efficacy and safety data, as well as the patient-focused perspective through quality-of-life data. Bosutinib has shown considerable and sustained efficacy in chronic myeloid leukemia, especially in the chronic phase, with resistance or intolerance to prior tyrosine kinase inhibitors. Bosutinib has distinct but manageable adverse events. In the absence of T315I and V299L mutations, there are no absolute contraindications for the use of bosutinib in this patient population

Chronic myeloid leukemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the presence of a signature hybrid oncogene, the BCR–ABL. The Philadelphia chromosome (Ph+) results from a reciprocal translocation between chromosome 9 and chromosome 22 that juxtaposes the two genes BCR and ABL and drives the leukemogenesis in CML. The ABL gene encodes for a nonreceptor tyrosine kinase that becomes deregulated and constitutively active after the juxtaposition of BCR. BCR–ABL is central in controlling downstream pathways involved in cell proliferation, regulation of cellular adhesion, and apoptosis.The understanding of the importance of this kinase activity in the pathophysiology of CML led to the development of tyrosine kinase inhibitors (TKI) that specifically target BCR–ABL. These agents became the mainstay of modern therapy in CML. CML has a triphasic clinical course, and the majority of patients (∼80%) are diagnosed during the early phase or the chronic phase (CP). However, and without effective treatment, CML invariably progresses to the advanced phases of the disease – the accelerated phase (AP) and the blast phase (BP). BP CML is a lethal refractory secondary leukemia with a short predicted survival.

Comprehensive molecular portraits of human breast tumors

 The Cancer Genome Atlas Network

Nature. 2012 October 4; 490(7418): 61–70. http://dx.doi.org/10.1038/nature11412.

We analyzed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, mRNA arrays, microRNA sequencing and reverse phase protein arrays. Our ability to integrate information across platforms provided key insights into previously-defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity.

Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at  > 10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the Luminal A subtype. We identified two novel protein expression-defined subgroups, possibly contributed by stromal/microenvironmental elements, and integrated analyses identified specific signaling pathways dominant in each molecular subtype including a HER2/p-HER2/HER1/p-HER1 signature within the HER2-Enriched expression subtype. Comparison of Basal-like breast tumors with high-grade Serous Ovarian tumors showed many molecular commonalities, suggesting a related etiology and similar therapeutic opportunities. The biologic finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biologic subtypes of breast cancer.

Most molecular studies of breast cancer have focused on just one or two high information content platforms, most frequently mRNA expression profiling or DNA copy number analysis, and more recently massively parallel sequencing. Supervised clustering of mRNA expression data has reproducibly established that breast cancers encompass several distinct disease entities, often referred to as the intrinsic subtypes of breast cancer. The recent development of additional high information content assays focused on abnormalities in DNA methylation, microRNA expression and protein expression, provide further opportunities to more completely characterize the molecular architecture of breast cancer.

Synbiology contribution and Nanotechnology

Synthetic RNAs Designed to Fight Cancer

Xiaowei Wang and his colleagues at  Washington University School of Medicine in St. Louis have designed synthetic molecules that combine the advantages of two experimental RNA therapies against cancer.  They have designed synthetic molecules that combine the advantages of two experimental RNA therapies against cancer.  RNA plays an important role in how genes are turned on and off in the body. Both siRNAs and microRNAs are snippets of RNA known to modulate a gene’s signal or shut it down entirely. Separately, siRNA and microRNA treatment strategies are in early clinical trials against cancer, but few groups have attempted to marry the two.

“We are trying to merge two largely separate fields of RNA research and harness the advantages of both,” said Xiaowei Wang, assistant professor of radiation oncology and a research member of the Siteman Cancer Center.  The study appears in the December issue of the journal RNA.

“We designed an artificial RNA that is a combination of siRNA and microRNA,” Wang said “our artificial RNA simultaneously inhibits both cell migration and proliferation.”  For therapeutic purposes, “small interfering” RNAs, or siRNAs, are designed and assembled in a lab and can be made to shut down– or interfere with– a single specific gene that drives cancer.  The siRNA molecules work extremely well at silencing a gene target because the siRNA sequence is made to perfectly complement the target sequence, thereby silencing a gene’s expression.

Though siRNAs are great at turning off the gene target, they also have potentially dangerous side effects: siRNAs inadvertently can shut down other genes that need to be expressed to carry out tasks that keep the body healthy.  The siRNAs interfere with off-target genesthat closely complement their “seed region,” a section of the siRNA  that governs binding to a gene target. “In the past, we tried to block the seed region in an attempt to reduce the side effects. Until now, we never tried to replace the seed region completely.”

Wang and his colleagues asked whether they could replace the siRNA’s seed region with the seed region from microRNA. Unlike siRNA, microRNA is a natural part of the body’s gene expression. And it can also shut down genes. As such, the microRNA seed region (with its natural targets) might reduce the toxic side effects caused by the artificial siRNA seed region. Plus, the microRNA seed region would add a new tool to shut down other genes that also may be driving cancer.

Wang’s group started with a bioinformatics approach, using a computer algorithm to design siRNA sequences against a common driver of cancer, a gene called AKT1 that encourages uncontrolled cell division. The program also selected siRNAs against AKT1 that had a seed region highly similar to the seed region of a microRNA known to inhibit a cell’s ability to move, thus potentially reducing the cancer’s ability to spread.

A Neutralizing RNA Aptamer

 Nucleic acid aptamers have been developed as high-affinity ligands that may act as antagonists of disease-associated proteins. Aptamers are non immunogenic and characterised by high specificity and low toxicity thus representing a valid alternative to antibodies or soluble ligand receptor traps/decoys to target specific cancer cell surface proteins in clinical diagnosis and therapy. The epidermal growth factor receptor (EGFR) has been implicated in the development of a wide range of human cancers including breast, glioma and lung. The observation that its inhibition can interfere with the growth of such tumors has led to the design of new drugs including monoclonal antibodies and tyrosine kinase inhibitors currently used in clinic. However, some of these molecules can result in toxicity and acquired resistance, hence the need to develop novel kinds of EGFR-targeting drugs with high specificity and low toxicity.

(CL Esposito, D Passaro, et al. A Neutralizing RNA Aptamer against EGFR Causes Selective Apoptotic Cell Death. PLoS ONE 6(9): e24071. http://dx.doi.org/10.1371/journal.pone.0024071)

Here we generated, by a cell-Systematic Evolution of  Ligands by EXponential enrichment (SELEX) approach, a nuclease resistant RNA-aptamer that specifically binds to EGFR with a binding constant of 10 nM. When applied to EGFR-expressing cancer cells the aptamer inhibits EGFR-mediated signal pathways causing selective cell death. Furthermore, at low doses it induces apoptosis even of cells that are resistant to the most frequently used EGFR-inhibitors, such as gefitinib and cetuximab, and inhibits tumor growth in a mouse xenograft model of human non-small-cell lung cancer (NSCLC). Interestingly, combined treatment with cetuximab and the aptamer shows clear synergy in inducing apoptosis in vitro and in vivo. In conclusion, we demonstrate that this neutralizing RNA aptamer is a promising bio-molecule that can be developed as a more effective alternative to the repertoire of already existing EGFR-inhibitors.

In-Silico Molecular Docking Analysis of Cancer Biomarkers

Currently, in the research scenario for cancer, the identification of anti-cancer drugs using immuno-modulatory proteins and other molecular agents to initiate apoptosis in cancer cells and to inhibit the signaling pathways of cancer biomarkers as a drug targeted therapy, for cancer cell proliferation assays by the researchers. In-Silico analysis is used to recognize anticancer compounds as a future prospective for In-Vitro and In-Vivo analysis. A large number of herbal remedies (e.g. garlic, mistletoe) are used by cancer patients for treating the cancer and/or reducing the toxicities of chemotherapeutic drugs. Some herbal medicines have shown potentially beneficial effects on cancer progression and may ameliorate chemotherapy-induced toxicities.  (K. Gowri Shankar et al., In-Silico Molecular Docking Analysis of Cancer Biomarkers with Bioactive Compounds of Tribulus terrestris. Intl J NOVEL TRENDS PHARMAL SCI. 2013; 3(4).

Tribulus terrestris is mentioned in ancient Indian Ayurvedic medical texts dating back thousands of years. Tribulus terrestris has been widely used in the Ayurvedic system of medicine for the treatment of sexualdysfunction and various urinary disorders. The aim of the present study is to evaluate the interactions of some bioactive compounds of Tribulus terrestris for In-Silico anticancer analysis with cancer biomarkers as targets. The targeted biomarkers for analysis include NSE-Lung cancer, Follistatin-Prostrate cancer, GGT Hepatocellular carcinoma, Human Prostasin-Ovarian cancer.

GC-MS analysis of Tribulus terrestris whole plant methanol extract revealed the existence of the major compound like 3,7,11,15-tetramethylhexadec-2-en-1-ol, 1,2-Benzenedicarboxylic acid, disooctyl ester, 9,12,15-Octadecatrienoic acid, (z,z,z)-, 9,12-Octadecadienoic acid (z,z)-, Hexadecadienoic acid, ethyl ester, n-Hexadecadienoic acid, Octadecanoic acid, Phytol, α-Amyrin are chosen as ligands. Hence, by analyzing the minimum binding energy of the ligand binding complex with the receptors by dockinganalysis using AutoDock tools will show effective nature of inhibition of these receptors by the unique ligands. Based on the results low minimum binding energy ligands are identified and used as a future studies can be done for specific receptors  docking.

Anti-Cancerous Effect of4,4′-Dihydroxychalcone ((2E,2′E)-3,3′-(1,4-Phenylene) Bis (1-(4-hydroxyphenyl) Prop-2-en-1-one)) on T47D Breast Cancer Cell Line

Narges Mahmoodi, T Besharati-Seidani, N Motamed, and NO Mahmoodi*
Annual Research & Review in Biology 2014; 4(12): 2045-2052
SCIENCEDOMAIN international    www.sciencedomain.org

Aims: The majority of human breast tumors are estrogen receptor α (ERα) positive. However, not all of the ERα+ breast cancers respond to anti-estrogens drugs for those women who do respond, initial positive responses can be of short duration. Thus, more effective drugs are needed to enhance the efficacy of anti-estrogens drugs or to be used separately in a period of time. In view of potential cytotoxicity associated with silybin as polyhydroxy compounds a synthetic 4-hydroxychalcones (bis-phenol) was considered to explore its anti-carcinogenic effects in comparison to silybin on ERα+ breast cancer cell line.

Methodology: We have studied the inhibitory effect of 4,4′-dihydroxychalcone on the T47D breast cancer cell line by MTT test and the IC50s were estimated using Pharm PCS.

Results: The 4,4′-dihydroxychalcone showed significant dose- and time-dependent cell growth inhibitory effects on T47D breast cancer cells. The IC50 of 4,4′-dihydroxychalcone on T47D cells after 24 and 48 hours was 160.88+/1 μM, 62.20+/1 μM and for silybin was 373.42+/-1 μM,176.98+/1 μM respectively.

Conclusion: Our results strongly suggests that this premade synthetic 4,4′-dihydroxychalcone can promote anti carcinogenic actions on T47D cell line. All 4,4′-dihydroxychalcone doses had a much larger inhibitory effect on cell viability than silybin doses in T47D cells. The ratio of the IC50 of 4,4′-dihydroxychalcone to silybin after 24 and 48 hours was 1: 2.3 and 1: 2.8 respectively.

Anticancer and multidrug resistance-reversal effects of solanidine analogs synthetized from pregnadienolone acetate.

István Zupkó, Judit Molnár, Borbála Réthy, Renáta Minorics, Eva Frank, et al.
Molecules (Impact Factor: 2.43). 01/2014; 19(2):2061-76.  http://dx.doi.org/10.3390/molecules19022061
Source: PubMed

ABSTRACT A set of solanidine analogs  with antiproliferative properties were recently synthetized from pregnadienolone acetate, which occurs in Nature. The aim of the present study was an in vitro characterization of their antiproliferative action and an investigation of their multidrug resistance-reversal activity on cancer cells. Six of the compounds elicited the accumulation of a hypodiploid population of HeLa cells, indicating their apoptosis-inducing character, and another one caused cell cycle arrest at the G2/M phase. The most effective agents inhibited the activity of topoisomerase I, as evidenced by plasmid supercoil relaxation assays. One of the most potent analogs down-regulated the expression of cell-cycle related genes at the mRNA level, including tumor necrosis factor alpha and S-phase kinase-associated protein 2, and induced growth arrest and DNA damage protein 45 alpha. Some of the investigated compounds inhibited the ABCB1 transporter and caused rhodamine-123 accumulation in murine lymphoma cells transfected by human MDR1 gene, expressing the efflux pump (L5178). One of the most active agents in this aspect potentiated the antiproliferative action of doxorubicin without substantial intrinsic cytostatic capacity. The current results indicate that the modified solanidine skeleton is a suitable substrate for the rational design and synthesis of further innovative drug candidates with anticancer activities.

Nutrition and Cancer

 Ascorbic Acid and Selenium Interaction: Its Relevance in Carcinogenesis

 Michael J. Gonzalez
Journal of Orthomolecular Medicine 1990; 5(2)

Ascorbic acid and selenium are two nutrients that seem to have a preventive potential in the process of carcinogenesis; because of a possible synergistic action that may produce an enhanced anticarcinogenic effect. Interaction between these nutrients have been reported. Results indicate that the protective effect of the inorganic form of selenium (Na Selenite) was nullified by ascorbic acid, whereas the chemopreventive action of the organic form (seleno-DL-methionine) was not affected.

A possibility exists that Selenite is reduced by ascorbic acid to elemental selenium and is therefore not available for tissue uptake. In experiments using Selenite; plasma and erythrocyte glutathione peroxidase enzyme activity was directly related to the level of ascorbic acid fed.

Complementary RNA and Protein Profiling Identifies Iron as a Key Regulator of Mitochondrial Biogenesis

J W. Rensvold, Shao-En On, A Jeevananthan, et al.
Cell Rep. 2013 January 31; 3(1): .   http://dx.doi.org/10.1016/j.celrep.2012.11.029

Mitochondria are centers of metabolism and signaling whose content and function must adapt to
changing cellular environments. The biological signals that initiate mitochondrial restructuring
and the cellular processes that drive this adaptive response are largely obscure. To better define
these systems, we performed matched quantitative genomic and proteomic analyses of mouse
muscle cells as they performed mitochondrial biogenesis. We find that proteins involved in
cellular iron homeostasis are highly coordinated with this process and that depletion of cellular
iron results in a rapid, dose-dependent decrease of select mitochondrial protein levels and
oxidative capacity. We further show that this process is universal across a broad range of cell
types and fully reversed when iron is reintroduced. Collectively, our work reveals that cellular iron
is a key regulator of mitochondrial biogenesis, and provides quantitative data sets that can be
leveraged to explore posttranscriptional and posttranslational processes that are essential for
mitochondrial adaptation.

Avemar outshines new cancer ‘breakthrough’ drug

by Michael Traub
Townsend Letter / Oct, 2010

Many of us in the cancer research community were happy to hear about progress against metastatic melanoma reported this June at the annual meeting of the American Society of Clinical
Oncology (ASCO). since there has not been an improvement in overall survival from chemotherapy in over three decades.
Data from a phase III clinical trial of the experimental monoclonal antibody ipilimumab (pronounced “ep-eh-lim-uemab”) showed that patients with melanoma survived longer if they were taking ipilimumab than if they were not, regardless of whether they also were taking the other drug in the study, an experimental cancer vaccine. (1)

A Closer Look: How Big an Improvement, at What Cost to Patients?

Overall Survival: the ‘Gold Standard’ for Judging Cancer Therapies

Overall survival (OS) is the length of time that a patient actuallysurvives a cancer after treatment. It can also be measured as the percentage of patients surviving a specific time. It is the gold
standard by which the usefulness of a cancer treatment should be determined. Many things can help a patient, but the most important goal of doctors and patients is for the cancer patient to live longer, with a decent quality of life (QOL).

Among patients taking ipilimumab with or without the experimental vaccine, median overall survival was about 10 months. That is compared with 6.4 months’ overall survival among patients receiving the vaccine by itself. About 45.6% of patients taking ipilimumab survived one year, an improvement of some 7% over the 38% seen in some earlier studies. This very modest improvement in survival comes at quite a price.

Severe Side Effects in More Than One in Four Ipilimumab Patients Ipilimumab has some side effects that can be “both severe and long-lasting,” according to the study report. Among patients taking ipilimumab by itself (without the vaccine), 19.1% had side effects requiring hospitalization or invasive intervention, 3.8% died from the effects of the drug, and another 33.8% had life-threatening or disabling side effects. All totaled, 26.7% of the patients taking ipilimumab by itself– more than 1 in 4-had side effects that were severe, very severe, or fatal. Severe side effects included diarrhea, nausea, constipation, vomiting, abdominal pain, fatigue, cough, and headache. Vernon Sondak, MD, of the H. Lee Moffitt Cancer and Research Institute, said that “using the drug requires the medical team to be on guard to manage toxicity at all times.” But even with its severe side effects, the researchers said that the drug should be welcomed because it can increase median survival from 6.4 months to 10.1 months. That is because any lengthening of lives is welcome in a disease that hasn’t seen a new drug that can do that in many years.

Fermented Wheat Germ (Avemar) Improves Melanoma Survival Without Harsh Side Effects

But what if there already were such a treatment available-not a drug, but a safe, natural substance shown in clinical trials to have a remarkably similar ability to lengthen the lives of melanoma patients, without the severe side effects of the new drug?
What if the other substance had no significant side effects at all?
What if, instead of causing severe and sometimes fatal side effects, that other substance actually helped prevent and reduce serious side effects caused by chemotherapy and radiotherapy?
In fact, there is just such a treatment available. It is known as fermented wheat germ extract (FWGE) and by its trade name Avemar. It has been approved as a medical nutriment for cancer
patients in Europe for years and is available in the US as a dietary supplement. It has been compared to dacarbazine (DTIC), standard melanoma therapy, in a clinical trial with longer
follow-up than the ipilimumab trial. And with better results.

In 2008, data were published in the research journal Cancer Biotherapy and Radiopharmaceuticals from seven years’ follow-up on a trial at the N. N. Blokhin Cancer Center in Moscow,
Russia, involving 52 patients who had taken or not taken Avemar while taking dacarbazine for the year following surgical removal of their stage III melanoma tumors. (2) Patients who got only dacarbazine survived 44.7 months. Those who got Avemar along with their dacarbazine survived 66.2 months. This is an improvement in overall survival time of over 48%. In the Russian study,
just as it has in other studies, Avemar reduced side effects of the chemotherapy. Among those taking only dacarbazine, 11 % experienced severe (grade 3 or grade 4) side effects that required hospitalization or invasive intervention. None of the Avemar patients had grade 3 or 4 side effects. Since it is difficult to compare length of survival between the recent ipilimumab study and the Avemar melanoma study, because the ipilimumab study tested mostly stage 4 melanoma patients and the Avemar study tested mostly stage 3 melanoma patients, it is most instructive to look at
the percentage improvement in overall survival from adding either treatment to the regimen. Ipilimumab and Avemar both produced very similar improvements in OS (56% vs. 48%, respectively),

Avemar Ameliorates Conventional Treatment Side Effects

The improvement of survival and the amelioration of chemotherapy side effects by Avemar seen in the Russian melanoma study is typical of Avemar’s effects when used in treating other cancers, including in combination with chemotherapy or radiotherapy. Among 170 colorectal cancer patients in a 2003 study published in the British journal of Cancer, Avemar improved overall survival
and reduced metastasis and recurrences after surgery, chemotherapy, and radiotherapy. (3) Taking Avemar for six months during and after those conventional treatments resulted in a 61.8% reduction in the death rate among those patients, compared with those who received only the conventional treatment. Those taking Avemar experienced lower rates of recurrences and metastases
as well, even though most patients in the Avemar group came into the study with more advanced disease, had more radiation earlier, and had been diagnosed longer. Side effects of Avemar, as in
other Avemar trials., were rare, mild, and transient, with no serious adverse events occurring.

In a 2004 study published in the journal of Pediatric Hematology and Oncology, childhood cancer patients taking Avemar during and after conventional therapies had a 42.8% reduction in the
low white blood cell counts and high fever known as febrile neutropenia, which can be a life-threatening consequence of chemotherapy and radiation. (4) This and similar results with
Avemar in other cancers are consistent with animal studies showing that Avemar helps the immune system recover a full white blood cell count after chemotherapy and radiation faster
than would otherwise happen. This study also demonstrated the safety of Avemar for children.

Why Avemar Works in Many Different Kinds of Cancer

Extensive studies in cells and animals have shown how Avemar works. Perhaps its most important action is to restrict cancer cells’ use of glucose. (5) Cancer cells use up to 50 times more glucose
than normal cells, a phenomenon known as the Warburg effect. (6) They use those enormous amounts of glucose to make ribose, the backbone sugar of DNA, much faster than normal cells can. To
do this, they must use a different series of biochemical reactions (“pathway”) than normal cells. Avemar makes this very difficult for cancer cells to do, because it inhibits the activity of the key enzyme in that pathway, transketolase (TK). (7) With the TK pathway blocked, cancer cells cannot use large amounts of glucose to make DNA fast enough to support the proliferation that makes them so dangerous.(8-10)

In experiments in the US and abroad, scientists have learned that Avemar has these additional effects. It:

* lowers the levels of a DNA repair enzyme known as poly (ADPribose) polymerase (PARP).” With this effect, cancer cells are forced to self-destruct, preventing them from proliferating and
producing a synergistic cancer-cell killing effect when given with chemotherapy, which also works to damage cancer cells’ DNA;
* reduces the number of molecules on cancer cells that identify them as originating within the body (MHC-1 molecules). (12) With cancer cells stripped of that protection, the immune system,
which recognizes the cancer cells as abnormal, no longer gives them the pass given to cells originating in the body. The cancer cells are attacked by the immune system’s natural killer (NK)
cells and destroyed;
* increases levels of molecules called intercellular adhesion molecule-1 (ICAM-1) on the blood vessels of cancer tumors. (13). The increase helps immune system cells pass through the walls of the blood vessels supplying the tumor blood flow, moving directly into the tumor to attack its cancer cells; increases the activity of the primary anticancer cytokine, tumor necrosis factor alpha (TNF-a), and produces a synergistic effect in interaction with other anticancer cytokines. (14) Cytokines are substances produced by cells to act directly on other cells. TNF-a helps force cancer cells into the programmed death known as apoptosis and inhibits tumorigenesis, the process through which new tumors are formed;
* inhibits the activity of ribonucleotide reductase (RR), a key enzyme that cells must have to make new DNA so that each cancer cell can divide to make two more like it. (15) With DNA
production slowed, increases in cancer cell growth and replication are inhibited.

Antimetastatic and Immune-Boosting Effects Are Key to Survival

Because the biochemical changes listed above have consistently been shown in both animal and human studies to be directly linked to reducing cancer’s ability to metastasize and to
improving the immune system’s ability to fight cancer, scientists count them as among the most likely main causes of improved survival seen in cancer patients when Avemar is used alone or,
more often, as an adjuvant in addition to standard-of-care therapies such as chemotherapy, radiotherapy, or the combination of the two. (16-23)

Extending Life: How Long, Exactly, and At What Cost in Quality of Life?

Any improvement in advanced melanoma survival, no matter how small, is certainly an achievement. But ipilimumab had severe side effects requiring hospitalization or invasive intervention in
over one-quarter of patients treated with it. And it increased median survival only by 3-plus months. On the other hand, Avemar added to dacarbazine improved survival very markedly, with no severe side effects. If actually improving overall survival substantially without significant side effects means that a drug should be considered as the new standard of care for first-line therapy, then there is no need to wait for further results. Avemar has already demonstrated very significant improvement in survival over chemotherapy alone and has a safety profile unmatched by
conventional therapies.

Michael Traub, ND, FABNO, is in private practice and serves as a member of Oncology Association of Naturopathic Physicians board of examiners.
Notes
(1.) Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010 Jun 14.
(2.) Demidov LV. Manziuk LV, Kharkevitch GY, Pirogova NA,  Artamonova EV. Adjuvant fermented wheat germ extract (Avemar) nutraceutical improves survival of high-risk skin
melanoma patients; a randomized, pilot, phase ll clinical study with a 7-year follow-up. Cancer Biother Radiopharm. 2008 Aug. 23(4):477-482. Erratum in: Cancer Biother Radiopharm. 2008
Oct;2315):669.
(3.) Jakab F, Shoenfeld Y, Balogh A. et al. A medical nutriment has supportive value in the treatment of colorectal cancer. Br J Cancer. 2001 Aug 4;89(3):465-9.
(4.) Garami M, Schuler D, Babosa M, et al. Fermented wheat germ extract reduces chemotherapy-induced febrile neutropenia in pediatric cancer patients, J Pediatr Hematol Oncol. 2004
Oct;26(10):631-635.
(5.) Boros I.G, Lapis K, Szende B, et al. Wheat germ extract decreases glucose uptake and RNA ribose formation but increases fatty acid synthesis in MIA pancreatic adenocarcinoma
cells. Pancreas. 2001 Aug:23(2):141-147.
(6.) Warburg, O. On the origin of cancer cells. Science. 1956 Feb 24; 123(31 91):309-314.
(7.) Boros LG, Lee VVN, Go VL., A metabolic hypothesis of cell growth and death in pancreatic cancer, Pancreas. 2002 Jan;
24:(1):26 33.
(8.) Boros LG, Lapis K, Szende B, et al. Op cit.
(9.) Comin-Anduix B, Boros LG, Marin S, et al. Fermented wheat germ extract inhibits glycolysis/pentose cycle enzymes and induces apoptosis through poly(ADP-ribose) polymerase
activation in Jurkat T-cell leukemia tumor cells. J Biol Chem. 2002 Nov 29;277 (48):46408-46414. Epub 2002 Sep 25.
(23.) Garami M, Schuler D, Babosa M, et al. Fermented wheat germ extract reduces chemotherapy-induced febrile neutropenia in pediatric cancer patients. J Pediatr Hematol Oncol. 2004 Oct;
26(10):631-635.

by Michael Traub, ND, FABNO
COPYRIGHT 2010 The Townsend Letter Group
COPYRIGHT 2010 Gale, Cengage Learning

Nanotechnology in Cancer Drug Delivery and Selective Targeting

Nanoparticles are rapidly being developed and trialed to overcome several limitations of traditional drug delivery systems and are coming up as a distinct therapeutics for cancer treatment. Conventional chemotherapeutics possess some serious side effects including damage of the immune system and other organs with rapidly proliferating cells due to nonspecific targeting, lack of solubility, and inability to enter the core of the tumors resulting in impaired treatment with reduced dose and with low survival rate.

Nanotechnology has provided the opportunity to get direct access of the cancerous cells selectively with increased drug localization and cellular uptake. Nanoparticles can be programmed for recognizing the cancerous cells and giving selective and accurate drug delivery avoiding interaction with the healthy cells. This review focuses on cell recognizing ability of nanoparticles by various strategies having unique identifying properties that distinguish them from previous anticancer therapies. It also discusses specific drug delivery by nanoparticles inside the cells illustrating many successful researches and how nanoparticles remove the side effects of conventional therapies with tailored cancer treatment.

(Kumar Bishwajit Sutradhar and Md. Lutful Amin. Hindawi Publ. Corp.  2014, Article ID 939378, 12 pages

http://dx.doi.org/10.1155/2014/939378)

Cancer, the uncontrolled proliferation of cells where apoptosis is greatly disappeared, requires very complex process of treatment. Because of complexity in genetic and phenotypic levels, it shows clinical diversity and therapeutic resistance. A variety of approaches are being practiced for the treatment of cancer each of which has some significant limitations and side effects. Cancer treatment includes surgical removal, chemotherapy, radiation, and hormone therapy. Chemotherapy, a  very common treatment, delivers anticancer drugs systemically to patients for quenching the uncontrolled proliferation of cancerous cells. Unfortunately, due to nonspecific targeting by anticancer agents, many side effects occur and poor drug delivery of those agents cannot bring out the desired outcome in most of the cases. Cancer drug development involves a very complex procedure which is associated with advanced polymer chemistry and electronic engineering.

The main challenge of cancer therapeutics is to differentiate the cancerous cells and the normal body cells. That is why the main objective becomes engineering the drug in such a way as it can identify the cancer cells to diminish their growth and proliferation. Conventional chemotherapy fails to target the cancerous cells selectively without interacting with the normal body cells. Thus they cause serious side effects including organ damage resulting in impaired  treatment with lower dose and ultimately low survival rates.

Nanotechnology is the science that usually deals with the size range from a few nanometers (nm) to several hundrednm, depending on their intended use. It has been the area of interest over the last decade for developing precise drug delivery systems as it offers numerous benefits to overcome the limitations of conventional formulations . It is very promising both in cancer diagnosis and treatment since it can enter the tissues at molecular level.

Cisplatin-incorporated nanoparticles of poly(acrylic acid-co-methyl methacrylate) copolymer

K Dong Lee, Young-Il Jeong,  DH Kim,  Gyun-Taek Lim,  Ki-Choon Choi.  Intl J Nanomedicine 2013:8 2835–2845.

Although cisplatin is extensively used in the clinical field, its intrinsic toxicity limits its clinical use. We investigated nanoparticle formations of poly(acrylic acid-co-methyl methacrylate) (PAA-MMA) incorporating cisplatin and their antitumor activity in vitro and in vivo.

Methods: Cisplatin-incorporated nanoparticles were prepared through the ion-complex for­mation between acrylic acid and cisplatin. The anticancer activity of cisplatin-incorporated nanoparticles was assessed with CT26 colorectal carcinoma cells.

Results: Cisplatin-incorporated nanoparticles have small particle sizes of less than 200 nm with spherical shapes. Drug content was increased according to the increase of the feeding amount of cisplatin and acrylic acid content in the copolymer. The higher acrylic acid content in the copolymer induced increase of particle size and decrease of zeta potential. Cisplatin-incorporated nanoparticles showed a similar growth-inhibitory effect against CT26 tumor cells in vitro. However, cisplatin-incorporated nanoparticles showed improved antitumor activity against an animal tumor xenograft model.

Conclusion: We suggest that PAA-MMA nanoparticles incorporating cisplatin are promising carriers for an antitumor drug-delivery system.

Researchers Say Molecule May Help Overcome Cancer Drug Resistance
By Estel Grace Masangkay

A group of researchers from the University of Delaware has discovered that a deubiquitinase (DUB) complex, USP1-UAF1, may present a key target in helping fight resistance to platinum-based anticancer drugs. The research team’s findings were published online in Nature Chemical Biology.

Zhihao Zhuang, associate professor in the Department of Chemistry and Biochemistry at UD, and his team studied a DNA damage tolerance mechanism called translesion synthesis (TLS). Enzymes known as TLS polymerases synthesize DNA over damaged nucleotide bases, followed by replication after lesion. The enzymes have been linked with building cancer cell resistance to certain cancer drugs including cisplatin. Cisplatin is used in treatment of ovarian, bladder, and testicular cancers which have spread.

“Cancer drugs like cisplatin work by damaging DNA and thereby preventing cancer cells from replicating the genomic DNA and dividing. However, cancer cells quickly develop resistance to cisplatin, and we and other researchers suspect that a polymerase known as Pol η is involved in overcoming cisplatin-induced lesions,” Professor Zhuang said.

The team found that USP1-UAF1 may play a crucial role in regulating DNA damage response. A new molecule ML323 can be used to inhibit processes such as translesion synthesis. Zhuang said, “Using ML323, we studied the cellular response to DNA damage and revealed new insights into the role of deubiquitination in both the TLS pathway and another one called the Fanconi anemia, or FA, pathway. We’re very encouraged by the fact that a single molecule is effective at inhibiting the USP1-UAF1 DUB complex and disrupting two essential DNA damage tolerance pathways.”

A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro

Cui-yan Han,  Li-ling Yue, Ling-yu Tai,  Li Zhou  et al.  Intl J Nanomedicine 2013:8 1541–1549

The discovery of suitable ligands that bind to cancer cells is important for drug delivery specifically targeted to tumors. Monoclonal antibodies and fragments that serve as ligands have specific targets. Natural ligands have strong mitogenic and neoangiogenic activities. Currently, small pep­tides are pursued as targeting moieties because of their small size, low immunogenicity, and their ability to be incorporated into certain delivery vectors.

The epidermal growth factor receptor (EGFR) serves an important function in the proliferation of tumors in humans and is an effective target for the treatment of cancer. The epidermal growth factor receptor (EGFR) is a transmembrane protein on the cell surface that is overexpressed in a wide variety of human cancers. EGFR is an effective tumor-specific target because of its significant functions in tumor cell growth, differentiation, and migration. EGFR-targeted small molecule peptides such as YHWYGYTPQNVI have been successfully identified using phage display library screening; by contrast, the peptide LARLLT has been generated using computer-assisted design (CAD).

These peptides can be conjugated to the surfaces of liposomes that are then delivered selectively to tumors by the specific and efficient binding of these peptides to cancer cells that express high levels of EGFR.

In this paper, we studied the targeting characteristics of small peptides (AEYLR, EYINQ, and PDYQQD) These small peptides were labeled with fluorescein isothiocyanate (FITC) and used the peptide LARLLT as a positive control, which bound to putative EGFR selected from a virtual peptide library by computer-aided design, and the independent peptide RALEL as a negative control.

Analyses with flow cytometry and an internalization assay using NCI-H1299 and K562 with high EGFR and no EGFR expression, respectively, indicated that FITC-AEYLR had high EGFR targeting activity. Biotin-AEYLR that was specifically bound to human EGFR proteins demonstrated a high affinity for human non-small-cell lung tumors.

We found that AEYLR peptide-conjugated, nanostructured lipid carriers enhanced specific cellular uptake in vitro during a process that was apparently mediated by tumor cells with high-expression EGFR. Analysis of the MTT assay indicated that the AEYLR peptide did not significantly stimulate or inhibit the growth activity of the cells. These findings suggest that, when mediated by EGFR, AEYLR may be a potentially safe and efficient delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy.

Arginine-based cationic liposomes for efficient in vitro plasmid DNA delivery with low cytotoxicity

SR Sarker  Y Aoshima,   R Hokama  T Inoue  et al. Intl J Nanomedicine 2013:8 1361–1375.

Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group.

 Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000.

 We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular.

Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity.

The gene delivery efficiency of amino acid-based cationic assemblies is influ­enced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions.

Molecularly targeted approaches herald a new era of non-small-cell lung cancer treatment

H Kaneda, T Yoshida,  I Okamoto.   Cancer Management and Research 2013:5 91–101.

The discovery of activating mutations in the epidermal growth-factor receptor (EGFR) gene in 2004 opened a new era of personalized treatment for non-small-cell lung cancer (NSCLC). EGFR mutations are associated with a high sensitivity to EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib. Treatment with these agents in EGFR-mutant NSCLC patients results in dramatically high response rates and prolonged progression-free survival compared with conventional standard chemotherapy. Subsequently, echinoderm microtubule-associated protein-like 4 (EML4)–anaplastic lymphoma kinase (ALK), a novel driver oncogene, has been found in 2007. Crizotinib, the first clinically available ALK tyrosine kinase inhibitor, appeared more effective compared with standard chemotherapy in NSCLC patients harboring EML4-ALK. The identification of EGFR mutations and ALK rearrangement in NSCLC has further accelerated the shift to personalized treatmentbased on the appropriate patient selection according to detailed molecular genetic characterization. This review summarizes these genetic biomarker-based approaches to NSCLC, which allow the instigation of individualized therapy to provide the desired clinical outcome.

Non-small-cell lung cancer (NSCLC) has a poor prognosis and remains the leading cause of death related to cancer worldwide. For most individuals with advanced, metastatic NSCLC, cytotoxic chemotherapy is the mainstay of treatment on the basis of the associated moderate improvement in survival and quality of life. However, the outcome of chemotherapy in such patients has reached a plateau in terms of overall response rate (25%–35%) and overall survival (OS; 8–10 months). This poor outcome, even for patients with advanced NSCLC who respond to such chemotherapy, has motivated a search for new therapeutic approaches.

Recent years have seen rapid progress in the development of new treatment strat­egies for advanced NSCLC, in particular the introduction of molecularly targeted therapiesand appropriate patient selection. First, the most important change has been customization of treatment according to patient selection based on the genetic profile of the tumor. Small-molecule tyrosine kinase inhibitors (TKIs) that target the epidermal growth-factor receptor (EGFR), such as gefitinib and erlotinib, are especially effective in the treatment of NSCLC patients who harbor activating EGFR mutations.

Surgical Nanorobotics using nanorobots made from advanced DNA origami and Synthetic Biology

Ido Bachelet’s moonshot to use nanorobotics for surgery has the potential to change lives globally. But who is the man behind the moonshot?

Ido graduated from the Hebrew University of Jerusalem with a PhD in pharmacology and experimental therapeutics. Afterwards he did two postdocs; one in engineering at MIT and one in synthetic biology in the lab of George Church at the Wyss Institute at Harvard.

Now, his group at Bar-Ilan University designs and studies diverse technologies inspired by nature.

They will deliver enzymes that break down cells via programmable nanoparticles.

Delivering insulin to tell cells to grow and regenerate tissue at the desired location.

Surgery would be performed by putting the programmable nanoparticles into saline and injecting them into the body to seek out remove bad cells and grow new cells and perform other medical work.

 

http://2.bp.blogspot.com/-bnAE6hL2RIE/Uy0wFB8pYPI/AAAAAAAAubM/BeSpFC4vLu0/s1600/screenshot-by-nimbus+(3).png

 

Robots killing and suppressing cancer cells

 

http://1.bp.blogspot.com/-LGsE1msGIrw/Uy0vKGoaQ3I/AAAAAAAAubE/2E1_lcAspao/s1600/screenshot-by-nimbus+(2).png

 

Robots delivering payload

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0

http://4.bp.blogspot.com/-kkfXlMyPRCI/Uy0wkYPMvBI/AAAAAAAAubU/0AQPpJpM5E4/s1600/screenshot-by-nimbus+(4).png

Molecular building blocks

 

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=236

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=283

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=287

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=292

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=333

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=397

http://2.bp.blogspot.com/-gCHiyZ2MBHg/Uy0ySRKw_II/AAAAAAAAubg/BeneEQ5bY-U/s1600/screenshot-by-nimbus+(5).png

 

Robot blocks neuron

http://4.bp.blogspot.com/-cbYNJnN_w7U/Uy0yrqyqebI/AAAAAAAAubo/b42r4WRMr8k/s1600/screenshot-by-nimbus+(6).png

 

automation of robotic surgery

 

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=470

Nanoparticles with computational logic has already been done

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=501

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=521

http://1.bp.blogspot.com/-rSyRzo7p50w/Uy0y5teQkDI/AAAAAAAAubw/8cxZ4t0WNHw/s1600/screenshot-by-nimbus+(7).png

 

 robotic algorithm

 

Load an ensemble of drugs into many particles for programmed release based on situation that is found in the body

http://1.bp.blogspot.com/-kc99CbOQYLs/Uy0zgUG13KI/AAAAAAAAub4/j6nM7hAVxUg/s1600/screenshot-by-nimbus+(8).png

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=572

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=577

 

robotic lung cancer Rx

 

chemotherapy regimen

 

Chemoprevention in Model Experiments

Effects of Two Disiloxanes ALIS-409 and ALIS-421 on Chemoprevention in Model Experiments

H TOKUDA,…. L AMARAL and J MOLNAR.ANTICANCER RESEARCH 33: 2021-2028 (2013).

ALIS

 

Figure 1. Chemical structures of ALIS-409 and ALIS-421.

Morpholino-disiloxane (ALIS-409) and piperazinodisiloxane (ALIS-421) compounds were developed as inhibitors of multidrug resistance of various types of cancer cells. In the present study, the effects of ALIS-409 and ALIS-421 compounds were investigated on cancer promotion and on co-existence of

tumor and normal cells. The two compounds were evaluated for their inhibitory effects on Epstein-Barr virus immediate early antigen (EBV-EA) expression induced by tetradecanoylphorbolacetate (TPA) in Raji cell cultures. The method is known as a primary screening test for antitumor effect, below the (IC50) concentration. ALIS-409 was more effective in inhibiting EBV-EA (100 μg/ml) and tumor promotion, than

ALIS-421, in the concentration range up to 1000 μg/ml. However, neither of the compounds were able to reduce tumor promotion significantly, expressed as inhibition of TPA-induced tumor antigen activation. Based on the in vitro results, the two disiloxanes were investigated in vivo for their effects on mouse skin tumors in a two-stage mouse skin carcinogenesis study.

 

 

 

 

 

 

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Author and Curator: Ritu Saxena, Ph.D.

Consultants: Aviva Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD

CONTENT:

Section I   : Mitochondrial diseases and molecular understanding

Section II  : Diagnosis and therapy of mitochondrial diseases

Section III: Mitochondria, metabolic syndrome and research

I. MITOCHONDRIAL DISEASES and MOLECULAR UNDERSTANDING

Mitochondrial cytopathy in adults – current understanding:

Mitochondrial cytopathies are a diverse group of inherited and acquired disorders that result in inadequate energy production leading to illnesses. Several syndromes have been linked to mutations in mitochondrial DNA. Some key features common to mitochondrial diseases are listed as follows:

  • Diverse manifestations of mitochondrial diseases: Although all mitochondrial diseases have the same characteristic of inadequate energy production as compared to the demand, they seem to show diverse manifestations in the form of organs being affected, age of onset and the rate of progression. Reason lies in the unique genetic makeup of mitochondria. The percentage of mtDNA carrying defects varies when the ovum divides and one daughter cells receiving more defective mtDNA and the other receiving less. Hence, successive divisions may lead to accumulation of defects in one of the developing organs or tissues. Since the process in which defective mtDNA becomes concentrated in an organ is random, this may account for the differing manifestations among patients with the same genetic defect. Also, somatic mutations and mutations occurring as a result of exposure to environmental toxins may cause mitochondrial diseases.

As stated by Robert K. Naviaux, founder and co-director of the Mitochondrial and Metabolic Disease Center (MMDC) at the University of California, San Diego;  

“It is a hallmark of mitochondrial diseases that identical mtDNA mutations may not produce identical diseases…the converse is also true, different mutations can lead to the same diseases.”

  • Postmitotic tissues are more vulnerable to mitochondrial diseases: Postmitotic tissues such as those in the brain, muscles, nerves, retinas, and kidneys, are vulnerable for several reasons. Apart from the fact that these tissues have high-energy demands, healthier neighboring cells unlike that observed in skin cannot replace the diseased cells. Thus, mutations in mtDNA accumulate over a period of time resulting in progressive dysfunction of individual cells and hence the organ itself.
  • High rate of mtDNA mutation: MtDNA mutates at rate that is six-seven times higher than the rate of mutation of nuclear DNA. First reason is the absence of histones on mtDNA and second is the exposure of mtDNA to free radicals due to their close proximity to electron transport chain. Additionally, lack of DNA repair enzymes results in mutant tRNA, rRNA and protein transcripts

Spectrum of mitochondrial diseases:

Following is the list of mitochondrial diseases occurring as a result of either mtDNA mutations, alteration in mitochondrial function or those diseases that sometimes might be associated with mitochondrial dysfunction.

  • Disorders associated with mtDNA mutations-

MELAS, MERRF, NARP, Myoneurogastrointestinal disorder and encephalopathy (MNGIE), Pearson Marrow syndrome Kearns-Sayre-CPEO, Leber hereditary optic neuropathy (LHON), Aminoglycoside-associated deafness, Diabetes with deafness

  • Mendelian disorders of mitochondrial function related to fuel homeostasis-

Luft disease, Leigh syndrome (Complex I, COX, PDH), Alpers Disease, MCAD, SCAD, SCHAD, VLCAD, LCHAD, Glutaric aciduria II, Lethal infantile cardiomyopathy, Friedreich ataxia, Maturity onset diabetes of young Malignant hyperthermia, Disorders of ketone utilization, mtDNA depletion syndrome, Reversible COX deficiency of infancy, Various defects of the Krebs Cycle, Pyruvate dehydrogenase deficiency, Pyruvate carboxylase deficiency, Fumarase deficiency, Carnitine palmitoyl transferase deficiency

  • Disorders sometimes associated with mitochondrial function-

Hemochromatosis, Wilson disease, Batten disease, Huntington disease, Menkes disease, Lesch-Nyhan syndrome, Aging, Type II diabetes mellitus, Atherosclerotic heart disease, Parkinson disease, Alzheimer dementia, Congestive heart failure, Niacin-responsive hypercholesterolemia, Postpartum cardiomyopathy, Alcoholic myopathy, Cancer metastasis, Irritable bowel syndrome Gastroparesis-GI dysmotility, Multiple sclerosis, Systemic lupus erythematosis, Rheumatoid arthritis.

II. DIAGNOSIS AND THERAPY OF MITOCHONDRIAL DISEASES

Diagnosis:

Owing to the diversity of symptoms, there is no accepted criterion for diagnosis. Also, due to overlapping symptoms of several diseases with those of mitochondrial dysfunction illnesses, it is important to evaluate the patient for other conditions. A diagnosis could involve combination of molecular genetic, pathologic, or biochemical data in a patient who has clinical features consistent with the diagnosis including mutational analysis on blood lymphocytes and possibly muscle biopsy for visual and biochemical analysis.

The two main biochemical features in most mtDNA disorders are:

  1. Respiratory chain deficiency and
  2. Lactic acidosis.

Skeletal muscle is chosen to study the pathogenic consequence of mtDNA mutations because of the formation of ragged-red fibers (RRF) through mitochondrial proliferation and massive mitochondrial accumulation in many pathogenic situations. RRF can be detected in two ways. Mitochondrial fibers in a subset of these fibers are shown by red or purple stained area by Gomori trichrome stain; the normal or less-affected fibers stain blue or turquoise. Deep purple areas show accumulations of mitochondria as activity of succinate dehydrogenase (SDH) in the case of mitochondrial mutation.

The primary care physician should remember this relatively simple rule of thumb: “When a common disease has features that set it apart from the pack, or involves 3 or more organ systems, think mitochondria.”

Treatment:

There are no cures for mitochondrial diseases; therefore, the treatment is focused on alleviating symptoms and enabling normal functioning of the affected organs. Most patients have used cofactor and vitamins; however, there is no overwhelming evidence that they are helpful in most patients.

  • Coenzyme Q10 (CoQ10) is the best-known cofactor used in treating mitochondrial cytopathies with no known side effects. CoQ10, residing in the inner mitochondrial membrane, functions as the mobile electron carrier and is a powerful antioxidant with benefits such as reduction in lactic acid levels, improved muscle strength, decreased muscle fatigue and so on.
  • Levocarnitine (L-carnitine, carnitine), is a cofactor required for the metabolism of fatty acids. Levocarnitine therapy improves strength, reversal of cardiomyopathy, and improved gastrointestinal motility, which can be a major benefit to those with poor motility due to their disease. Intestinal cramping and pain are the major side effects.
  • Creatine phosphate, synthesized from creatine can accumulate in small amounts in the body, and can act as storage for a high-energy phosphate bond. Muscular creatine may be depleted in mitochondrial cytopathy, and supplemental creatine phosphate has been shown to be helpful in some patients with weakness due to their disease.
  • B Vitamin, are necessary for the function of several enzymes associated with energy production. The need for supplemental B vitamin therapy is not proven, aside from rare cases of thiamine (vitamin B1)-responsive pyruvate dehydrogenase deficiency.

Research – Restriction enzyme for gene therapy of Mitochondria diseases:

Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in humans and defects in this genome are now recognized as important causes of various diseases. Presently, there is no effective treatment for patients suffering from diseases that harbor mutations in mtDNA.

Tanaka et al discovered a gene therapy method to treat a mitochondrial disease associated with mtDNA heteroplasmy. Heteroplasmy is where mutant and wild-type mtDNA molecules co-exist within cells. This syndrome of neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) is caused by mutations in mtDNA leading to amino acid replacement in the resulting protein that codes for a subunit of mitochondrial ATP synthase. Level of mutant mtDNA is crucial for the disease as above a certain threshold level of mtDNA, the disease becomes biochemically and clinically apparent. Authors hypothesized that a possible method to treat patients was by selectively destroying mutant mtDNA, thereby only allowing propagation of wild-type mtDNA. Since restriction endonucleases can recognize highly specific sequences, they were utilized for gene therapy. Tanaka et al utilized Sma1, a restriction endonuclease to destroy mutant mtDNA, leading to increase in wild-type mtDNA levels.

Thus, authors concluded, “ the present results indicate that the use of a mitochondrion-targeted restriction enzyme which specifically recognizes a mutant mtDNA provides a novel strategy for gene therapy of mitochondrial diseases.”

III. MITOCHONDRIA, METABOLIC SYNDROME & RESEARCH

Mitochondria:

Mitochondria are double-membrane organelles located in the cytoplasm and often referred to as the “powerhouse” of the cell. In simple terms, they convert energy into forms that are usable by the cell. Mitochondria are semi-autonomous in that they are only partially dependent on the cell to replicate and grow. They have their own DNA, ribosomes, and can make their own proteins. They are the sites of cellular respiration that generates fuel for the cell’s activities. Mitochondria are also involved in other cell processes such as cell division, cellular growth and cell death. Multiple essential cellular functions are mediated by thousands of mitochondrial-specific proteins, encoded by both the nuclear and mitochondrial genomes.

Interestingly, mitochondria take on many different shapes and along with serving several different metabolic functions. In fact, each mitochondrion’s shape is characteristic of the specialized cell in which it resides. The number of mitochondria too varies in difference cell types, with as high as 500-2000 in some nucleated cells and as low as zero in RBCs and 2-6 in platelets.

The standard sequence to which all human mtNDNA is compared is referred to as the “Cambridge Sequence.” It was sequenced from several different human mtDNAs by a Medical Research Council (MRC) labora- tory based at Cambridge, UK, in 1981 and as a part of this work, Fred Sanger, the received his second Nobel Prize. Several variations in the form of polymorphisms are observed from the Cambridge sequence in the mtDNA of different individuals.

Metabolic syndrome:

Metabolic syndrome is a cluster of conditions — increased blood pressure, a high blood sugar level, excess body fat around the waist or abnormal cholesterol levels — that occur together, increasing your risk of heart disease, stroke and diabetes. Metabolic syndrome is becoming more and more common in the United States. In the future, it may overtake smoking as the leading risk factor for heart disease. In general, a person who has metabolic syndrome is twice as likely to develop heart disease and five times as likely to develop diabetes as someone who doesn’t have metabolic syndrome.

The five conditions described below are metabolic risk factors. You must have at least three metabolic risk factors to be diagnosed with metabolic syndrome.

  • A large waistline. This also is called abdominal obesity or “having an apple shape.” Excess fat in the stomach area is a greater risk factor for heart disease than excess fat in other parts of the body, such as on the hips.
  • A high triglyceride level (or you’re on medicine to treat high triglycerides). Triglycerides are a type of fat found in the blood.
  • A low HDL cholesterol level (or you’re on medicine to treat low HDL cholesterol). HDL sometimes is called “good” cholesterol. This is because it helps remove cholesterol from your arteries. A low HDL cholesterol level raises your risk for heart disease.
  • High blood pressure (or you’re on medicine to treat high blood pressure). Blood pressure is the force of blood pushing against the walls of your arteries as your heart pumps blood. If this pressure rises and stays high over time, it can damage your heart and lead to plaque buildup.
  • High fasting blood sugar (or you’re on medicine to treat high blood sugar). Mildly high blood sugar may be an early sign of diabetes.

Role of Mitochondria in Metabolic Syndrome & Diabetes:

Impaired mitochondrial function has recently emerged as a potential causes of insulin resistance and/or diabetes progression, risk factors of metabolic syndrome.

Mitochondria plays several key functions including generation of ATP, and generating metabolites via Tricarboxylic acid cycle that function in cytosolic pathways, oxidative catabolism of amino acids, ketogenesis, urea cycle; the generation of reactive oxygen species (ROS); the control of cytoplasmic calcium; and the synthesis of all cellular Fe/S clusters, protein cofactors essential for cellular functions such as protein translation and DNA repair. These roles define the mitochondria to be involved in metabolic homeostasis and hence, a major candidate for metabolic syndrome and its associated risk factor including diabetes, obesity and insulin resistance.

Research and Therapeutic relevance:

Understanding the underlying molecular mechanism of aberrant role of mitochondria is important in developing therapeutic agents for mitochondria-associated diseases. In the recent issue of Mitonews, several papers have been published using the products of MitoSciences, which describe research pertaining to the importance of mitochondria in obesity and diabetes. Some recent research articles based on mitochondrial research (also mentioned in MitoNews) have been briefly discussed here:

  • Metabolic inflexibility and Metabolic syndrome: Metabolic inflexibility is defined as the failure of insulin-resistant patients to appropriately adjust mitochondrial fuel selection in response to nutritional cues. Although the phenomenon has been emphasized an important aspect of metabolic syndrome, the molecular mechanisms have not yet been fully deciphered. In a recent article by Muoio et al, published in Cell Metabolism journal, essential role of the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT) has been identified in regulating substrate switching and glucose tolerance. CrAT regulates mitochondrial and intracellular Carbon trafficking by converting acetyl-CoA to its membrane permeant acetylcarnitine ester. Using muscle muscle-specific Crat knockout mice, primary human skeletal myocytes, and human subjects undergoing L-carnitine supplementation, authors have suggested a model wherein CrAT combats nutrient stress, promotes metabolic flexibility, and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility.
  • Rosiglitazone and obesity: Eepicardial adipose tissue (EAT) has been described in humans as a functioning brown adipose tissue (BAT) and has been shown in animal models to have a lower glucose oxidation rate and higher fatty acid (FA) metabolism. In obese individuals, epicardial adipose tissue (EAT) is “hypertrophied”. EAT is a source of BAT may be a source of proinflamatory cytokines. Distel et al published their studies using a rat model of obesity and insulin resistance treated with rosiglitazone. They observed that rosiglitazone, promoted a BAT phenotype in the EAT depot characterized by an increase in the expression levels of genes encoding proteins involved in mitochondrial processing and density PPARγ coactivator 1 alpha (PGC-1α), NADH dehydrogenase 1 and cytochrome oxidase (COX4) resulting in significant up-regulation of PGC1-α and COX4 protein. The authors concluded that PPAR-γ agonist could induce a rapid browning of the EAT that probably contributes to the increase in lipid turnover. Thus, important insights into the mechanism of fat metabolism and involvement of mitochondrial proteins with a therapy were presented in the article.
  • Mitochondrial dysfunction and diabetic neuropathy: Animal models of diabetic neuropathy show that mitochondrial dysfunction occurs in sensory neurons that may contribute to distal axonopathy. The adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signalling axis senses the metabolic demands of cells and regulates mitochondrial function. Studies in muscle, liver and cardiac tissues have shown that the activity of AMPK and PGC-1α is decreased under hyperglycaemia. Chowdhury et al using type 1 and type 2 diabetic rat and mice models studied the hypothesis that deficits in AMPK/PGC-1 signalling in sensory neurons underlie impaired axonal plasticity, suboptimal mitochondrial function and development of neuropathy. The authors have shown there is a significant reduction in phospho-AMPK, phopho-ACC, total PGC-1α, NDUFS3and COXIV in sensory neurons of the dorsal root ganglia of 14 week old diabetic mice with marked signs of thermal hypoalgesia. These results were associated with an impaired neuronal bioenergetic profile and a decrease in the activity of mitochondrial complex I, complex IV and citrate synthase. The fact that resveratrol treatment reversed the changes observed in vitro and in vivo suggest that the development of distal axonopathy in diabetic neuropathy is linked to nutrient excess and mitochondrial dysfunction via defective signalling of the AMPK/PGC-1α pathway.
  • ROS and diabetes: Mitochondria generated reactive oxygen species (ROS) has been associated with kidney damage occurring in diabetes. Rosca et al, published an article investigating the source and site of ROS production by kidney cortical tubule mitochondria in streptozotocin-induced type 1 diabetes in rats. The authors observed that in diabetic mitochondria, the fatty acid oxidation enzymes were elevated with increased oxidative phosphorylation and increased ROS production. The authors observed ROS production with fatty acid oxidation remained unchanged by limiting electron flow in ETC complexes, changes in ETC substrate processing and that the ROS supported by pyruvate also remained unaltered. The authors hence concluded that mitochondrial fatty acid oxidation is the source of increased ROS production in kidney cortical tubules in early diabetes

Sources:

http://www.ncbi.nlm.nih.gov/pubmed/11453081

http://health.cat/open.php?url=http://biochemgen.ucsd.edu/mmdc/ep-3-10.pdf

http://findarticles.com/p/articles/mi_go2827/is_n6_v27/ai_n28687375/

http://www.columbiamitodiagnostics.org/images/Mitobrochure.pdf

http://www.ncbi.nlm.nih.gov/pubmed?term=12372991

http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0004546/

http://www.mayoclinic.com/health/metabolic%20syndrome/DS00522

http://www.nhlbi.nih.gov/health/health-topics/topics/ms/

http://www.ncbi.nlm.nih.gov/pubmed?term=22560225

http://www.ncbi.nlm.nih.gov/pubmed?term=%20%20%20%2022575275

http://www.ncbi.nlm.nih.gov/pubmed?term=%20%20%20%2022561641

http://www.mitosciences.com/mitonews_08_06.html

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