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Posts Tagged ‘Warburg Effect’


Tumor Ammonia Recycling: How Cancer Cells Use Glutamate Dehydrogenase to Recycle Tumor Microenvironment Waste Products for Biosynthesis

Reporter: Stephen J. Williams, PhD

A feature of the tumorigenic process is the rewiring of the metabolic processes that provides a tumor cell the ability to grow and thrive in conditions of limiting nutrients as well as the ability to utilize waste products in salvage pathways for production of new biomass (amino acids, nucleic acids etc.) required for cellular growth and division 1-8.  A Science article from Spinelli et al. 9 (and corresponding Perspective article in the same issue by Dr. Chi V. Dang entitled Feeding Frenzy for Cancer Cells 10) describes the mechanism by which estrogen-receptor positive (ER+) breast cancer cells convert glutamine to glutamate, release ammonia  into the tumor microenvironment, diffuses into tumor cells and eventually recycle this ammonia by reductive amination of a-ketoglutarate by glutamate dehydrogenase (GDH) to produce glutamic acid and subsequent other amino acids needed for biomass production.   Ammonia can accumulate in the tumor microenvironment in poorly vascularized tumor. Thus ammonia becomes an important nitrogen source for tumor cells.

Mammalian cells have a variety of mechanisms to metabolize ammonia including

  • Glutamate synthetase (GS) in the liver can incorporate ammonia into glutamate to form glutamine
  • glutamate dehydrogenase (GDH) converts glutamate to a-ketoglutarate and ammonia under allosteric regulation (discussed in a post on this site by Dr. Larry H. Berstein; subsection Drugging Glutaminolysis)
  • the reverse reaction of GDH, which was found to occur in ER+ breast cancer cells, a reductive amination of a-ketoglutarate to glutamate11, is similar to the reductive carboxylation of a-ketoglutarate to citrate by isocitrate dehydrogenase (IDH) for fatty acid synthesis (IDH is overexpressed in many tumor types including cancer stem cells 12-15), and involved in immune response and has been developed as a therapeutic target for various cancers. IDH mutations were shown to possess the neomorphic activity to generate the oncometabolite, 2-hydroxyglutarate (2HG) 16-18. With a single codon substitution, the kinetic properties of the mutant IDH isozyme are significantly altered, resulting in an obligatory sequential ordered reaction in the reverse direction 19.

 

In the Science paper, Spinelli et al. report that ER+ breast cancer cells have the ability to utilize ammonia sources from their surroundings in order to produce amino acids and biomass as these ER+ breast cancer cells have elevated levels of GS and GDH with respect to other breast cancer histotypes.

GDH was elevated in ER+ luminal cancer cells and the quiescent epithelial cells in organoid culture

However proliferative cells were dependent on transaminases, which transfers nitrogen from glutamate to pyruvate or oxaloacetate to form a-ketoglutarate and alanine or aspartate. a-ketoglutarate is further metabolized in the citric acid cycle.

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1.    Reductive amination and transamination reactions of glutamic acid.  Source http://www.biologydiscussion.com/organism/metabolism-organism/incorporation-of-ammonia-into-organic-compounds/50870

Spinelli et al. showed GDH is necessary for ammonia reductive incorporation into a-ketoglutarate and also required for ER+ breast cancer cell growth in immunocompromised mice.

In addition, as commented by Dr. Dang in his associated Perspectives article, (quotes indent)

The metabolic tumor microenvironment produced by resident cells, such as fibroblasts and macrophages, can create an immunosuppressive environment 20.  Hence, it will be of great interest to further understand whether products such as ammonia could affect tumor immunity or induce autophagy  (end quote indent)

 

 

 

Figure 2.  Tumor ammonia recycling.  Source:  From Chi V. Dang Feeding Frenzy for cancer cells.  Rights from RightsLink (copyright.com)

Metabolic recycling of ammonia via glutamate dehydrogenase supports breast cancer biomass

Jessica B. Spinelli1,2, Haejin Yoon1, Alison E. Ringel1, Sarah Jeanfavre2, Clary B. Clish2, Marcia C. Haigis1 *

1.      1Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. 2.      2Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

* *Corresponding author. Email: marcia_haigis@hms.harvard.edu

Science  17 Nov 2017:Vol. 358, Issue 6365, pp. 941-946 DOI: 10.1126/science.aam9305

Abstract

Ammonia is a ubiquitous by-product of cellular metabolism; however, the biological consequences of ammonia production are not fully understood, especially in cancer. We found that ammonia is not merely a toxic waste product but is recycled into central amino acid metabolism to maximize nitrogen utilization. In our experiments, human breast cancer cells primarily assimilated ammonia through reductive amination catalyzed by glutamate dehydrogenase (GDH); secondary reactions enabled other amino acids, such as proline and aspartate, to directly acquire this nitrogen. Metabolic recycling of ammonia accelerated proliferation of breast cancer. In mice, ammonia accumulated in the tumor microenvironment and was used directly to generate amino acids through GDH activity. These data show that ammonia is not only a secreted waste product but also a fundamental nitrogen source that can support tumor biomass.

 

 

References

1          Strickaert, A. et al. Cancer heterogeneity is not compatible with one unique cancer cell metabolic map. Oncogene 36, 2637-2642, doi:10.1038/onc.2016.411 (2017).

2          Hui, S. et al. Glucose feeds the TCA cycle via circulating lactate. Nature 551, 115-118, doi:10.1038/nature24057 (2017).

3          Mashimo, T. et al. Acetate is a bioenergetic substrate for human glioblastoma and brain metastases. Cell 159, 1603-1614, doi:10.1016/j.cell.2014.11.025 (2014).

4          Sousa, C. M. et al. Erratum: Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion. Nature 540, 150, doi:10.1038/nature19851 (2016).

5          Sousa, C. M. et al. Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion. Nature 536, 479-483, doi:10.1038/nature19084 (2016).

6          Commisso, C. et al. Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells. Nature 497, 633-637, doi:10.1038/nature12138 (2013).

7          Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 57-70 (2000).

8          Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646-674, doi:10.1016/j.cell.2011.02.013 (2011).

9          Spinelli, J. B. et al. Metabolic recycling of ammonia via glutamate dehydrogenase supports breast cancer biomass. Science 358, 941-946, doi:10.1126/science.aam9305 (2017).

10        Dang, C. V. Feeding frenzy for cancer cells. Science 358, 862-863, doi:10.1126/science.aaq1070 (2017).

11        Smith, T. J. & Stanley, C. A. Untangling the glutamate dehydrogenase allosteric nightmare. Trends in biochemical sciences 33, 557-564, doi:10.1016/j.tibs.2008.07.007 (2008).

12        Metallo, C. M. et al. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 481, 380-384, doi:10.1038/nature10602 (2011).

13        Garrett, M. et al. Metabolic characterization of isocitrate dehydrogenase (IDH) mutant and IDH wildtype gliomaspheres uncovers cell type-specific vulnerabilities. Cancer & metabolism 6, 4, doi:10.1186/s40170-018-0177-4 (2018).

14        Calvert, A. E. et al. Cancer-Associated IDH1 Promotes Growth and Resistance to Targeted Therapies in the Absence of Mutation. Cell reports 19, 1858-1873, doi:10.1016/j.celrep.2017.05.014 (2017).

15        Sciacovelli, M. & Frezza, C. Metabolic reprogramming and epithelial-to-mesenchymal transition in cancer. The FEBS journal 284, 3132-3144, doi:10.1111/febs.14090 (2017).

16        Dang, L. et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 462, 739-744, doi:10.1038/nature08617 (2009).

17        Gross, S. et al. Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations. The Journal of experimental medicine 207, 339-344, doi:10.1084/jem.20092506 (2010).

18        Ward, P. S. et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer cell 17, 225-234, doi:10.1016/j.ccr.2010.01.020 (2010).

19        Rendina, A. R. et al. Mutant IDH1 enhances the production of 2-hydroxyglutarate due to its kinetic mechanism. Biochemistry 52, 4563-4577, doi:10.1021/bi400514k (2013).

20        Zhang, X. et al. IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligand expression. Neuro-oncology 18, 1402-1412, doi:10.1093/neuonc/now061 (2016).

 

Other articles on this Open Access Journal on Cancer Metabolism Include:

 

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

 

Accumulation of 2-hydroxyglutarate is not a biomarker for malignant progression of IDH-mutated low grade gliomas

 

 

Protein-binding, Protein-Protein interactions & Therapeutic Implications [7.3]

Is the Warburg effect an effect of deregulated space occupancy of methylome?

Therapeutic Implications for Targeted Therapy from the Resurgence of Warburg ‘Hypothesis’

New Insights on the Warburg Effect [2.2]

The Inaugural Judith Ann Lippard Memorial Lecture in Cancer Research: PI 3 Kinase & Cancer Metabolism

Renal (Kidney) Cancer: Connections in Metabolism at Krebs cycle and Histone Modulation

Warburg Effect and Mitochondrial Regulation- 2.1.3

Refined Warburg Hypothesis -2.1.2

 

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Is the Warburg effect an effect of deregulated space occupancy of methylome?

Larry H. Bernstein and Radoslav Bozov, co-curation

LPBI

 

 

It would appear that pyruvate is directly used by cancer cell machinery for sustaining genome independence, and that CRISP-Cas9 system is essentially a modified CAD protein for making small bases.

13C-labeled biochemical probes for the study of cancer metabolism with dynamic nuclear polarization-enhanced magnetic resonance imaging

Lucia Salamanca-Cardona and Kayvan R. Keshari

Cancer & Metabolism 2015; 3:9          http://dx.doi.org:/10.1186/s40170-015-0136-2

In recent years, advances in metabolic imaging have become dependable tools for the diagnosis and treatment assessment in cancer. Dynamic nuclear polarization (DNP) has recently emerged as a promising technology in hyperpolarized (HP) magnetic resonance imaging (MRI) and has reached clinical relevance with the successful visualization of [1-13C] pyruvate as a molecular imaging probe in human prostate cancer. This review focuses on introducing representative compounds relevant to metabolism that are characteristic of cancer tissue: aerobic glycolysis and pyruvate metabolism, glutamine addiction and glutamine/glutamate metabolism, and the redox state and ascorbate/dehydroascorbate metabolism. In addition, a brief introduction of probes that can be used to trace necrosis, pH changes, and other pathways relevant to cancer is presented to demonstrate the potential that HP MRI has to revolutionize the use of molecular imaging for diagnosis and assessment of treatments in cancer.

 

Since the hallmark discovery of the Warburg effect in cancer cells in the 1920s, it has been widely accepted that the metabolic properties of cancer cells are vastly different from those of normal cells [1]. Starting from the observation that many cancerous (neoplastic) cells have higher rates of glucose utilization and lactate production, the development of tools and methods to correlate specific cellular metabolic processes to different types of cancer cells has received increased research focus [2, 3]. Several imaging techniques are currently in use for this purpose, including radiography, scintigraphy, positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance (MR) [4, 5].

For more than 30 years, MR has been a revolutionary diagnostic tool, used in a wide range of settings from the central nervous system to cardiomyopathies and cancers. MR imaging (MRI) can outline molecular and cellular processes with high spatial resolution. Typically, MRI of body tissues is achieved via contrast visualization of the protons (1H) of water, which are present in high abundance in living systems. This can be extended to MR spectroscopy (MRS), which can further differentiate between less abundant, carbon-bearing, biological metabolites in vivo utilizing 1Hs of these compounds [6, 7]. However, despite its usefulness in imaging whole body tissues, 1H MRS has low spectral resolution and poor sensitivity for these less abundant metabolites. In addition,13C MRS is increasingly difficult, in comparison to 1H MRS, in that both the gyromagnetic ratio (approximately 25 % of 1H) and natural abundance (1.1 % of 1H) are significantly lower, making the detection of carbon-bearing compounds difficult [8, 9]. The low spectral resolution of 1H MRS for metabolites can be addressed by using 13C-enriched compounds, and with this direct 13C MRS, metabolic processes can be traced, utilizing enriched tags on specific carbons in a given metabolite [10]. While enrichment of molecules in 13C can also moderately address the sensitivity limitation of MRS, recent work in hyperpolarization (HP) provides a means of dramatically increasing sensitivity and enhancing signals, well beyond that of the equilibrium state obtained via MRS. [11, 12]. The focus of this review will be the introduction of this approach in the setting of cancer metabolism, delineating probes of interest, which have been applied to study metabolic processes in vivo.

Obtaining a hyperpolarized probe

In MR, a desired target is placed in a magnetic field where the nuclear spins of molecules are aligned with or against the direction of the magnetic field. The nuclear spins have thus different energies, and an MR signal is detected upon relaxation of nuclear spins of higher energy. At thermal equilibrium, the number of spins aligned with the magnetic field nearly equals the number of spins opposing the direction of the magnetic field. Thus, at thermal equilibrium, spin polarization is in the order of >0.0005 % resulting in a limited signal. Signal increases on the order of 100,000-fold can be achieved by hyperpolarizing the system via the redistribution of the spin population levels found at equilibrium [10, 13]. There are several techniques that have been used to achieve hyperpolarization of various nuclei: spin-exchange optical pumping of 3He and 129Xe, parahydrogen-induced polarization (PHIP), and dissolution dynamic nuclear polarization (DNP) [11,14, 15]. Both PHIP and DNP techniques can polarize biologically relevant nuclei like 13C and 15N, although there is a wider range of molecules that can be targeted for hyperpolarization using dissolution DNP [14, 1618].

The goal of DNP is the transfer of polarization from highly polarized unpaired electron spins to the nuclear spins of a desired target compound. This is achieved by applying an external magnetic field to a free-radical agent in order to polarize electron spins, followed by saturating the electron spin resonance via microwave irradiation in order to obtain polarization transfer. The free-radical agent is generally a stable organic compound that is compatible with aqueous buffers, which are used as solvent in order to obtain a homogeneous distribution of the radical [13]. Nearly 100 % of the electrons on the free-radical agent are polarized when the free-radical/solvent mixture is subjected to high magnetic fields (≥3.3 T) followed by rapid freezing to 1 K using liquid helium in order to obtain a sample frozen to an amorphous state, which is necessary for retention and transfer of polarization [18]. For biological applications, after transfer of electron spin polarization to the nuclei of interest has occurred, the preparation must exist in solution, which can be achieved utilizing a dissolution process in which the solid sample is rapidly melted via injection of a hot solvent, typically a biologically compatible buffer, into the frozen sample [13]. The dissolution process results in a liquid sample at room temperature, while still preserving the enhanced polarization obtained by the microwave irradiation of the frozen sample [8]. Additionally, the use of chelating agents (e.g., EDTA) with the solvent to eliminate trace metals and more recently the use of gadolinium (Gd) chelates with the DNP sample have been used to further enhance and retain polarization in the liquid sample, albeit with caution over potential toxic effects when applied in vivo and the potential for loss of hyperpolarization due to T 1 shortening [11, 19, 20]. More in-depth exploration of the technical aspects of probe development has been previously reviewed [8, 11].

Considerations in probe selection and current research

The usefulness of a molecule for hyperpolarized MRS is dependent on the polarization lifetime of the nucleus of interest, and this property is determined by the spin-lattice relaxation constant (T1) [21]. Dipolar coupling, the magnetic field range, and molecular size can also affect the T1 of a given nucleus. In general, high magnetic fields and large molecular weights decrease the T1. Dipole-dipole coupling of 13C with 1H is common in biologically relevant molecules, and it shortens relaxation times; therefore, carbon atoms directly bound to 1H are generally not useful as probes for HP. For example, all carbons present in glucose (an important substrate in cancer cells) have relaxation times shorter than 2 s [22]. On the other hand, carbonyl carbons of biologically relevant molecules generally have T1’s above 20 s even at high magnetic fields like [1-13C] pyruvic acid, which has relaxation times of 67, 48, and 44 s at 3, 11.7, and 14.1 T, respectively [2325]. Even carbons that are less oxidized than carbonyls, like the hemi-ketal in [2-13C] fructose have T1’s one order of magnitude higher than glucose carbons. Short spin-lattice relaxation times can sometimes be increased by deuterium enrichment of the sample. With this technique, protons that are directly bound to carbons are exchanged for deuterium atoms which results in the reduction of dipole-dipole relaxation, further preserving the hyperpolarized state [26]. This has resulted in increased T1’s of 13C nuclei in molecules such as glucose (T1 increased from 2 s to 10–14 s), providing the possibility of utilizing them in future metabolic studies [2729]. Despite the effect of deuterium enrichment, research efforts have largely focused on developing carbonyl-bearing molecules as molecular imaging probes. The focus of this review is to introduce representative compounds relevant to metabolism that are characteristic of cancer tissue and have been applied in the work of multiple groups: aerobic glycolysis, glutamine addiction, and the redox state.

Pyruvate and aerobic glycolysis

Of particular interest to cancer metabolism is the increased conversion of glucose to lactate as a result of modulated aerobic glycolysis. This process, also known as the Warburg effect, is characteristic of many tumors with altered metabolism where pyruvate generated from glucose metabolism via glycolysis is preferentially converted to lactate by lactate dehydrogenase (LDH) as opposed to entering the tricarboxylic acid cycle [1]. With this phenotype, cancer cells show a preference for lactate fermentation even in the presence of oxygen, thus bypassing oxidative respiration for ATP generation. Because of this, pyruvate has been the preferred probe for HP MRS research since it is an intermediate metabolite in pathways characteristic of aberrant metabolism in cancer cells, including increased lactate production as a result of aerobic glycolysis where detection of HP pyruvate-derived lactate can be used as a marker for cancer and response to treatment [30, 31] as well as an intermediate in amino acid metabolism (e.g., interconversion to alanine via transamination with glutamate) (Fig. 1). In addition, as mentioned before, carbonyl carbons in pyruvate have long relaxation times where even the methyl carbon can have T1’s above 50 s after deuterium enrichment [32]. The interconversion of pyruvate to lactate has been exploited for MRI by using [1-13C] pyruvate and detecting the accumulation of increased lactate in cancerous tissue as compared to surrounding benign tissue. Increased conversion of pyruvate to lactate and alanine has been demonstrated to precede MYC-driven tumorigenesis by using HP [1-13C] pyruvate in murine models [33]. Furthermore, in the same study, a decrease in the flux of alanine was observed at the tumor stage while a decrease in lactate conversion was indicative of tumor regression [33]. In transgenic adenocarcinoma of mouse prostate (TRAMP) models, in vivo studies using HP [1-13C] pyruvate demonstrated that hyperpolarized pyruvate and its metabolic products can be used non-invasively and with high specificity to obtain a profile of the histologic grade of prostate cancers [34]. In vivo imaging following hyperpolarized pyruvate has also been used to evaluate the role of glutaminase and LDH in human lymphoma models [35] as well as to elucidate metabolism of pyruvate in breast cancer [36] and renal cell carcinoma with treatment [30, 37].
https://i2.wp.com/static-content.springer.com/image/art%3A10.1186%2Fs40170-015-0136-2/MediaObjects/40170_2015_136_Fig1_HTML.gif
Flux of hyperpolarized [1-13C] pyruvate to [1-13C] lactate in prostate regions. a MR image from patient with prostate cancer showing regions of cancerous tissue and surrounding normal tissue. bd Localized dynamic hyperpolarized [1-13C]pyruvate and [1-13C]lactate spectral from voxels overlapping the contralateral region of prostate (turquoise), a region of prostate cancer (yellow), and a vessel outside the prostate (green). Adapted with permission from ref. [43]

Early work that utilized HP pyruvate to assess the response of tumors to treatment was conducted in mice xenografted with EL-4 lymphoma cells and treated with etoposide, a topoisomerase inhibitor that causes rapid cell death [38, 39]. In this study, tumor necrosis was correlated to a decrease in the flux of hyperpolarized lactate which was suggested to be due to a decrease in NAD+ and NADH in the intracellular pool as well as loss of LDH function. More recently, HP [1-13C] pyruvate has been used as a biomarker to evaluate early response to radiation therapy in glioma tumors by observing a decrease in hyperpolarized lactate suggested to be a result of changes in tumor perfusion which can be detected between 24 and 96 h following treatment [40, 41]. HP [1-13C] pyruvate has also been used to detect early response to temozolomide (TMZ) treatment on human glioblastoma rat models [42]. The study successfully showed for the first time detection of response to TMZ therapy 1 day after TMZ administration. The continued reports on using HP pyruvate as an imaging probe for assessing treatment response indicate the potential of the compound to become a standard in the field. Moreover, these studies demonstrate the usefulness of HP [1-13C] pyruvate as a tool for early assessment of therapy response, which can improve treatment selection at the clinical level. Pyruvate has also been validated as a metabolic imaging marker for use in humans [43]. In a two-phase study, patients with biopsy-proven prostate cancer of various histological grades were injected with HP [1-13C] pyruvate. In the first phase, a maximum dose level was determined to establish pharmacological safety of the HP probe while still injecting enough pyruvate to allow visualization. This addressed one of the major challenges faced in translating HP MRI to clinical applications: the potential toxicity of compounds that must be injected into patients. In the second phase, metabolism of pyruvate was visualized in real time and differences in the ratio of [1-13C] lactate to [1-13C] pyruvate between identified cancerous regions and normal tissue regions were successfully observed (Fig. 1ad). [1-13C] lactate in regions that did not contain tumor was not detected, confirming previous biopsy and preclinical studies that demonstrated low flux of [1-13C] pyruvate to lactate and low concentrations of lactate in benign prostate tissues [44, 45]. Preliminary results indicated the possibility of detecting previously unobserved cancerous regions by HP [1-13C] pyruvate, later confirmed to be Gleason 4+3 cancer by biopsy, though further investigation into the relationship between grade and metabolism in prostate cancer patients is needed. While there are challenges associated with translation to clinical use for HP [1-13C] pyruvate, the first in human study demonstrated the feasibility of hyperpolarization technology as a safe diagnostic tool and provides the potential for utilizing this approach in preclinical models with direct translation to the clinic.

Glutamine metabolism

Glutamine is an amino acid that plays an important cellular role as nitrogen donor in the form of an amide group for purine and pyrimidine biosynthesis, leaving a glutamate molecule in the process although glutamine can also be converted to glutamate by glutaminase in a reaction independent of nucleotide biosynthesis. Glutamate is the primary nitrogen donor for the biosynthesis of non-essential amino acids. Transaminases catalyze the transfer of the amine group from glutamate to α-ketoacids to synthesize alanine, aspartate (precursor for asparagine), serine (precursor for glycine and cysteine), ornithine (precursor for arginine), and proline which is derived from the glutamate carbon backbone. Glutamine is considered a non-essential amino acid as it can be recycled from glutamate and ammonia in a reaction catalyzed by glutamine synthetase; however, some cancer cells show increase consumption of glutamine and are unable to grow in the absence of exogenous glutamine [46, 47]. This metabolic characteristic of cells to require exogenous glutamine for growth has been termed “glutamine addiction” and has generated extensive research interest as an indicator of development of cancerous tissues [48]. In particular to the field of HP MRI, the conversion rate of glutamine to glutamate (Fig. 2) was explored in hepatocellular carcinoma (HCC) using a [5-13C] glutamine probe (Fig. 2) [49]. Using the ratio between [5-13C] glutamine and [5-13C] glutamate, it was demonstrated that HCC cells convert glutamine at a higher rate than normal cells supporting the notion of glutamine addiction. One important aspect of this study was the choice of [5-13C] glutamine as a probe as opposed to [1-13C] glutamine, which has a longer T1 (16.1 vs. 24.6 s at 9.4 T) [49, 50]. [5-13C] glutamine was selected because the chemical shift change obtained from [1-13C] in glutamine and glutamate is far too small, which could prevent proper identification and quantification of the peaks. This highlights the importance of understanding not only the target compound to be hyperpolarized but also the metabolic products to be detected and their resulting spectra in MR. This is further emphasized with studies that demonstrate the usefulness of [1-13C] glutamine as a source for [1-13C] glutamate in order to follow the metabolism of α-ketoglutarate to observe the metabolic state of the TCA cycle in transformed cells [51]. Furthermore, [1-13C] α-ketoglutarate has been hyperpolarized and used to visualize other metabolic events involving [1-13C] glutamate such as mutations in IDH1 expression in glioma tumors and pathways dependent on hypoxia-inducible factor (HIF) [5153]. More recently, [5-13C] glutamine has been used to visualize the metabolism of liver cancer in vivo and in vitro, as well as the treatment response of prostate cancer cells in vitro [54]. Based on the promise of glutamine as a biomarker for cancer diagnosis and treatment response, extending the spin-lattice relaxation time of the [5-13C] glutamine has been researched and successfully accomplished. The facile synthesis of [5-13C-4-2H2] glutamine has been reported, and its study showed that by relying on the effect of deuterium enrichment to lessen dipolar coupling effects, the T1 of [5-13C] glutamine could be increased from approximately 15 to 30 s [55]. Visualization of real-time conversion of glutamine to glutamate in SF188 cells was achieved using this probe, demonstrating the promise of [5-13C-4-2H2] glutamine as a probe for molecular imaging of metabolic events in real time. Further investigation of this probe applied to in vivo preclinical models will lay the foundation for its clinical translational potential in the future.
https://i1.wp.com/static-content.springer.com/image/art%3A10.1186%2Fs40170-015-0136-2/MediaObjects/40170_2015_136_Fig2_HTML.gif
Metabolism of [5-13C] glutamine to [5-13C] glutamate. a Time-dependent spectral data following conversion of [5-13C] glutamine to [5-13C] glutamate. The signals are from 13C-enriched [5-13C]glutamate at 181.5 ppm and [5-13C]glutamine at 178.5 ppm and from natural abundance 13C label in [1-13C]glutamate at 175.2 ppm and [1-13C]glutamine at 174.7 ppm. b Plot of the ratio of the signal intensities of [5-13C]glutamate/[5-13C]glutamine showing the ratio in hepatoma cells (shaded circle), cell lysate (square), and control (triangle). These results demonstrated that hepatoma cancer cells convert glutamine to glutamine at a higher rate than normal cells. Adapted with permission from ref. [49]

Dehydroascorbate as a redox sensor

Reactive oxygen species (ROS) like the hydroxyl radical, superoxide, and hydrogen peroxide have been shown to cause DNA damage and can lead to mutations that transform normal cells into cancerous cells [56]. The reduction/oxidation (redox) state, which is dependent on the balance between oxidizing equivalents like ROS and reducing cofactors, can provide insight into the physiological condition of the cell with respect to potential cancer transformations. Furthermore, the presence of ROS in tissue has been implicated to be a factor in developing resistance to radiation therapies [57]. During oxidative stress (i.e., when there is an increase in ROS), redox homeostasis is maintained by the action of antioxidant compounds, such as ascorbate (or vitamin C, VitC), which can scavenge for ROS and reduce the compounds to rid the cells of damaging agents [58]. In this process, ascorbate that is available to cells in high concentrations can be oxidized to dehydroascorbate (DHA) while reducing ROS. DHA can then be transported into the cell where DHA is reduced back to ascorbate resulting in a process of recycling ascorbate and DHA (Fig. 3) [59]. In this sense, the ratio of DHA to ascorbate can be used as a molecular marker to investigate the redox state and thus the physiological state of tissues. Additionally, conversion of DHA to ascorbate can be enzymatically catalyzed in an NADPH-dependent manner or via oxidation of glutathione (GSH) to glutathione sulfide (GSSG); thus, visualization of ascorbate/DHA metabolism offers a method for probing in vivo metabolism of NADPH as well as determination of GSSG to GSH ratio, both of which have been implicated to be indicators of oxidative stress in the cells, particularly for neurodegenerative, cardiovascular, and cancer diseases [6062]. Hyperpolarized [1-13C] DHA was successfully used in murine models to detect increased reducing capacity in prostate cancer with the purpose of developing a non-invasive, early diagnostic tool for improving selection of treatment therapies [62, 63]. DHA demonstrates a relatively long T1 at clinically relevant field strengths (>50 s at 3 T) and adequate chemical shift separation between it and its metabolic product ascorbate (δ = 3.8 ppm). Increased reduction of HP [1-13C] DHA to ascorbate was observed in tumor tissue compared to normal tissue as well as other metabolic organs (Fig. 3). This was additionally demonstrated in lymphoma cells, further supporting the potential for using DHA as a probe in living systems [64]. A following study validated these results, and the correlation between increased DHA reduction and glutathione was established in vivo, thus showing the utility of [1-13C] DHA as a molecular imaging probe to detect events that go beyond the direct metabolism of DHA [63]. Notwithstanding the potential of HP DHA as a diagnostic probe, the toxicity of DHA remains to be validated. Earlier studies on mammalian cells showed DHA toxicity starting at 10 mM, while a study carried on rats demonstrated neurological effects of DHA starting at injections of 50 mg/kg [65, 66]. However, as outlined above, successful use of DHA injections in rats and mice for hyperpolarization has been demonstrated without reported side effects on the animals. More research is needed to determine the parameters regarding the toxicity of DHA in larger animal models using pure formulations to assess its potential for clinical trials. Further work in DHA could demonstrate its applicability for the study of ROS and redox changes in model systems.
https://i1.wp.com/static-content.springer.com/image/art%3A10.1186%2Fs40170-015-0136-2/MediaObjects/40170_2015_136_Fig3_HTML.gif
Determination of redox state by imaging of HP [1-13C] ascorbate (VitC) and [1-13C] dehydroascorbate (DHA). Oxidative stress caused by ROS (1.) can be alleviated by oxidation of ascorbate to DHA (2.), and recycling of DHA to ascorbate can occur indirectly with oxidation of glutathione (3.) or directly with oxidation of NADH (4.). The ratio of [ascorbate] to [DHA] has been successfully used in mice models as a biomarker to determine pH in vivo. Adapted with permission from ref. [62]

Other metabolic imaging probes

While the three probes discussed earlier are the most well studied in metabolic events that are characteristic of cancer cells in general, other molecules have been evaluated in their potential to be used as biomarkers. Hyperpolarized bicarbonate (H13CO3) has been successfully used to determine the pH in extracellular matrix of lymphoma tumors in mice, and a correlation between acidic environments and cancer was established [67]. The relaxation times for bicarbonate compounds at 3 T are between 34 and 50 s, which is enough time to detect the rapid conversion of H13CO3 and 13CO2 catalyzed by carbonic anhydrase [23]. The attractive feature of this probe is based on how ubiquitous acidic extracellular environments are to a wide variety of diseases; thus, HP bicarbonate has the potential for clinical translation beyond cancer research, though extensive work will be necessary to generate a preparation which will result in an adequate dose for the clinic [68, 69]. More recently, the potential of α-ketoisocaproate (KIC) as a molecular probe for in vivo detection of branched chain amino acid transaminase (BCAT) has been explored. BCAT catalyzes the conversion of KIC to leucine, and its expression has been suggested to correlate to genetic characterization of certain tumors. In a pilot study, HP α-keto-[1-13C]-isocaproate was shown to have a T1 of 100 s so its metabolism can be sensitively traced for over a minute after injection [70]. In the same study, metabolism of HP [1-13C] KIC to [1-13C] leucine by BCAT was observed in murine lymphoma tumor tissue but was absent in rat mammary adenocarcinoma with a correlation between BCAT expression and [1-13C] leucine signal detection [70]. Additionally, in the same models, [1-13C] pyruvate conversion to [1-13C] lactate and [1-13C] alanine was detected in both types of tumors. These findings show the promise of using [1-13C] KIC as a discriminative probe in addition to pyruvate in order to diagnose different types of cancer [71, 72]. Furthermore, the correlation between BCAT expression and [1-13C] leucine detection was also shown in rat brain tissue, confirming the usefulness of HP [1-13C] KIC in assessing BCAT activity in vivo [73]. Choline is another compound that has been evaluated as a molecular imaging probe since elevated choline and choline-derived metabolites have been correlated by 1H-MRS imaging to cancer in the brain, breast, colon, cervix, and prostate [7476]. Despite its potential as a global marker for cancer because of the long T1 relaxation times that can be achieved with deuterium and 15N enrichment [77, 78], HP applications of 13C enriched choline are limited due to the small change in chemical shifts of choline and choline-derived metabolites as well as its potential toxicity [16, 79, 80]. It has been shown that choline toxicity occurs at doses of 53 mg/kg in mice, although a recent study successfully detected HP 13C choline in vivo without adverse effects in rats at doses of 50 mg/kg by using atropine to prevent cholinergic intoxication [81, 82] though metabolic products have been difficult to visualize in vivo. As mentioned earlier, the usefulness of glucose as a probe is limited due to the short relaxation times of all the carbons present in the molecule and although the T1’s can be increased through deuterium enrichment, the lifetime of the probe remains a hurdle for clinical applications [27, 28]. Thus, fructose (a pentose analog of glucose) has been successfully used as an alternative to probe glycolytic pathways [83] in TRAMP models where differences in HP [2-13C] fructose uptake and metabolism was visualized in tumor regions compared to surrounding normal tissues. Like choline, the limiting factor in the usefulness of [2-13C] fructose for in vivo studies is in small chemical shifts between the metabolite and its phosphorylated product. Finally, tumor necrosis can be used as a measure of treatment response, particularly early necrosis. HP [1,4-,13C] malate has been visualized in lymphoma mice models after injection of HP [1,4-13C] fumarate [84]. In normal cells, fumarate has a slow rate of transport into the mitochondria; however, in necrotic cells where the mitochondrial membrane is degraded, fumarase has access to the HP fumarate and its ubiquitous cofactor, water, thus facilitating rapid conversion to malate. Preliminary studies have shown the potential for its use in animal models though further work is required to determine the necessary density of necrotic cells for detection and the timings required for adequate visualization in patients.

Conclusions

The application of hyperpolarized 13C imaging has been extensively investigated in preclinical models, and the successful demonstration of HP [1-13C] pyruvate in patients with prostate cancer has validated the potential of HP MRI as a safe diagnostic and treatment assessment tool. Application of other probes beyond pyruvate is still in its infancy, particularly because of the need to further study the currently developed models under conditions that are relevant to a clinical setting (i.e., lower magnetic fields) as well as to study the necessary parameters (probe toxicity dose limits, safety limits for rapid injection) to withstand the necessary hurdles to translation. Nevertheless, these vast research findings are promising and indicate an eventual translation to humans. Furthermore, there is a large variety of biologically relevant molecules that have the potential to be hyperpolarized (Fig. 4), and molecular imaging of metabolic events in real time using not only one single probe but a combination of relevant probes could become an invaluable tool in elucidating so far undiscovered metabolic and proteomic interactions that play a role in cancer development and treatment. This gives HP MRI the great potential to revolutionize current molecular imaging technologies.
https://i1.wp.com/static-content.springer.com/image/art%3A10.1186%2Fs40170-015-0136-2/MediaObjects/40170_2015_136_Fig4_HTML.gif
Metabolic pathways with compounds that can be used as molecular imaging probes for HP MRI. A wide variety of metabolic pathways have already been visualized or have the potential to be visualized using hyperpolarization technology that can be applied to different pathological states of the cell including cardiovascular disease and a large variety of cancers. 1. Metabolism of C1 (red dots) in pyruvate. Theasterisks on selected compounds represent enrichment of 13C in the second pass of pyruvate in TCA cycle. 2. Metabolism of C1 (brown dots) in DHA using a pool of NADPH derived from the pentose phosphate pathway. 3. Metabolism of C1 (blue dots) and C5 (green dots) of glutamine. 4. Metabolism of C1 and C4 (purple dots) of fumarate unrelated to TCA metabolites. 5. Metabolism of extracellular bicarbonate (gray dots). MTC1 monocarboxylate transporter 1, MTC4 monocarboxylate transporter 4,System ASC amino acid transporter, GLUTs glucose transporters, DCT dicarboxylate transporter, DHARdehydroascorbate reductase, GR glutathione reductase, GSH glutathione, GSSG glutathione disulfide,LDH lactate dehydrogenase, ALT alanine transaminase, CA carbonic anhydrase, PC pyruvate carboxylase,PDH pyruvate dehydrogenase, CS citrate synthase, GLS glutaminase, GLDH glutamate dehydrogenase,IDH isocitrate dehydrogenase, OGDC oxoglutarate dehydrogenase complex, SCS succinyl CoA synthetase, SQR succinate dehydrogenase, FH fumarate hydratase, MDH malate dehydrogenase, FUMfumarase. Cofactors have been omitted for brevity

Abbreviations

ALT:   alanine transaminase;   BCAT:  branched chain amino acid transaminase;   DHA:  dehydroascorbate;   DNP:  dynamic nuclear polarization;   EDTA:  ethylenediaminetetraacetic acid;   GSH:  glutathione;   GSSG:   glutathione sulfide;   HCC:  hepatocellular carcinoma;   HIF:  hypoxia-inducible factor;   HP:  hyperpolarized/hyperpolarization;   IDH:  isocitrate dehydrogenase;   KIC:  ketoisocaproate;   LDH:  lactate dehydrogenase;   MR: magnetic resonance;   MRI:  Magnetic resonance imaging;   MRS:  magnetic resonance spectroscopy;   NAD(H):  nicotinamide adenine dinucleotide;   NADP(H):  nicotinamide adenine dinucleotide phosphate;   PET:  positron emission tomography;   ROS:  reactive oxygen species;   SPECT:  single-photon emission computed tomography;   TRAMP:  transgenic adenocarcinoma of mouse prostate

References

  1. Warburg O. On the origin of cancer cells. Science. 1956;123:309–14.View ArticlePubMed
  2. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer. 2004;4(11):891–9.View ArticlePubMed
  3. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324:1029–33.View ArticlePubMed CentralPubMed
  4. Shie P, Cardarelli R, Brandon D, Erdman W, AbdulRahim N. Meta-analysis: comparison of F-18 fluorodeoxyglucose-positron emission tomography and bone scintigraphy in the detection of bone metastases in patients with breast cancer. Clin Nucl Med. 2008;33:97–101.View ArticlePubMed
  5. Frangioni JV. New technologies for human cancer imaging. J Clin Oncol. 2008;26:4012–21.View ArticlePubMed CentralPubMed
  6. Castillo M, Kwock L, Mukherji SK. Clinical applications of proton MR spectroscopy. Am J Neuroradiol. 1996;17:1–16.PubMed
  7. Barker PB, Bizzi A, De Stefano N, Gullapalli R, Lin DD. Clinical MR spectroscopy: techniques and applications. Cambridge University Press; 2009.
  8. Comment A, Merritt ME. Hyperpolarized magnetic resonance as a sensitive detector of metabolic function. Biochemistry. 2014;53:7333–57.View ArticlePubMed
  9. Rider OJ, Tyler DJ. Clinical implications of cardiac hyperpolarized magnetic resonance imaging. J Cardiov Magn Reson. 2013;15:93.View Article
  10. Golman K, Olsson LE, Axelsson O, Månsson S, Karlsson M, Petersson JS. Molecular imaging using hyperpolarized 13C. Br J Radiol. 2003;76 Suppl 2:S118–S27.View ArticlePubMed

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sjwilliamspa commented on Is the Warburg effect an effect of deregulated space occupancy of methylome?

Is the Warburg effect an effect of deregulated space occupancy of methylome? Larry H. Bernstein and Radoslav Bozov, …

It would be an interesting figure, although I am not sure anyone has been able to measure it, is the spatial distribution of lactate and pyruvate over the tumor as a function of diffusion distance such as a heat map to see if pyruvate and lactate levels have a gradiant over a solid tumor. I am not sure it would but interesting to see where tumor cells, which undergo Warburg type metabolic phenotype actually exist, if it is a function of angiogenesis or a function of the proliferative capacity of cells in situ.

Response by LHB…

Radoslav Bozov has repeatedly referred to the real problem of space/time in the required experimental view that is intractable, as seen by Erwin Schroedinger.  It is confounded by
the restrictions imposed by research, and to an extent also the dilemma of location and velocity.

I think it is to an extent also inherent in the modern revelations of autophagy and apoptosis that were not part of the view in the mid 20th century.  However, the work of B. Chance led to a substantially better understanding of the hydride transfer in the NAD/NADH.  What is overlooked is the important role cited by NO Kaplan of NADPH/NADP vs NADH/NAD associated with synthetic and, alternatively, catabolic processes in the cell. What role the pyridine nucleotide transhydrogenase would play is anyones guess.   In any case the proliferation of malignant cells is dependent on NADPH.  This would limit the NAD/NADH related reactions. The effect in the cytoplasm is PYR –> LAC, with generation of NAD from NADH.  In addition, the type of isoenzyme favored should be consequential.  For instance, the M-type LDH does not form an abortive ternary complex LDH*NAD+*PYR. In addition, Bernstein, Everse and Grisham showed that in cancer there is an aberrant cytoplasmic MDH.

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H2S-mediated protein sulfhydration in stress reveals metabolic reprogramming

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the Integrated Stress Response

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>Bo-JhihGuan, 

Ilya Bederman
Department of Pediatrics, Case Western Reserve University, Cleveland, United States
No competing interests declared

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>IlyaBederman, 

Mithu Majumder
Department of Pharmacology, Case Western Reserve University, Cleveland, United States
No competing interests declared

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>MithuMajumder, et al.
eLife 2015;10.7554/eLife.10067    

http://elifesciences.org/content/early/2015/11/23/eLife.10067http://dx.doi.org/10.7554/eLife.10067

The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the Integrated Stress Response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.
Posttranslational modification is a fundamental mechanism in the regulation of structure and function of proteins. The covalent modification of specific amino acid residues influences diverse biological processes and cell physiology across species. Reactive cysteine residues in proteins have high nucleophilicity and low pKa values and serve as a major target for oxidative modifications, which can vary depending on the subcellular environment, including the type and intensity of intracellular or environmental cues. Oxidative environments cause different post-translational cysteine modifications, including disulfide bond formation (-S-S-), sulfenylation (-S-OH), nitrosylation (-S-NO), glutathionylation (-S-SG), and sulfhydration (-S-SH) (also called persulfidation) (Finkel, 2012; Mishanina et al., 2015). In the latter, an oxidized cysteine residue included glutathionylated, 60 sulfenylated and nitrosylated on a protein reacts with the sulfide anion to form a cysteine persulfide. The reversible nature of this modification provides a mechanism to fine tune biological processes in different cellular redox states. Sulfhydration coordinates with other post-translational protein modifications such as phosphorylation and nitrosylation to regulate cellular functions (Altaany et al., 2014; Sen et al., 2012). Despite great progress in bioinformatics and advanced mass spectroscopic techniques (MS), identification of different cysteine-based protein modifications has been slow compared to other post-translational modifications. In the case of sulfhydration, a small number of proteins have been identified, among them the glycolytic enzyme glyceraldehyde phosphate dehydrogenase, GAPDH (Mustafa et al., 2009). Sulfhydrated GAPDH at Cys150 exhibits an increase in its catalytic activity, in contrast to the inhibitory effects of nitrosylation or glutathionylation of the same cysteine residue (Mustafa et al., 2009; Paul and Snyder, 2012). The biological significance of the Cys150 modification by H2S is not well-studied, but H2S could serve as a biological switch for protein function acting via oxidative modification of specific cysteine residues in response to redox homeostasis (Paul and Snyder, 2012). Understanding the physiological significance of protein sulfhydration requires the development of genome-wide innovative experimental approaches. Current methodologies based on the modified biotin switch technique do not allow detection of a broad spectrum of sulfhydrated proteins (Finkel, 2012). Guided by a previously reported strategy (Sen et al., 2012), we developed an experimental approach that allowed us to quantitatively evaluate the sulfhydrated proteome and the physiological consequences of H2S synthesis during chronic ER stress. The new methodology allows a quantitative, close-up view of the integrated cellular response to environmental and intracellular cues, and is pertinent to our understanding of human disease development.
The ER is an organelle involved in synthesis of proteins followed by various modifications. Disruption of this process results in the accumulation of misfolded proteins, causing ER stress (Tabas and Ron, 2011; Walter and Ron, 2011), which is associated with development of many diseases ranging from metabolic dysfunction to neurodegeneration (Hetz, 2012). ER stress induces transcriptional, translational, and metabolic reprogramming, all of which are interconnected through the transcription factor Atf4. Atf4 increases expression of genes promoting adaptation to stress via their protein products. One such gene is the H2S-producing enzyme, γ-cystathionase (CTH), previously shown to be involved in the signaling pathway that negatively regulates the activity of the protein tyrosine phosphatase 1B (PTP1B) via sulfhydration (Krishnan et al., 2011). We therefore hypothesized that low or even modest levels of reactive oxygen species (ROS) during ER stress may reprogram cellular metabolism via H2S-mediated protein sulfhydration (Figure 1A).
In summary, sulfhydration of specific cysteines in proteins is a key function of H2S (Kabil and Banerjee, 2010; Paul and Snyder, 2012; Szabo et al., 2013). Thus, the development of tools that can quantitatively measure genome-wide protein sulfhydration in physiological or pathological conditions is of central importance. However, a significant challenge in studies of the biological significance of protein sulfhydration is the lack of an approach to selectively detect sulfhydrated cysteines from other modifications (disulfide bonds, glutathionylated thiols and sulfienic acids) in complex biological samples. In this study, we introduced the BTA approach that allowed the quantitative assessment of changes in the sulfhydration of specific cysteines in the proteome and in individual proteins. BTA is superior to other reported methodologies that aimed to profile cysteine modifications, such as the most commonly used, a modified biotin switch technique (BST). BST was originally designed to study protein nitrosylation and postulated to differentiate free thiols and persulfides (Mustafa et al., 2009). A key advantage of BTA over the existing methodologies, is that the experimental approach has steps to avoid false-positive and negative results, as target proteins for sulfhydration. BST is commonly generating such false targets for cysteine modifications (Forrester et al., 2009; Sen et al., 2012). Using mutiple validations, our data support the specificity and reliability of the BTA assay for analysis of protein sulfhydration both in vitro and in vivo. With this approach, we found that ATF4 is the master regulator of protein sulfhydration in pancreatic β cells during ER stress, by means of its function as a transcription factor. A large number of protein targets have been discovered to undergo sulfhydration in β cells by the BTA approach. Almost 1,000 sulfhydrated cysteine- containing peptides were present in the cells under the chronic ER stress condition of treatment with Tg for 18 h. Combined with the isotopic-labeling strategy, almost 820 peptides on more than 500 proteins were quantified in the 405 cells overexpressing ATF4. These data show the potential of the BTA method for further systematic studies of biological events. To our knowledge, the current dataset encompasses most known sulfhydrated cysteine residues in proteins in any organism. Our bioinformatics analyses revealed sulfhydrated cysteine residues located on a variety of structure-function domains, suggesting the possibility of regulatory mechanism(s) mediated by protein sulfhydration. Structure and sequence analysis revealed consensus motifs that favor sulfhydration; an arginine residue and alpha-helix dipoles are both contributing to stabilize sulfhydrated cysteine thiolates in the local environment.
Pathway analyses showed that H2S-mediated sulfhydration of cysteine residues is that part of the ISR with the highest enrichment in proteins involved in energy metabolism. The metabolic flux revealed that H2S promotes aerobic glycolysis associated with decreased oxidative phosphorylation in mitochondria during ER stress in β cells. The TCA cycle revolves by the action of the respiratory chain that requires oxygen to operate. In response to ER stress, mitochondrial function and cellular respiration are down-regulated to limit oxygen demand and to sustain mitochondria. When ATP production from the TCA cycle becomes limited and glycolytic flux increases, there is a risk of accumulation of lactate from pyruvate. One way to escape accumulation of lactate is the mitochondrial conversion of pyruvate to oxalacetic acid (OAA) by pyruvate carboxylase. This latter enzyme was found to be sulfhydrated, consistent with the notion that sulfhydration is linked to metabolic reprogramming towards glycolysis.
The switch of energy production from mitochondria to glycolysis is known as a signature of hypoxic conditions. This metabolic switch has also been observed in many cancer cells characterized as the Warburg effect, which contributes to tumor growth. The Warburg effect provides advantages to cancer cell survival via the rapid ATP production through glycolysis, as well as the increased conversion of glucose into anabolic biomolecules (amino acid, nucleic acid and lipid biosynthesis) and reducing power (NADPH) for regeneration of antioxidants. This metabolic response of tumor cells contributes to tumor growth and metastasis (Vander Heiden et al., 2009). By analogy, the aerobic glycolysis trigged by increased H2S production could give β cells the capability to acquire ATP and nutrients to adapt their cellular metabolism towards maintaining ATP levels in the ER (Vishnu et al., 2014), increasing synthesis of glycerolphospholipids, glycoproteins and protein (Krokowski et al., 2013b), all important components of the ISR. Similar to hypoxic conditions, a phenotype associated with most tumors, the decreased mitochondria function in β cells during ER stress, can also be viewed as an adaptive response by limiting mitochondria ROS and mitochondria-mediated apoptosis. We therefore view that the H2S-mediated increase in glycolysis is an adaptive mechanism for survival of β cells to chronic ER stress, along with the improved ER function and insulin production and folding, both critical factors controlling hyperglycemia in diabetes. Future work should determine which are the key proteins targeted by H2S and thus contributing to metabolic reprogramming of β cells, and if and how insulin synthesis and secretion is affected by sulfhydration of these proteins during ER stress.
Abnormal H2S metabolism has been reported to occur in various diseases, mostly through the deregulation of gene expression encoding for H2S-generating enzymes (Wallace and Wang, 2015). An increase of their levels by stimulants is expected to have similar effects on sulfhydration of proteins like the ATF4- induced CTH under conditions of ER stress. It is the levels of H2S under oxidative conditions that influence cellular functions. In the present study, ER stress in β cells induced elevated Cth levels, whereas CBS was unaffected. The deregulated oxidative modification at cysteine residues by H2S may be a major contributing factor to disease development. In this case, it would provide a rationale for the design of therapeutic agents that would modulate the activity of the involved enzymes.

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Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception


Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception

Curator: Larry H. Bernstein, MD, FCAP

Pasteur Effect – Warburg Effect – What its history can teach us today. 

José Eduardo de Salles Roselino

The Warburg effect, in reality the “Pasteur-effect” was the first example of metabolic regulation described. A decrease in the carbon flux originated at the sugar molecule towards the end of the catabolic pathway, with ethanol and carbon dioxide observed when yeast cells were transferred from an anaerobic environmental condition to an aerobic one. In Pasteur´s studies, sugar metabolism was measured mainly by the decrease of sugar concentration in the yeast growth media observed after a measured period of time. The decrease of the sugar concentration in the media occurs at great speed in yeast grown in anaerobiosis (oxygen deficient) and its speed was greatly reduced by the transfer of the yeast culture to an aerobic condition. This finding was very important for the wine industry of France in Pasteur’s time, since most of the undesirable outcomes in the industrial use of yeast were perceived when yeasts cells took a very long time to create, a rather selective anaerobic condition. This selective culture media was characterized by the higher carbon dioxide levels produced by fast growing yeast cells and by a higher alcohol content in the yeast culture media.

However, in biochemical terms, this finding was required to understand Lavoisier’s results indicating that chemical and biological oxidation of sugars produced the same calorimetric (heat generation) results. This observation requires a control mechanism (metabolic regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative processes (metabolism). In addition, Lavoisier´s results were the first indications that both processes happened inside similar thermodynamics limits. In much resumed form, these observations indicate the major reasons that led Warburg to test failure in control mechanisms in cancer cells in comparison with the ones observed in normal cells.

[It might be added that the availability of O2 and CO2 and climatic conditions over 750 million years that included volcanic activity, tectonic movements of the earth crust, and glaciation, and more recently the use of carbon fuels and the extensive deforestation of our land masses have had a large role in determining the biological speciation over time, in sea and on land. O2 is generated by plants utilizing energy from the sun and conversion of CO2. Remove the plants and we tip the balance. A large source of CO2 is from beneath the earth’s surface.]

Biology inside classical thermodynamics places some challenges to scientists. For instance, all classical thermodynamics must be measured in reversible thermodynamic conditions. In an isolated system, increase in P (pressure) leads to increase in V (volume), all this occurring in a condition in which infinitesimal changes in one affects in the same way the other, a continuum response. Not even a quantic amount of energy will stand beyond those parameters.

In a reversible system, a decrease in V, under same condition, will led to an increase in P. In biochemistry, reversible usually indicates a reaction that easily goes either from A to B or B to A. For instance, when it was required to search for an anti-ischemic effect of Chlorpromazine in an extra hepatic obstructed liver, it was necessary to use an adequate system of increased biliary system pressure in a reversible manner to exclude a direct effect of this drug over the biological system pressure inducer (bile secretion) in Braz. J. Med. Biol. Res 1989; 22: 889-893. Frequently, these details are jumped over by those who read biology in ATGC letters.

Very important observations can be made in this regard, when neutral mutations are taken into consideration since, after several mutations (not affecting previous activity and function), a last mutant may provide a new transcript RNA for a protein and elicit a new function. For an example, consider a Prion C from lamb getting similar to bovine Prion C while preserving  its normal role in the lamb when its ability to change Human Prion C is considered (Stanley Prusiner).

This observation is good enough, to confirm one of the most important contributions of Erwin Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

After Hans Krebs, description of the cyclic nature of the citrate metabolism and after its followers described its requirement for aerobic catabolism two major lines of research started the search for the understanding of the mechanism of energy transfer that explains how ADP is converted into ATP. One followed the organic chemistry line of reasoning and therefore, searched for a mechanism that could explain how the breakdown of carbon-carbon link could have its energy transferred to ATP synthesis. One of the major leaders of this research line was Britton Chance. He took into account that relatively earlier in the series of Krebs cycle reactions, two carbon atoms of acetyl were released as carbon dioxide ( In fact, not the real acetyl carbons but those on the opposite side of citrate molecule). In stoichiometric terms, it was not important whether the released carbons were or were not exactly those originated from glucose carbons. His research aimed at to find out an intermediate proteinaceous intermediary that could act as an energy reservoir. The intermediary could store in a phosphorylated amino acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its phosphate group to ADP producing ATP. A key intermediate involved in the transfer was identified by Kaplan and Lipmann at John Hopkins as acetyl coenzyme A, for which Fritz Lipmann received a Nobel Prize.

Alternatively, under possible influence of the excellent results of Hodgkin and Huxley a second line of research appears. The work of Hodgkin & Huxley indicated that the storage of electrical potential energy in transmembrane ionic asymmetries and presented the explanation for the change from resting to action potential in excitable cells. This second line of research, under the leadership of Peter Mitchell postulated a mechanism for the transfer of oxide/reductive power of organic molecules oxidation through electron transfer as the key for the energetic transfer mechanism required for ATP synthesis.
This diverted the attention from high energy (~P) phosphate bond to the transfer of electrons. During most of the time the harsh period of the two confronting points of view, Paul Boyer and followers attempted to act as a conciliatory third party, without getting good results, according to personal accounts (in L. A. or Latin America) heard from those few of our scientists who were able to follow the major scientific events held in USA, and who could present to us later. Paul  Boyer could present how the energy was transduced by a molecular machine that changes in conformation in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite direction in order to produce ADP plus Pi from ATP (reversibility).

However, earlier, a victorious Peter Mitchell obtained the result in the conceptual dispute, over the Britton Chance point of view, after he used E. Coli mutants to show H+ gradients in the cell membrane and its use as energy source, for which he received a Nobel Prize. Somehow, this outcome represents such a blow to Chance’s previous work that somehow it seems to have cast a shadow over very important findings obtained during his earlier career that should not be affected by one or another form of energy transfer mechanism.  For instance, Britton Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and the idea of control of energy metabolism that brings us back to Pasteur.

This metabolic alternative result seems to have been neglected in the recent years of obesity epidemics, which led to a search for a single molecular mechanism required for the understanding of the accumulation of chemical (adipose tissue) reserve in our body. It does not mean that here the role of central nervous system is neglected. In short, in respiring mitochondria the rate of electron transport linked to the rate of ATP production is determined primarily by the relative concentrations of ADP, ATP and phosphate in the external media (cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when the yield of ATP is high as it is in aerobiosis and the cellular use of ATP is not changed, the oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene expression), throttled down to the resting state. The dependence of respiratory rate on ADP concentration is also seen in intact cells. A muscle at rest and using no ATP has a very low respiratory rate.   [When skeletal muscle is stressed by high exertion, lactic acid produced is released into the circulation and is metabolized aerobically by the heart at the end of the activity].

This respiratory control of metabolism will lead to preservation of body carbon reserves and in case of high caloric intake in a diet, also shows increase in fat reserves essential for our biological ancestors survival (Today for our obesity epidemics). No matter how important this observation is, it is only one focal point of metabolic control. We cannot reduce the problem of obesity to the existence of metabolic control. There are numerous other factors but on the other hand, we cannot neglect or remove this vital process in order to correct obesity. However, we cannot explain obesity ignoring this metabolic control. This topic is so neglected in modern times that we cannot follow major research lines of the past that were interrupted by the emerging molecular biology techniques and the vain belief that a dogmatic vision of biology could replace all previous knowledge by a new one based upon ATGC readings. For instance, in order to display bad consequences derived from the ignorance of these old scientific facts, we can take into account, for instance, how ion movements across membranes affects membrane protein conformation and therefore contradicts the wrong central dogma of molecular biology. This change in protein conformation (with unchanged amino acid sequence) and/or the lack of change in protein conformation is linked to the factors that affect vital processes as the heart beats. This modern ignorance could also explain some major pitfalls seen in new drugs clinical trials and in a small scale on bad medical practices.

The work of Britton Chance and of Peter Mitchell have deep and sound scientific roots that were made with excellent scientific techniques, supported by excellent scientific reasoning and that were produced in a large series of very important intermediary scientific results. Their sole difference was to aim at very different scientific explanations as their goals (They have different Teleology in their minds made by their previous experiences). When, with the use of mutants obtained in microorganisms P Mitchell´s goal was found to survive and B Chance to succumb to the experimental evidence, all those excellent findings of B Chance and followers were directed to the dustbin of scientific history as an example of lack of scientific consideration.  [On the one hand, the Mitchell model used a unicellular organism; on the other, Chance’s work was with eukaryotic cells, quite relevant to the discussion.]

We can resume the challenge faced by these two great scientists in the following form: The first conceptual unification in bioenergetics, achieved in the 1940s, is inextricably bound up with the name of Fritz Lipmann. Its central feature was the recognition that adenosine triphosphate, ATP, serves as a universal energy  “currency” much as money serves as economic currency. In a nutshell, the purpose of metabolism is to support the synthesis of ATP. In microorganisms, this is perfect! In humans or mammals, or vertebrates, by the same reason that we cannot consider that gene expression is equivalent to protein function (an acceptable error in the case of microorganisms) this oversimplifies the metabolic requirement with a huge error. However, in case our concern is ATP chemistry only, the metabolism produces ATP and the hydrolysis of ATP pays for the performance of almost, all kinds of works. It is possible to presume that to find out how the flow of metabolism (carbon flow) led to ATP production must be considered a major focal point of research of the two contenders. Consequently, what could be a minor fall of one of the contenders, in case we take into account all that was found during their entire life of research, the real failure in B Chance’s final goal was amplified far beyond what may be considered by reason!

Another aspect that must be taken into account: Both contenders have in the scientific past a very sound root. Metabolism may produce two forms of energy currency (I personally don´t like this expression*) and I use it here because it was used by both groups in order to express their findings. Together with simplistic thermodynamics, this expression conveys wrong ideas): The second kind of energy currency is the current of ions passing from one side of a membrane to the other. The P. Mitchell scientific root undoubtedly have the work of Hodgkin & Huxley, Huxley &  Huxley, Huxley & Simmons

*ATP is produced under the guidance of cell needs and not by its yield. When glucose yields only 2 ATPs per molecule it is oxidized at very high speed (anaerobiosis) as is required to match cellular needs. On the other hand, when it may yield (thermodynamic terms) 38 ATP the same molecule is oxidized at low speed. It would be similar to an investor choice its least money yield form for its investment (1940s to 1972) as a solid support. B. Chance had the enzymologists involved in clarifying how ATP could be produced directly from NADH + H+ oxidative reductive metabolic reactions or from the hydrolysis of an enolpyruvate intermediary. Both competitors had their work supported by different but, sound scientific roots and have produced very important scientific results while trying to present their hypothetical point of view.

Before the winning results of P. Mitchell were displayed, one line of defense used by B. Chance followers was to create a conflict between what would be expected by a restrictive role of proteins through its specificity ionic interactions and the general ability of ionic asymmetries that could be associated with mitochondrial ATP production. Chemical catalyzed protein activities do not have perfect specificity but an outstanding degree of selective interaction was presented by the lock and key model of enzyme interaction. A large group of outstanding “mitochondriologists” were able to show ATP synthesis associated with Na+, K+, Ca2+… asymmetries on mitochondrial membranes and any time they did this, P. Mitchell have to display the existence of antiporters that exchange X for hydrogen as the final common source of chemiosmotic energy used by mitochondria for ATP synthesis.

This conceptual battle has generated an enormous knowledge that was laid to rest, somehow discontinued in the form of scientific research, when the final E. Coli mutant studies presented the convincing final evidence in favor of P. Mitchell point of view.

Not surprisingly, a “wise anonymous” later, pointed out: “No matter what you are doing, you will always be better off in case you have a mutant”

(Principles of Medical Genetics T D Gelehrter & F.S. Collins chapter 7, 1990).

However, let’s take the example of a mechanical wristwatch. It clearly indicates when the watch is working in an acceptable way, that its normal functioning condition is not the result of one of its isolated components – or something that can be shown by a reductionist molecular view.  Usually it will be considered that it is working in an acceptable way, in case it is found that its accuracy falls inside a normal functional range, for instance, one or two standard deviations bellow or above the mean value for normal function, what depends upon the rigor wisely adopted. While, only when it has a faulty component (a genetic inborn error) we can indicate a single isolated piece as the cause of its failure (a reductionist molecular view).

We need to teach in medicine, first the major reasons why the watch works fine (not saying it is “automatic”). The functions may cross the reversible to irreversible regulatory limit change, faster than what we can imagine. Latter, when these ideas about normal are held very clear in the mind set of medical doctors (not medical technicians) we may address the inborn errors and what we may have learn from it. A modern medical technician may cause admiration when he uses an “innocent” virus to correct for a faulty gene (a rather impressive technological advance). However, in case the virus, later shows signals that indicate that it was not so innocent, a real medical doctor will be called upon to put things in correct place again.

Among the missing parts of normal evolution in biochemistry a lot about ion fluxes can be found. Even those oscillatory changes in Ca2+ that were shown to affect gene expression (C. De Duve) were laid to rest since, they clearly indicate a source of biological information that despite the fact that it does not change nucleotides order in the DNA, it shows an opposing flux of biological information against the dogma (DNA to RNA to proteins). Another, line has shown a hierarchy, on the use of mitochondrial membrane potential: First the potential is used for Ca2+ uptake and only afterwards, the potential is used for ADP conversion into ATP (A. L. Lehninger). In fact, the real idea of A. L. Lehninger was by far, more complex since according to him, mitochondria works like a buffer for intracellular calcium releasing it to outside in case of a deep decrease in cytosol levels or capturing it from cytosol when facing transient increase in Ca2+ load. As some of Krebs cycle dehydrogenases were activated by Ca2+, this finding was used to propose a new control factor in addition to the one of ADP (B. Chance). All this was discontinued with the wrong use of calculus (today we could indicate bioinformatics in a similar role) in biochemistry that has established less importance to a mitochondrial role after comparative kinetics that today are seen as faulty.

It is important to combat dogmatic reasoning and restore sound scientific foundations in basic medical courses that must urgently reverse the faulty trend that tries to impose a view that goes from the detail towards generalization instead of the correct form that goes from the general finding well understood towards its molecular details. The view that led to curious subjects as bioinformatics in medical courses as training in sequence finding activities can only be explained by its commercial value. The usual form of scientific thinking respects the limits of our ability to grasp new knowledge and relies on reproducibility of scientific results as a form to surpass lack of mathematical equation that defines relationship of variables and the determination of its functional domains. It also uses old scientific roots, as its sound support never replaces existing knowledge by dogmatic and/or wishful thinking. When the sequence of DNA was found as a technical advance to find amino acid sequence in proteins it was just a technical advance. This technical advance by no means could be considered a scientific result presented as an indication that DNA sequences alone have replaced the need to study protein chemistry, its responses to microenvironmental changes in order to understand its multiple conformations, changes in activities and function. As E. Schrodinger correctly describes the chemical structure responsible for the coded form stored of genetic information must have minimal interaction with its microenvironment in order to endure hundreds and hundreds years as seen in Hapsburg’s lips. Only magical reasoning assumes that it is possible to find out in non-reactive chemical structures the properties of the reactive ones.

For instance, knowledge of the reactions of the Krebs cycle clearly indicate a role for solvent that no longer could be considered to be an inert bath for catalytic activity of the enzymes when the transfer of energy include a role for hydrogen transport. The great increase in understanding this change on chemical reaction arrived from conformational energy.

Again, even a rather simplistic view of this atomic property (Conformational energy) is enough to confirm once more, one of the most important contribution of E. Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

In a very simplistic view, while energy manifests itself by the ability to perform work conformational energy as a property derived from our atomic structure can be neutral, positive or negative (no effect, increased or decreased reactivity upon any chemistry reactivity measured as work)

Also:

“I mean the fact that we, whose total being is entirely based on a marvelous interplay of this very kind, yet if all possess the power of acquiring considerable knowledge about it. I think it possible that this knowledge may advance to little just a short of a complete understanding -of the first marvel. The second may well be beyond human understanding.”

In fact, scientific knowledge allows us to understand how biological evolution may have occurred or have not occurred and yet does not present a proof about how it would have being occurred. It will be always be an indication of possible against highly unlike and never a scientific proven fact about the real form of its occurrence.

As was the case of B. Chance in its bioenergetics findings, we may get very important findings that indicates wrong directions in the future as was his case, or directed toward our past.

The Skeleton of Physical Time – Quantum Energies in Relative Space of S-labs

By Radoslav S. Bozov  Independent Researcher

WSEAS, Biology and BioSystems of Biomedicine

Space does not equate to distance, displacement of an object by classically defined forces – electromagnetic, gravity or inertia. In perceiving quantum open systems, a quanta, a package of energy, displaces properties of wave interference and statistical outcomes of sums of paths of particles detected by a design of S-labs.

The notion of S-labs, space labs, deals with inherent problems of operational module, R(i+1), where an imagination number ‘struggles’ to work under roots of a negative sign, a reflection of an observable set of sums reaching out of the limits of the human being organ, an eye or other foundational signal processing system.

While heavenly bodies, planets, star systems, and other exotic forms of light reflecting and/or emitting objects, observable via naked eye have been deduced to operate under numerical systems that calculate a periodic displacement of one relative to another, atomic clocks of nanospace open our eyes to ever expanding energy spaces, where matrices of interactive variables point to the problem of infinity of variations in scalar spaces, however, defining properties of minute universes as a mirror image of an astronomical system. The first and furthermost problem is essentially the same as those mathematical methodologies deduced by Isaac Newton and Albert Einstein for processing a surface. I will introduce you to a surface interference method by describing undetermined objective space in terms of determined subjective time.

Therefore, the moment will be an outcome of statistical sums of a numerical system extending from near zero to near one. Three strings hold down a dual system entangled via interference of two waves, where a single wave is a product of three particles (today named accordingly to either weak or strong interactions) momentum.

The above described system emerges from duality into trinity the objective space value of physical realities. The triangle of physical observables – charge, gravity and electromagnetism, is an outcome of interference of particles, strings and waves, where particles are not particles, or are strings strings, or  are waves waves of an infinite character in an open system which we attempt to define to predict outcomes of tomorrow’s parameters, either dependent or independent as well as both subjective to time simulations.

We now know that aging of a biological organism cannot be defined within singularity. Thereafter, clocks are subjective to apparatuses measuring oscillation of defined parameters which enable us to calculate both amplitude and a period, which we know to be dependent on phase transitions.

The problem of phase was solved by the applicability of carbon relative systems. A piece of diamond does not get wet, yet it holds water’s light entangled property. Water is the dark force of light. To formulate such statement, we have been searching truth by examining cooling objects where the Maxwell demon is translated into information, a data complex system.

Modern perspectives in computing quantum based matrices, 0+1 =1 and/or 0+0=1, and/or 1+1 =0, will be reduced by applying a conceptual frame of Aladdin’s flying anti-gravity carpet, unwrapping both past and future by sending a photon to both, placing present always near zero. Thus, each parallel quantum computation of a natural system approaching the limit of a vibration of a string defining 0 does not equal 0, and 1 does not equal 1. In any case, if our method 1+1 = 1, yet, 1 is not 1 at time i+1. This will set the fundamentals of an operational module, called labris operator or in simplicity S-labs. Note, that 1 as a result is an event predictable to future, while interacting parameters of addition 1+1 may be both, 1 as an observable past, and 1 as an imaginary system, or 1+1 displaced interactive parameters of past observable events. This is the foundation of Future Quantum Relative Systems Interference (QRSI), taking analytical technologies of future as a result of data matrices compressing principle relative to carbon as a reference matter rational to water based properties.

Goedel’s concept of loops exist therefore only upon discrete relative space uniting to parallel absolute continuity of time ‘lags’. ( Goedel, Escher and Bach: An Eternal Golden Braid. A Metaphorical Fugue on Minds and Machines in the Spirit of Lewis Carroll. D Hofstadter.  Chapter XX: Strange Loops, Or Tangled Hierarchies. A grand windup of many of the ideas about hierarchical systems and self-reference. It is concerned with the snarls which arise when systems turn back on themselves-for example, science probing science, government investigating governmental wrongdoing, art violating the rules of art, and finally, humans thinking about their own brains and minds. Does Gödel’s Theorem have anything to say about this last “snarl”? Are free will and the sensation of consciousness connected to Gödel’s Theorem? The Chapter ends by tying Gödel, Escher, and Bach together once again.)  The fight struggle in-between time creates dark spaces within which strings manage to obey light properties – entangled bozons of information carrying future outcomes of a systems processing consciousness. Therefore, Albert Einstein was correct in his quantum time realities by rejecting a resolving cube of sugar within a cup of tea (Henri Bergson 19th century philosopher. Bergson’s concept of multiplicity attempts to unify in a consistent way two contradictory features: heterogeneity and continuity. Many philosophers today think that this concept of multiplicity, despite its difficulty, is revolutionary.) However, the unity of time and space could not be achieved by deducing time to charge, gravity and electromagnetic properties of energy and mass.

Charge is further deduced to interference of particles/strings/waves, contrary to the Hawking idea of irreducibility of chemical energy carrying ‘units’, and gravity is accounted for by intrinsic properties of   anti-gravity carbon systems processing light, an electromagnetic force, that I have deduced towards ever expanding discrete energy space-energies rational to compressing mass/time. The role of loops seems to operate to control formalities where boundaries of space fluctuate as a result of what we called above – dark time-spaces.

Indeed, the concept of horizon is a constant due to ever expanding observables. Thus, it fails to acquire a rational approach towards space-time issues.

Richard Feynman has touched on issues of touching of space, sums of paths of particle traveling through time. In a way he has resolved an important paradigm, storing information and possibly studying it by opening a black box. Schroedinger’s cat is alive again, but incapable of climbing a tree when chased by a dog. Every time a cat climbs a garden tree, a fruit falls on hedgehogs carried away parallel to living wormholes whose purpose of generating information lies upon carbon units resolving light.

In order to deal with such a paradigm, we will introduce i+1 under square root in relativity, therefore taking negative one ( -1 = sqrt (i+1), an operational module R dealing with Wheelers foam squeezed by light, releasing water – dark spaces. Thousand words down!

What is a number? Is that a name or some kind of language or both? Is the issue of number theory possibly accountable to the value of the concept of entropic timing? Light penetrating a pyramid holding bean seeds on a piece of paper and a piece of slice of bread, a triple set, where a church mouse has taken a drop of tear, but a blood drop. What an amazing physics! The magic of biology lies above egoism, above pride, and below Saints.

We will set up the twelve parameters seen through 3+1 in classic realities:

–              discrete absolute energies/forces – no contradiction for now between Newtonian and Albert Einstein mechanics

–              mass absolute continuity – conservational law of physics in accordance to weak and strong forces

–              quantum relative spaces – issuing a paradox of Albert Einstein’s space-time resolved by the uncertainty principle

–              parallel continuity of multiple time/universes – resolving uncertainty of united space and energy through evolving statistical concepts of scalar relative space expansion and vector quantum energies by compressing relative continuity of matter in it, ever compressing flat surfaces – finding the inverse link between deterministic mechanics of displacement and imaginary space, where spheres fit within surface of triangles as time unwraps past by pulling strings from future.

To us, common human beings, with an extra curiosity overloaded by real dreams, value happens to play in the intricate foundation of life – the garden of love, its carbon management in mind, collecting pieces of squeezed cooling time.

The infinite interference of each operational module to another composing ever emerging time constrains unified by the Solar system, objective to humanity, perhaps answers that a drop of blood and a drop of tear is united by a droplet of a substance separating negative entropy to time courses of a physical realities as defined by an open algorithm where chasing power subdue to space becomes an issue of time.

Jose Eduardo de Salles Roselino

Some small errors: For intance an increase i P leads to a decrease in V ( not an increase in V)..

 

Radoslav S. Bozov  Independent Researcher

If we were to use a preventative measures of medical science, instruments of medical science must predict future outcomes based on observable parameters of history….. There are several key issues arising: 1. Despite pinning a difference on genomic scale , say pieces of information, we do not know how to have changed that – that is shift methylome occupying genome surfaces , in a precise manner.. 2. Living systems operational quo DO NOT work as by vector gravity physics of ‘building blocks. That is projecting a delusional concept of a masonry trick, who has not worked by corner stones and ever shifting momenta … Assuming genomic assembling worked, that is dealing with inferences through data mining and annotation, we are not in a position to read future in real time, and we will never be, because of the rtPCR technology self restriction into data -time processing .. We know of existing post translational modalities… 3. We don’t know what we don’t know, and that foundational to future medicine – that is dealing with biological clocks, behavior, and various daily life inputs ranging from radiation to water systems, food quality, drugs…

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Therapeutic Implications for Targeted Therapy from the Resurgence of Warburg ‘Hypothesis’

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

(Note that each portion of the discussion is followed by a reference)

It is now a time to pause after almost a century of a biological scientific discoveries that have transformed the practice of medicine and impacted the lives of several generations of young minds determined to probe the limits of our knowledge.  In the century that we have entered into the scientific framework of medicine has brought together a difficult to grasp evolution of the emergence of human existence from wars, famine, droughts, storms, infectious diseases, and insect born pestilence with betterment of human lives, only unevenly divided among societal classes that have existed since time immemorial. In this short time span there have emerged several generations of physicians who have benefited from a far better medical education that their forebears could have known. In this expansive volume on cancer, we follow an incomplete and continuing challenge to understand cancer, a disease that has become associated with longer life spans in developed nations.

While there are significant improvements in the diagnosis and treatment of cancers, there is still a personal as well as locality factor in the occurrence of this group of diseases, which has been viewed incorrectly as a “dedifferentiation” of mature tissue types and the emergence of a cell phenotype that is dependent on glucose, reverts to a cancer “stem cell type” (loss of stemness), loses cell to cell adhesion, loses orderly maturation, and metastasizes to distant sites. At the same time, physician and nurses are stressed in the care of patients by balancing their daily lives and maintaining a perspective.

The conceptual challenge of cancer diagnosis and management has seemed insurmountable, but owes much to the post World War I activities of Otto Heinrich Warburg. It was Warburg who made the observation that cancer cells metabolize glucose by fermentation in much the way Pasteur 60 years earlier observed fermentation of yeast cells. This metabolic phenomenon occurs even in the presence of an oxygen supply, which would provide a huge deficit in ATP production compared with respiration. The cancer cell is “addicted to glucose” and produced lactic acid. Warburg was awarded the Nobel Prize in Medicine for this work in 1931.

In the last 15 years there has been a resurgence of work on the Warburg effect that sheds much new light on the process that was not previously possible, with significant therapeutic implications.  In the first place, the metabolic mechanism for the Warburg effect was incomplete even at the beginning of the 21st century.  This has been partly rectified with the enlightening elucidation of genome modifications, cellular metabolic regulation, and signaling pathways.

The following developments have become central to furthering our understanding of malignant transformation.

  1. There is usually an identifiable risk factor, such as, H. pylori, or of a chronic inflammatory state, as in the case of Barrett’s esophagus.
  2. There are certain changes in glucose metabolism that have been unquestionably been found in the evolution of this disease. The changes are associated with major changes in metabolic pathways, miRN signaling, and the metabolism geared to synthesis of cells with an impairment of the cell death cycle. In these changes, mitochondrial function is central to both the impaired respiration and the autophagy geared to the synthesis of cancer cells.

The emergence of this cell prototype is characterized by the following, again related to the Warburg effect:

  1. Cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis
  2. 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.
  3. Cancer cells tend to express a partially inhibited splice variant of pyruvate kinase (PK-M2), leading to decreased pyruvate production.
  4. The two proteins that mediate pyruvate conversion to lactate and its export, M-type lactate dehydrogenase and the monocarboxylate transporter MCT-4, are commonly upregulated in cancer cells leading to decreased pyruvate oxidation.
  5. The enzymatic step following mitochondrial entry is the conversion of pyruvate to acetyl-CoA by the pyruvate dehydrogenase (PDH) complex. Cancer cells frequently exhibit increased expression of the PDH kinase PDK1, which phosphorylates and inactivates PDH. This PDH regulatory mechanism is required for oncogene induced transformation and reversed in oncogene-induced senescence.
  6. The PDK inhibitor dichloroacetate has shown some clinical efficacy, which correlates with increased pyruvate oxidation. One of the simplest mechanisms to explain decreased mitochondrial pyruvate oxidation in cancer cells, a loss of mitochondrial pyruvate import, has been observed repeatedly over the past 40 years. This process has been impossible to study at a molecular level until recently, however, as the identities of the protein(s) that mediate mitochondrial pyruvate uptake were unknown.
  7. The mitochondrial pyruvate carrier (MPC) as a multimeric complex that is necessary for efficient mitochondrial pyruvate uptake. The MPC contains two distinct proteins, MPC1 and MPC2; the absence of either leads to a loss of mitochondrial pyruvate uptake and utilization in yeast, flies, and mammalian cells.

A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth

John C. Schell, Kristofor A. Olson, Lei Jiang, Amy J. Hawkins, et al.
Molecular Cell Nov 6, 2014; 56: 400–413.
http://dx.doi.org/10.1016/j.molcel.2014.09.026

In addition to the above, the following study has therapeutic importance:

Glycolysis has become a target of anticancer strategies. Glucose deprivation is sufficient to induce growth inhibition and cell death in cancer cells. The increased glucose transport in cancer cells has been attributed primarily to the upregulation of glucose transporter 1 (Glut1),  1 of the more than 10 glucose transporters that are responsible for basal glucose transport in almost all cell types. Glut1 has not been targeted until very recently due to the lack of potent and selective inhibitors.

First, Glut1 antibodies were shown to inhibit cancer cell growth. Other Glut1 inhibitors and glucose transport inhibitors, such as fasentin and phloretin, were also shown to be effective in reducing cancer cell growth. A group of inhibitors of glucose transporters has been recently identified with IC50 values lower than 20mmol/L for inhibiting cancer cell growth. However, no animal or detailed mechanism studies have been reported with these inhibitors.

Recently, a small molecule named STF-31 was identified that selectively targets the von Hippel-Lindau (VHL) deficient kidney cancer cells. STF-31 inhibits VHL deficient cancer cells by inhibiting Glut1. It was further shown that daily intraperitoneal injection of a soluble analogue of STF-31 effectively reduced the growth of tumors of VHL-deficient cancer cells grafted on nude mice. On the other hand, STF-31 appears to be an inhibitor with a narrow cell target spectrum.

These investigators recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation–like mechanism. These compounds target Glut1 and are efficacious in vivo as anticancer agents. A novel representative compound WZB117 not only inhibited cell growth in cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin. Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which express Glut1 as their sole glucose transporter. Cancer cell treatment with WZB117 led to decreases in levels of Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATP sensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued compound-treated cancer cells, suggesting that the reduction of intracellular ATP plays an important role in the anticancer mechanism of the molecule.

A Small-Molecule Inhibitor of Glucose Transporter 1 Downregulates Glycolysis, Induces Cell-Cycle Arrest, and Inhibits Cancer Cell Growth In Vitro and In Vivo

Yi Liu, Yanyan Cao, Weihe Zhang, Stephen Bergmeier, et al.
Mol Cancer Ther Aug 2012; 11(8): 1672–82
http://dx.doi.org://10.1158/1535-7163.MCT-12-0131

Alterations in cellular metabolism are among the most consistent hallmarks of cancer. These investigators have studied the relationship between increased aerobic lactate production and mitochondrial physiology in tumor cells. To diminish the ability of malignant cells to metabolize pyruvate to lactate, M-type lactate dehydrogenase levels were knocked down by means of LDH-A short hairpin RNAs. Reduction in LDH-A activity resulted in stimulation of mitochondrial respiration and decrease of mitochondrial membrane potential. It also compromised the ability of these tumor cells to proliferate under hypoxia. The tumorigenicity of the LDH-A-deficient cells was severely diminished, and this phenotype was reversed by complementation with the human ortholog LDH-A protein. These results demonstrate that LDH-A plays a key role in tumor maintenance.

The results are consistent with a functional connection between alterations in glucose metabolism and mitochondrial physiology in cancer. The data also reflect that the dependency of tumor cells on glucose metabolism is a liability for these cells under limited-oxygen conditions. Interfering with LDH-A activity as a means of blocking pyruvate to lactate conversion could be exploited therapeutically. Because individuals with complete deficiency of LDH-A do not show any symptoms under ordinary circumstances, the genetic data suggest that inhibition of LDH-A activity may represent a relatively nontoxic approach to interfere with tumor growth.

Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance

Valeria R. Fantin Julie St-Pierre and Philip Leder
Cancer Cell Jun 2006; 9: 425–434.
http://dx.doi.org:/10.1016/j.ccr.2006.04.02

The widespread clinical use of positron-emission tomography (PET) for the detection of aerobic glycolysis in tumors and recent findings have rekindled interest in Warburg’s theory. Studies on the physiological changes in malignant conversion provided a metabolic signature for the different stages of tumorigenesis; during tumorigenesis, an increase in glucose uptake and lactate production have been detected. The fully transformed state is most dependent on aerobic glycolysis and least dependent on the mitochondrial machinery for ATP synthesis.

Tumors ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumors and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumors leads to enhanced, oxygen-independent glucose usage and a lactate based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumor growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualized cancer therapy based on the determination of metabolic changes in tumors that might enable the targeted inhibition of invasion and metastasis.

Other important links between cancer-causing genes and glucose metabolism have been already identified. Activation of the oncogenic kinase Akt has been shown to stimulate glucose uptake and metabolism in cancer cells and renders these cells susceptible to death in response to glucose withdrawal. Such tumor cells have been shown to be dependent on glucose because the ability to induce fatty acid oxidation in response to glucose deprivation is impaired by activated Akt. In addition, AMP-activated protein kinase (AMPK) has been identified as a link between glucose metabolism and the cell cycle, thereby implicating p53 as an essential component of metabolic cell-cycle control.

Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted

S Langbein, M Zerilli, A zur Hausen, W Staiger, et al.
British Journal of Cancer (2006) 94, 578–585.
http://dx.doi.org:/10.1038/sj.bjc.6602962

The unique metabolic profile of cancer (aerobic glycolysis) might confer apoptosis resistance and be therapeutically targeted. Compared to normal cells, several human cancers have high mitochondrial membrane potential (DJm) and low expression of the K+ channel Kv1.5, both contributing toapoptosis resistance. Dichloroacetate (DCA) inhibits mitochondrial pyruvate dehydrogenase kinase (PDK), shifts metabolism from glycolysis to glucose oxidation, decreases DJm, increases mitochondrial H2O2, and activates Kv channels in all cancer, but not normal, cells; DCA upregulates Kv1.5 by an NFAT1-dependent mechanism. DCA induces apoptosis, decreases proliferation, and inhibits tumor growth, without apparent toxicity. Molecular inhibition of PDK2 by siRNA mimics DCA. The mitochondria-NFAT-Kv axis and PDK are important therapeutic targets in cancer; the orally available DCA is a promising selective anticancer agent.

Cancer progression and its resistance to treatment depend, at least in part, on suppression of apoptosis. Although mitochondria are recognized as regulators of apoptosis, their importance as targets for cancer therapy has not been adequately explored or clinically exploited. In 1930, Warburg suggested that mitochondrial dysfunction in cancer results in a characteristic metabolic phenotype, that is, aerobic glycolysis (Warburg, 1930). Positron emission tomography (PET) imaging has now confirmed that most malignant tumors have increased glucose uptake and metabolism. This bioenergetic feature is a good marker of cancer but has not been therapeutically pursued..

The small molecule DCA is a metabolic modulator that has been used in humans for decades in the treatment of lactic acidosis and inherited mitochondrial diseases. Without affecting normal cells, DCA reverses the metabolic electrical remodeling that we describe in several cancer lines (hyperpolarized mitochondria, activated NFAT1, downregulated Kv1.5), inducing apoptosis and decreasing tumor growth. DCA in the drinking water at clinically relevant doses for up to 3 months prevents and reverses tumor growth in vivo, without apparent toxicity and without affecting hemoglobin, transaminases, or creatinine levels. The ease of delivery, selectivity, and effectiveness  make DCA an attractive candidate for proapoptotic cancer therapy which can be rapidly translated into phase II–III clinical trials.

A Mitochondria-K+ Channel Axis Is Suppressed in Cancer and Its Normalization Promotes Apoptosis and Inhibits Cancer Growth

Sebastien Bonnet, Stephen L. Archer, Joan Allalunis-Turner, et al.

Cancer Cell Jan 2007; 11: 37–51.
http://dx.doi.org:/10.1016/j.ccr.2006.10.020

Tumor cells, just as other living cells, possess the potential for proliferation, differentiation, cell cycle arrest, and apoptosis. There is a specific metabolic phenotype associated with each of these conditions, characterized by the production of both energy and special substrates necessary for the cells to function in that particular state. Unlike that of normal living cells, the metabolic phenotype of tumor cells supports the proliferative state. Aim: To present the metabolic hypothesis that (1) cell transformation and tumor growth are associated with the activation of metabolic enzymes that increase glucose carbon utilization for nucleic acid synthesis, while enzymes of the lipid and amino acid synthesis pathways are activated in tumor growth inhibition, and (2) phosphorylation and allosteric and transcriptional regulation of intermediary metabolic enzymes and their substrate availability together mediate and sustain cell transformation from one condition to another. Conclusion: Evidence is presented that demonstrates opposite changes in metabolic phenotypes induced by TGF-β, a cell transforming agent, and tumor growth-inhibiting phytochemicals such as genistein and Avemar, or novel synthetic antileukemic drugs such as STI571 (Gleevec).  Intermediary metabolic enzymes that mediate the growth signaling pathways and promote malignant cell transformation may serve as high efficacy nongenetic novel targets for cancer therapies.

A Metabolic Hypothesis of Cell Growth and Death in Pancreatic Cancer

Laszlo G. Boros, Wai-Nang Paul Lee, and Vay Liang W. Go
Pancreas 2002; 24(1):26–33

Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of kidney cancer. Here, we integrated an unbiased genome-wide RNA interference screen for ccRCC survival regulators with an analysis of recurrently overexpressed genes in ccRCC to identify new therapeutic targets in this disease. One of the most potent survival regulators, the monocarboxylate transporter MCT4 (SLC16A3), impaired ccRCC viability in all eight ccRCC lines tested and was the seventh most overexpressed gene in a meta-analysis of five ccRCC expression datasets.

MCT4 silencing impaired secretion of lactate generated through glycolysis and induced cell cycle arrest and apoptosis. Silencing MCT4 resulted in intracellular acidosis, and reduction in intracellular ATP production together with partial reversion of the Warburg effect in ccRCC cell lines. Intra-tumoral heterogeneity in the intensity of MCT4 protein expression was observed in primary ccRCCs.

MCT4 protein expression analysis based on the highest intensity of expression in primary ccRCCs was associated with poorer relapse-free survival, whereas modal intensity correlated with Fuhrman nuclear grade. Consistent with the potential selection of subclones enriched for MCT4 expression during disease progression, MCT4 expression was greater at sites of metastatic disease. These data suggest that MCT4 may serve as a novel metabolic target to reverse the Warburg effect and limit disease progression in ccRCC.

Clear cell carcinoma (ccRCC) is the commonest subtype of renal cell carcinoma, accounting for 80% of cases. These tumors are highly resistant to cytotoxic chemotherapy and until recently, systemic treatment options for advanced ccRCC were limited to cytokine based therapies, such as interleukin-2 and interferon-α. Recently, anti-angiogenic drugs and mTOR inhibitors, all targeting the HIF–VEGF axis which is activated in up to 91% of ccRCCs through loss of the VHL tumor suppressor gene [1], have been shown to be effective in metastatic ccRCC [2–5]. Although these drugs increase overall survival to more than 2 years [6], resistance invariably occurs, making the identification of new molecular targets a major clinical need to improve outcomes in patients with metastatic ccRCC.

Genome-wide RNA interference analysis of renal carcinoma survival regulators identifies MCT4 as a Warburg effect metabolic target

Marco Gerlinger, Claudio R Santos, Bradley Spencer-Dene, et al.
J Pathol 2012; 227: 146–156
http://dx.doi.org:/10.1002/path.4006

Hypoxia-inducible factor 1 (HIF-1) plays a key role in the reprogramming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which take up glucose and convert it to lactate; pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; and BNIP3, which triggers selective mitochondrial autophagy. The shift from oxidative to glycolytic metabolism allows maintenance of redox homeostasis and cell survival under conditions of prolonged hypoxia. Many metabolic abnormalities in cancer cells increase HIF-1 activity. As a result, a feed-forward mechanism can be activated that drives HIF-1 activation and may promote tumor progression.

Metastatic cancer is characterized by reprogramming of cellular metabolism leading to increased uptake of glucose for use as both an anabolic and a catabolic substrate. Increased glucose uptake is such a reliable feature that it is utilized clinically to detect metastases by positron emission tomography using 18F-fluorodeoxyglucose (FDG-PET) with a sensitivity of >90% [1]. As with all aspects of cancer biology, the details of metabolic reprogramming differ widely among individual tumors. However, the role of specific signaling pathways and transcription factors in this process is now understood in considerable detail. This review will focus on the involvement of hypoxia-inducible factor 1 (HIF-1) in both mediating metabolic reprogramming and responding to metabolic alterations. The placement of HIF-1 both upstream and downstream of cancer metabolism results in a feed-forward mechanism that may play a major role in the development of the invasive, metastatic, and lethal cancer phenotype.

O2 concentrations are significantly reduced in many human cancers compared with the surrounding normal tissue. The median PO2 in breast cancers is 10 mmHg, as compared with65 mmHg in normal breast tissue. Reduced O2 availability induces HIF-1, which regulates the transcription of hundreds of genes that encode proteins involved in every aspect of cancer biology, including: cell immortalization and stem cell maintenance; genetic instability; glucose and energy metabolism; vascularization; autocrine growth factor signaling; invasion and metastasis; immune evasion; and resistance to chemotherapy and radiation therapy.

HIF-1 is a transcription factor that consists of an O2 regulated HIF-1a and a constitutively expressed HIF-1b subunit. In well-oxygenated cells, HIF-1a is hydroxylated on proline residue 402 (Pro-402) and/or Pro-564 by prolyl hydroxylase domain protein 2 (PHD2), which uses O2 and a-ketoglutarate as substrates in a reaction that generates CO2 and succinate as byproducts. Prolylhydroxylated HIF-1a is bound by the von Hippel–Lindau tumor suppressor protein (VHL), which recruits an E3-ubiquitin ligase that targets HIF-1a for proteasomal degradation (Figure 1a). Asparagine 803 in the transactivation domain is hydroxylated in well-oxygenated cells by factor inhibiting HIF-1 (FIH-1), which blocks the binding of the coactivators p300 and CBP. Under hypoxic conditions, the prolyl and asparaginyl hydroxylation reactions are inhibited by substrate (O2) deprivation and/or the mitochondrial generation of reactive oxygen species (ROS), which may oxidize Fe(II) present in the catalytic center of the hydroxylases.

The finding that acute changes in PO2 increase mitochondrial ROS production suggests that cellular respiration is optimized at physiological PO2 to limit ROS generation and that any deviation in PO2 – up or down – results in increased ROS generation. If hypoxia persists, induction of HIF-1 leads to adaptive mechanisms to reduce ROS and re-establish homeostasis, as described below. Prolyl and asparaginyl hydroxylation provide a molecular mechanism by which changes in cellular oxygenation can be transduced to the nucleus as changes in HIF-1 activity.

HIF-1: upstream and downstream of cancer metabolism

Gregg L Semenza
Current Opinion in Genetics & Development 2010, 20:51–56

This review comes from a themed issue on Genetic and cellular mechanisms of oncogenesis Edited by Tony Hunter and Richard Marais

http://dx.doi.org:/10.1016/j.gde.2009.10.009

Hypoxia-inducible factor 1 (HIF-1) regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, genetic instability, vascularization, metabolic reprogramming, autocrine growth factor signaling, invasion/metastasis, and treatment failure. In animal models, HIF-1 overexpression is associated with increased tumor growth, vascularization, and metastasis, whereas HIF-1 loss-of-function has the opposite effect, thus validating HIF-1 as a target. In further support of this conclusion, immunohistochemical detection of HIF-1a overexpression in biopsy sections is a prognostic factor in many cancers. A growing number of novel anticancer agents have been shown to inhibit HIF-1 through a  variety of molecular mechanisms. Determining which combination of drugs to administer to any given patient remains a major obstacle to improving cancer treatment outcomes.

Intratumoral hypoxia The majority of locally advanced solid tumors contain regions of reduced oxygen availability. Intratumoral hypoxia results when cells are located too far from a functional blood vessel for diffusion of adequate amounts of O2 as a result of rapid cancer cell proliferation and the formation of blood vessels that are structurally and functionally abnormal. In the most extreme case, O2 concentrations are below those required for survival, resulting in cell death and establishing a selection for cancer cells in which apoptotic pathways are inactivated, anti-apoptotic pathways are activated, or invasion/metastasis pathways that promote escape from the hypoxic microenvironment are activated. This hypoxic adaptation may arise by alterations in gene expression or by mutations in the genome or both and is associated with reduced patient survival.

Hypoxia-inducible factor 1 (HIF-1) The expression of hundreds of genes is altered in each cell exposed to hypoxia. Many of these genes are regulated by HIF-1. HIF-1 is a heterodimer formed by the association of an O2-regulated HIF1a subunit with a constitutively expressed HIF-1b subunit. The structurally and functionally related HIF-2a protein also dimerizes with HIF-1b and regulates an overlapping battery of target genes. Under nonhypoxic conditions, HIF-1a (as well as HIF-2a) is subject to O2-dependent prolyl hydroxylation and this modification is required for binding of the von Hippel–Lindau tumor suppressor protein (VHL), which also binds to Elongin C and thereby recruits a ubiquitin ligase complex that targets HIF-1a for ubiquitination and proteasomal degradation. Under hypoxic conditions, the rate of hydroxylation and ubiquitination declines, resulting in accumulation of HIF-1a. Immunohistochemical analysis of tumor biopsies has revealed high levels of HIF-1a in hypoxic but viable tumor cells surrounding areas of necrosis.

Genetic alterations in cancer cells increase HIF-1 activity In the majority of clear-cell renal carcinomas, VHL function is lost, resulting in constitutive activation of HIF-1. After re-introduction of functional VHL, renal carcinoma cell lines are no longer tumorigenic, but can be made tumorigenic by expression of HIF2a in which the prolyl residues that are subject to hydroxylation have been mutated. In addition to VHL loss-of-function, many other genetic alterations that inactivate tumor suppressors

Evaluation of HIF-1 inhibitors as anticancer agents

Gregg L. Semenza
Drug Discovery Today Oct 2007; 12(19/20).
http://dx.doi.org:/10.1016/j.drudis.2007.08.006

Hypoxia-inducible factor-1 (HIF-1), which is present at high levels in human tumors, plays crucial roles in tumor promotion by upregulating its target genes, which are involved in anaerobic energy metabolism, angiogenesis, cell survival, cell invasion, and drug resistance. Therefore, it is apparent that the inhibition of HIF-1 activity may be a strategy for treating cancer. Recently, many efforts to develop new HIF-1-targeting agents have been made by both academic and pharmaceutical industry laboratories. The future success of these efforts will be a new class of HIF-1-targeting anticancer agents, which would improve the prognoses of many cancer patients. This review focuses on the potential of HIF-1 as a target molecule for anticancer therapy, and on possible strategies to inhibit HIF-1 activity. In addition, we introduce YC-1 as a new anti-HIF-1, anticancer agent. Although YC-1 was originally developed as a potential therapeutic agent for thrombosis and hypertension, recent studies demonstrated that YC-1 suppressed HIF-1 activity and vascular endothelial growth factor expression in cancer cells. Moreover, it halted tumor growth in immunodeficient mice without serious toxicity during the treatment period. Thus, we propose that YC-1 is a good lead compound for the development of new anti-HIF-1, anticancer agents.

Although many anticancer regimens have been introduced to date, their survival benefits are negligible, which is the reason that a more innovative treatment is required. Basically, the identification of the specific molecular features of tumor promotion has allowed for rational drug discovery in cancer treatment, and drugs have been screened based upon the modulation of specific molecular targets in tumor cells. Target-based drugs should satisfy the following two conditions.

First, they must act by a described mechanism.

Second, they must reduce tumor growth in vivo, associated with this mechanism.

Many key factors have been found to be involved in the multiple steps of cell growth signal-transduction pathways. Targeting these factors offers a strategy for preventing tumor growth; for example, competitors or antibodies blocking ligand–receptor interaction, and receptor tyrosine kinase inhibitors, downstream pathway inhibitors (i.e., RAS farnesyl transferase inhibitors, mitogen-activated protein kinase and mTOR inhibitors), and cell-cycle arresters (i.e., cyclin-dependent kinase inhibitors) could all be used to inhibit tumor growth.

In addition to the intracellular events, tumor environmental factors should be considered to treat solid tumors. Of these, hypoxia is an important cancer-aggravating factor because it contributes to the progression of a more malignant phenotype, and to the acquisition of resistance to radiotherapy and chemotherapy. Thus, transcription factors that regulate these hypoxic events are good targets for anticancer therapy and in particular HIF-1 is one of most compelling targets. In this paper, we introduce the roles of HIF-1 in tumor promotion and provide a summary of new anticancer strategies designed to inhibit HIF-1 activity.

New anticancer strategies targeting HIF-1

Eun-Jin Yeo, Yang-Sook Chun, Jong-Wan Park
Biochemical Pharmacology 68 (2004) 1061–1069
http://dx.doi.org:/10.1016/j.bcp.2004.02.040

Classical work in tumor cell metabolism focused on bioenergetics, particularly enhanced glycolysis and suppressed oxidative phosphorylation (the ‘Warburg effect’). But the biosynthetic activities required to create daughter cells are equally important for tumor growth, and recent studies are now bringing these pathways into focus. In this review, we discuss how tumor cells achieve high rates of nucleotide and fatty acid synthesis, how oncogenes and tumor suppressors influence these activities, and how glutamine metabolism enables macromolecular synthesis in proliferating cells.

Otto Warburg’s demonstration that tumor cells rapidly use glucose and convert the majority of it to lactate is still the most fundamental and enduring observation in tumor metabolism. His work, which ushered in an era of study on tumor metabolism focused on the relationship between glycolysis and cellular bioenergetics, has been revisited and expanded by generations of tumor biologists. It is now accepted that a high rate of glucose metabolism, exploited clinically by 18FDGPET scanning, is a metabolic hallmark of rapidly dividing cells, correlates closely with transformation, and accounts for a significant percentage of ATP generated during cell proliferation. A ‘metabolic transformation’ is required for tumorigenesis. Research over the past few years has reinforced this idea, revealing the conservation of metabolic activities among diverse tumor types, and proving that oncogenic mutations can promote metabolic autonomy by driving nutrient uptake to levels that often exceed those required for cell growth and proliferation.

In order to engage in replicative division, a cell must duplicate its genome, proteins, and lipids and assemble the components into daughter cells; in short, it must become a factory for macromolecular biosynthesis. These activities require that cells take up extracellular nutrients like glucose and glutamine and allocate them into metabolic pathways that convert them into biosynthetic precursors (Figure 1). Tumor cells can achieve this phenotype through changes in the expression of enzymes that determine metabolic flux rates, including nutrient transporters and enzymes [8– 10]. Current studies in tumor metabolism are revealing novel mechanisms for metabolic control, establishing which enzyme isoforms facilitate the tumor metabolic phenotype, and suggesting new targets for cancer therapy.

The ongoing challenge in tumor cell metabolism is to understand how individual pathways fit together into the global metabolic phenotype of cell growth. Here we discuss two biosynthetic activities required by proliferating tumor cells: production of ribose-5 phosphate for nucleotide biosynthesis and production of fatty acids for lipid biosynthesis. Nucleotide and lipid biosynthesis share three important characteristics.

  • First, both use glucose as a carbon source.
  • Second, both consume TCA cycle intermediates, imposing the need for a mechanism to replenish the cycle.
  • Third, both require reductive power in the form of NADPH.

In this Essay, we discuss the possible drivers, advantages, and potential liabilities of the altered metabolism of cancer cells (Figure 1, not shown). 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.

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).

Blood vessels recruited to the tumor microenvironment, however, are disorganized, may not deliver blood effectively, and therefore do not completely alleviate hypoxia (reviewed in Gatenby and Gillies, 2004). 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. Therefore, the Warburg effect—that is, an uncoupling of glycolysis from oxygen levels—cannot be explained solely by upregulation of HIF. Other molecular mechanisms are likely to be important, such as the metabolic changes induced by oncogene activation and tumor suppressor loss.

Oncogene Activation Drives Changes in Metabolism Not only may the tumor microenvironment select for a deranged metabolism, but oncogene status can also drive metabolic changes. Since Warburg’s time, the biochemical study of cancer metabolism has been overshadowed by efforts to identify the mutations that contribute to cancer initiation and progression. Recent work, however, 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; Ramanathan 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.

Cancer Cell Metabolism: Warburg & Beyond

Hsu PP & Sabatini DM
Cell  Sep 5, 2008; 134, 703-705
http://dx.doi.org:/10.1016/j.cell.2008.08.021

Tumor cells respond to growth signals by the activation of protein kinases, altered gene expression and significant modifications in substrate flow and redistribution among biosynthetic pathways. This results in a proliferating phenotype with altered cellular function. These transformed cells exhibit unique anabolic characteristics, which includes increased and preferential utilization of glucose through the non-oxidative steps of the pentose cycle for nucleic acid synthesis but limited de novo fatty  acid   synthesis   and   TCA   cycle   glucose   oxidation. This  primarily nonoxidative anabolic profile reflects an undifferentiated highly proliferative aneuploid cell phenotype and serves as a reliable metabolic biomarker to determine cell proliferation rate and the level of cell transformation/differentiation in response to drug treatment.

Novel drugs effective in particular cancers exert their anti-proliferative effects by inducing significant reversions of a few specific non-oxidative anabolic pathways. Here we present evidence that cell transformation of various mechanisms is sustained by a unique disproportional substrate distribution between the two branches of the pentose cycle for nucleic acid synthesis, glycolysis and the TCA cycle for fatty acid synthesis and glucose oxidation. This can be demonstrated by the broad labeling and unique specificity of [1,2-13C2]glucose to trace a large number of metabolites in the metabolome. Stable isotope-based dynamic metabolic profiles (SIDMAP) serve the drug discovery process by providing a powerful new tool that integrates the metabolome into a functional genomics approach to developing new drugs. It can be used in screening kinases and their metabolic targets, which can therefore be more efficiently characterized, speeding up and improving drug testing, approval and labeling processes by saving trial and error type study costs in drug testing.

Metabolic Biomarker and Kinase Drug Target Discovery in Cancer Using Stable Isotope-Based Dynamic Metabolic Profiling (SIDMAP)

László G. Boros, Daniel J. Brackett and George G. Harrigan
Current Cancer Drug Targets, 2003, 3, 447-455 447

Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150 kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, while silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.

A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila , and Humans

Daniel K. Bricker, Eric B. Taylor, John C. Schell, Thomas Orsak, et al.
Science Express 24 May 2012
http://dx.doi.org:/10.1126/science.1218099

Adenosine deaminase acting on RNA (ADAR) enzymes convert adenosine (A) to inosine (I) in double-stranded (ds) RNAs. Since Inosine is read as Guanosine, the biological consequence of ADAR enzyme activity is an A/G conversion within RNA molecules. A-to-I editing events can occur on both coding and non-coding RNAs, including microRNAs (miRNAs), which are small regulatory RNAs of ~20–23 nucleotides that regulate several cell processes by annealing to target mRNAs and inhibiting their translation. Both miRNA precursors and mature miRNAs undergo A-to-I RNA editing, affecting the miRNA maturation process and activity. ADARs can also edit 3′ UTR of mRNAs, further increasing the interplay between mRNA targets and miRNAs. In this review, we provide a general overview of the ADAR enzymes and their mechanisms of action as well as miRNA processing and function. We then review the more recent findings about the impact of ADAR-mediated activity on the miRNA pathway in terms of biogenesis, target recognition, and gene expression regulation.

Review ADAR Enzyme and miRNA Story: A Nucleotide that Can Make the Difference 

Sara Tomaselli, Barbara Bonamassa, Anna Alisi, Valerio Nobili, Franco Locatelli and Angela Gallo
Int. J. Mol. Sci. 19 Nov 2013; 14, 22796-22816 http://dx.doi.org:/10.3390/ijms141122796

The fermented wheat germ extract (FWGE) nutraceutical (Avemar™), manufactured under “good manufacturing practice” conditions and, fulfilling the self-affirmed “generally recognized as safe” status in the United States, has been approved as a “dietary food for special medical purposes for cancer patients” in Europe. In this paper, we report the adjuvant use of this nutraceutical in the treatment of high-risk skin melanoma patients. Methods: In a randomized, pilot, phase II clinical trial, the efficacy of dacarbazine (DTIC)-based adjuvant chemotherapy on survival parameters of melanoma patients was compared to that of the same treatment supplemented with a 1-year long administration of FWGE. Results: At the end of an additional 7-year-long follow-up period, log-rank analyses (Kaplan-Meier estimates) showed significant differences in both progression-free (PFS) and overall survival (OS) in favor of the FWGE group. Mean PFS: 55.8 months (FWGE group) versus 29.9 months (control group), p  0.0137. Mean OS: 66.2 months (FWGE group) versus 44.7 months (control group), p < 0.0298. Conclusions: The inclusion of Avemar into the adjuvant protocols of high-risk skin melanoma patients is highly recommended.

Adjuvant Fermented Wheat Germ Extract (Avemar™) Nutraceutical Improves Survival of High-Risk Skin Melanoma Patients: A Randomized, Pilot, Phase II Clinical Study with a 7-Year Follow-Up

LV Demidov, LV Manziuk, GY Kharkevitch, NA Pirogova, and EV Artamonova
Cancer Biotherapy & Radiopharmaceuticals 2008; 23(4)
http://dx.doi.org:/10.1089/cbr.2008.0486

Cancer cells possess unique metabolic signatures compared to normal cells, including shifts in aerobic glycolysis, glutaminolysis, and de novo biosynthesis of macromolecules. Targeting these changes with agents (drugs and dietary components) has been employed as strategies to reduce the complications associated with tumorigenesis. This paper highlights the ability of several food components to suppress tumor-specific metabolic pathways, including increased expression of glucose transporters, oncogenic tyrosine kinase, tumor-specific M2-type pyruvate kinase, and fatty acid synthase, and the detection of such effects using various metabonomic technologies, including liquid chromatography/mass spectrometry (LC/MS) and stable isotope-labeled MS. Stable isotope-mediated tracing technologies offer exciting opportunities for defining specific target(s) for food components. Exposures, especially during the early transition phase from normal to cancer, are critical for the translation of knowledge about food components into effective prevention strategies. Although appropriate dietary exposures needed to alter cellular metabolism remain inconsistent and/or ill-defined, validated metabonomic biomarkers for dietary components hold promise for establishing effective strategies for cancer prevention.

Bioactive Food Components and Cancer-Specific Metabonomic Profiles

Young S. Kim and John A. Milner
Journal of Biomedicine and Biotechnology 2011, Art ID 721213, 9 pages
http://dx.doi.org:/10.1155/2011/721213

This reviewer poses the following observation.  The importance of the pyridine nucleotide reduced/oxidized ratio has not been alluded to here, but the importance cannot be understated. It has relevance to the metabolic functions of anabolism and catabolism of the visceral organs.  The importance of this has ties to the pentose monophosphate pathway. The importance of the pyridine nucleotide transhydrogenase reaction remains largely unexplored.  In reference to the NAD-redox state, the observation was made by Nathan O. Kaplan that the organs may be viewed with respect to their primary functions in anabolic or high energy catabolic activities. Thus we find that the endocrine organs are largely tied to anabolic functioning, and to NADP, whereas cardiac and skeletal muscle are highly dependent on NAD. The consequence of this observed phenomenon appears to be related to a difference in the susceptibility to malignant transformation.  In the case of the gastrointestinal tract, the rate of turnover of the epithelium is very high. However, with the exception of the liver, there is no major activity other than cell turnover. In the case of the liver, there is a major commitment to synthesis of lipids, storage of fuel, and synthesis of proteins, which is largely anabolic, but there is also a major activity in detoxification, which is not.  In addition, the liver has a double circulation. As a result, a Zahn infarct is uncommon.  Now we might also consider the heart.  The heart is a muscle syncytium with a high need for oxygen.  Cutting of the oxygen supply makes the myocytes vulnerable to ischemic insult and abberant rhythm abnormalities.  In addition, the cardiomyocyte can take up lactic acid from the circulation for fuel, which is tied to the utilization of lactate from vigorous skeletal muscle activity.  The skeletal muscle is tied to glycolysis in normal function, which has a poor generation of ATP, so that the recycling of excess lactic acid is required by cardiac muscle and hepatocytes.  This has not been a part of the discussion, but this reviewer considers it important to remember in considering the organ-specific tendencies to malignant transformation.

Comment (Aurelian Udristioiu):

Otto Warburg observed that many cancers lose their capacity for mitochondrial respiration, limiting ATP production to anaerobic glycolytic pathways. The phenomenon is particularly prevalent in aggressive malignancies, most of which are also hypoxic [1].
Hypoxia induces a stochastic imbalance between the numbers of reduced mitochondrial species vs. available oxygen, resulting in increased reactive oxygen species (ROS) whose toxicity can lead to apoptotic cell death.
Mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration-.generation in the presence of intact tricarboxylic acid (TCA) enzyme.
One mitochondrial adaptation to increased ROS is over-expression of the uncoupling protein 2 (UCP2) that has been reported in multiple human cancer cell lines [2-3]. Increased UCP2 expression was also associated with reduced ATP production in malignant oxyphilic mouse leukemia and human lymphoma cell lines [4].
Hypoxia reduces the ability of cells to maintain their energy levels, because less ATP is obtained from glycolysis than from oxidative phosphorylation. Cells adapt to hypoxia by activating the expression of mutant genes in glycolysis.
-Severe hypoxia causes a high mutation rate, resulting in point mutations that may be explained by reduced DNA mismatch repairing activity.
The most direct induction of apoptosis caused by hypoxia is determined by the inhibition of the electron carrier chain from the inner membrane of the mitochondria. The lack of oxygen inhibits the transport of protons and thereby causes a decrease in membrane potential. Cell survival under conditions of mild hypoxia is mediated by phosphoinositide-3 kinase (PIK3) using severe hypoxia or anoxia, and then cells initiate a cascade of events that lead to apoptosis [5].
After DNA damage, a very important regulator of apoptosis is the p53 protein. This tumor suppressor gene has mutations in over 60% of human tumors and acts as a suppressor of cell division. The growth-suppressive effects of p53 are considered to be mediated through the transcriptional trans-activation activity of the protein. In addition to the maturational state of the clonal tumor, the prognosis of patients with CLL is dependent of genetic changes within the neoplastic cell population.

1.Warburg O. On the origin of cancer cells. Science 1956; 123 (3191):309-314
PubMed Abstract ; Publisher Full Text

2.Giardina TM, Steer JH, Lo SZ, Joyce DA. Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages. Biochim Biophys Acta 2008, 1777(2):118-129. PubMed Abstract | Publisher Full Text

3. Horimoto M, Resnick MB, Konkin TA, Routhier J, Wands JR, Baffy G. Expression of uncoupling protein-2 in human colon cancer. Clin Cancer Res 2004; 10 (18 Pt1):6203-6207. PubMed Abstract | Publisher Full Text

4. Randle PJ, England PJ, Denton RM. Control of the tricarboxylate cycle and it interactions with glycolysis during acetate utilization in rat heart. Biochem J 1970; 117(4):677-695. PubMed Abstract | PubMed Central Full Text

5. Gillies RJ, Robey I, Gatenby RA. Causes and consequences of increased glucose metabolism of cancers. J Nucl Med 2008; 49(Suppl 2):24S-42S. PubMed Abstract | Publisher Full Text

Shortened version of Comment –

Hypoxia induces a stochastic imbalance between the numbers of reduced mitochondrial species vs. available oxygen, resulting in increased reactive oxygen species (ROS) whose toxicity can lead to apoptotic cell death.
Mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration-.generation in the presence of intact tricarboxylic acid (TCA) enzyme.
One mitochondrial adaptation to increased ROS is over-expression of the uncoupling protein 2 (UCP2) that has been reported in multiple human cancer cell lines. Increased UCP2 expression was also associated with reduced ATP production in malignant oxyphilic mouse leukemia and human lymphoma cell lines.
Severe hypoxia causes a high mutation rate, resulting in point mutations that may be explained by reduced DNA mismatch repairing activity.

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Metastatic Disease (4.3)

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

In the preceding discussions the hematological and nonhematological cancers were elaborated.  These were tumors of blood or solid tumors that are malignant.  Malignant solid tumors have a loss of normal architecture.  Malignant cancers of the blood forming organ also have a disruption of the architecture in the blood forming organs, and they are circulating elements that are either acutely increased in number or chronically increased to very high circulating counts as well as many cells in the marrow.  The diagnosis depends on the type of cell elements and the stage of maturation.  In the case of blood cell cancers, one might consider an intermediate stage that has a long course that is in the case of the myelogenous series, myeloid dysplasia, which includes myelofibrosis, which in either case is not a benign course. In the case of solid tumors, there is an anatomic structure of the cancer site.

The usual structure for a carcinoma is either adjoining cells surrounding a vascular supply, as in the liver, a parenchymal gland, as in pancreas, a tubular structure, as the gastrointestinal tract and lungs (which are embryologically and outpouching of the gut), or a skin surface.  In the case of carcinomas, the cells mature from a basement membrane of small flattened cells that overlie a fibrovascular matrix and an underlying myxoid stroma, perhaps beneath which is a muscular organ, then covered by a flat layer of cells. In the case of all epithelial structures there is an orderly maturation of epithelium from the basal layer to the mature epithelial cells that are elongate, have a brush border, and secrete into the glandular structure.  The cell maturation becomes disrupted and disorderly to different degrees in the development of malignancy from a dysplasia to low grade malignancy, to high grade anaplastic cancer.

The development of a cancer implies the loss of tissue architecture, the replication of cells, the development of a neoplasms circulation (which is the topic of vascular endothelial growth factor (VEGF)), the overgrowth of the circulation so that the tumor has insufficient blood supply, and vascular invasion.  We refer to the Warburg Hypothesis with respect to the malignancy relying on glycolysis in the presence of oxygen (aerobic glycolysis), but it may be questionable to imply that there is sufficient oxygen supply.  In some cases a cancer may occur from a longstanding inflammatory focus.  This has been seen to occur in osteomyelitis and in gastrointestinal fistulas.  The growth of a neoplasm, when it exceeds its blood supply, requires adaptive changes. The most obvious to consider would be a decreased reliance of mitochondrial respiration.  Warburg refer to the increase production of lactic acid as analogous to Pasteur observation of fermentation in yeast (Pasteur effect).  He measured the lactic acid production by various tissues, and the consumption with the oxygen consumption showed that in many tissues approximately two molecules of lactate are prevented from appearing when one molecule of oxygen is consumed – a relationship that Meyerhof had found in muscle. This he expressed as the “Meyerhof quotient”:

Anaerobic glycolysis – aerobic glycolysis/oxygen consumption

Ref: Otto Warburg: Cell Physiologist, Biochemist, and Eccentric
Hans Krebs in collaboration with Roswitha Schmid
Clarendon Press, Oxford, 1981. Pp 19-25.

The special feature of cancer cells was the high rate of glycolysis in the presence of oxygen, whereas muscle can form lactate from carbohydrate in the absence of oxygen. This led to the discovery that all animal tissues are capable of glycolysis both aerobically and anaerobically.  Pasteur had established 60 years earlier that the rates of fermentation are generally hiugh anaerobically, but low aerobically. This led Warburg to the conclusion that cancer cells are distinguished from noncancer cells by their failure to suppress glycolysis in the presence of oxygen. He discovered in 1926 that the link between respiration and fermentation can be severed by a specific inhibitor, ethylcarbylamine. He looked at carbylethylamine as an inhibitor of the ‘Pasteur effect’, and determined that the catalyst was a heavy metal ion. But the proposed mechanism was shown not to be correct by Engelhardt, Lynen, Bucher, Lowry, Racker, and Sols.The activity of the enzyme phospofructokinase is regulated by the concentrations of ATP, ADP and inorganic phosphate (Pi). The “allosteric properties” of PFK could account for the ‘Pasteur effect’.  ATP inactivates PFK, while ADP and Pi activate it. Further, etylcarbylamine was found to be an uncoupler of oxidative phosphorylation (OxPhos), but Warburg was right in postulating that a heavy metal was involved since heavy metals are involved in
OxPhos.  The explanation for this is now that when malignant transformation occurs, the cells’ energy supply is redirected from their normal function to growth. This change was found to be irreversible upon restoration of oxygen supply.

The topic of discussion is metastasis. What does it have to do with malignancy and respiration? Metastasis is the other key feature of cancer cells. What it has to do with respiration would probably tie in with the change in the cells’ energy supply that is directed toward proliferation. As the cell metabolism is reconfigured, there is also a change in the cell signaling with respect to apoptosis and the events regarding autophagy.  This has to extend beyond the mitochondria, mainly because autophagy involves mitophagy, the ER and the entire cytoskeleton.  This means that the cytoplasmic relationship to the intercellular matrix and the fibroblast stroma would have to be affected, as the cell breaks away from its close association with adjacent cells.  Cells can migrate to adjacent lymphatic structures, and either enter the circulation by way of the lymphatics or by invasion of the venous circulation directly. In any case, entry into the circulation allows for transport to distant sites.  With respect to migration to distant sites, we recall the hypothesis of Paget that the cells metastasize directly into the circulating blood, and they may ‘seed’ to favorable organs.

The discussion now turns to the assessment of apoptosis as a means to inhibition of cancer cell lines, which proliferate if unchecked and migrate away from the primary site.  I use a few examples from a symposium volume of the Annals of the New York Academy of Sciences:

Apoptosis: From Signaling Pathways to Therapeutic Tools.
Ed, Mark Diederich
ANYAA9 2003; 1010:1-799
The role of β-glucuronidase in induction of apoptosis by Genistein Combined Polysaccharide (GCP) in xenogenetic mice bearing human mammary cancer cells.
Yuan L, Wagatsuma C, Sun B, Kim Jung-Hwan, Surh Young-Joon
Ann NY Acad Sci 2003;1010: 347-349.
http://dx.doi.org:/10.1196/annals.1299.063

  • GCP inhibits tumor cell growth through multiple mechanisms, including induction of tumor apoptosis
  • The biological activities of genistein (aglycon) are more evident in tumor tissues than in normal tissues.
  • Hiugh doses of genistein administration rarely induces toxicity to normal tissues.
  • Higher levels of β-glucuronidase expression in tumor tissues results in more genistein aglycon, leading to tumor destruction.

Induction of apoptosis in human pancreatic cancer cells by docosahexanoic acid
Merendino N, Molinari R, Loppi B, Pessina G, D’Aquino M, Tomassi G, Velotti F.
Ibid 361-364. http://dx.doi.org:/10.1196/annals.1299.143

Polyunsaturated fatty acids have been indicated to induce anti-proliferative and/or apoptotic effects in various tumor cells. We showed that, at a 200-μM concentration, both alpha-linoleic (18:2 n-6; LA) or docosahexaenoic (22:6 n-3; DHA) acid inhibited cell growth, while only DHA induced apoptosis in the human Paca-44 pancreatic cancer cell line. Investigating the mechanism underlying DHA-induced apoptosis, we showed that DHA induced a rapid and dramatic (>60%) intracellular depletion of reduced glutathione (GSH), without affecting oxidized glutathione (GSSG). Moreover, using two specific inhibitors of carrier-mediated GSH extrusion, cystathionine or methionine, we observed that GSH depletion occurred via an active GSH extrusion, and that inhibition of GSH efflux completely reversed apoptosis. These results provide the first evidence for a possible causative role of GSH depletion in DHA-induced apoptosis.

Opposite phenotypes of cancer and aging arise from alternative regulation of common signaling pathways.
Ukraintseva SV1, Yashin AI.
Ann N Y Acad Sci. 2003 Dec; 1010:489-92.
http://dx.doi.org:/10.1196/annals.1299.089

Phenotypic features of malignant and senescent cells are in many instances opposite. Cancer cells do not “age”; their metabolic, proliferative, and growth characteristics are opposite to those observed with cellular aging (both replicative and functional). In many such characteristics cancer cells resemble embryonic cells. One can say that cancer manifests itself as a local, uncontrolled “rejuvenation” in an organism. Available evidence from human and animal studies suggests that the opposite phenotypic features of aging and cancer arise from the opposite regulation of genes participating in apoptosis/growth arrest or growth signal transduction pathways in cells. This fact may be applicable in the development of new anti-aging treatments. Genes that are contrarily regulated in cancer and aging cells (e.g., proto-oncogenes or tumor suppressors) could be candidate targets for anti-aging interventions. Their “cancer-like” regulation, if strictly controlled, might help to rejuvenate the human organism.

CUGBP2 Plays a Critical Role in Apoptosis of Breast Cancer Cells in Response to Genotoxic Injury
Mukhopadhyay D, Jung J, Murmu N, Houchen CW, Dieckgraefe BK, Anant A
Ibid 504–509. http://dx.doi.org:/10.1196/annals.1299.093

 Posttranscriptional control of gene expression plays a key role in regulating gene expression in cells undergoing apoptosis. Cyclooxygenase-2 (COX-2) is a crucial enzyme in the conversion of arachidonic acid to prostaglandin E2 (PGE2) and is significantly upregulated in many types of adenocarcinomas. COX-2 overexpression leads to increased PGE2 production, resulting in increased cellular proliferation. PGE2 enhances the resistance of cells to ionizing radiation. Accordingly, understanding mechanisms regulating COX-2 expression may lead to important therapeutic advances. Besides transcriptional control, COX-2 expression is significantly regulated by mRNA stability and translation. We have previously demonstrated that RNA binding protein CUGBP2 binds AU-rich sequences to regulate COX-2 mRNA translation. In the current study, we have determined that expression of both COX-2 mRNA and CUGBP2 mRNA are induced in MCF-7 cells, a breast cancer cell line, following exposure to 12 Gy γ-irradiation. However, only CUGBP2 protein is induced, but COX-2 protein levels were not altered. Silencer RNA (siRNA)-mediated inhibition of CUGBP2 reversed the block in COX-2 protein expression. Furthermore, MCF-7 cells underwent apoptosis in response to radiation injury, which was also reversed by CUGBP2 siRNAs. These data suggest that CUGBP2 is a critical regulator of the apoptotic response to genotoxic injury in breast cancer cells.

Multiple and synergistic deregulations of apoptosis-controlling genes in pancreatic carcinoma cells
A Trauzold,1 S Schmiedel,1 C Röder,1 C Tams,1 M Christgen,1 S Oestern,1 A Arlt,2 S Westphal,1 M Kapischke,1 H Ungefroren,1 and H Kalthoff1,
Ibid 510-513.  Br J Cancer. 2003 Nov 3; 89(9): 1714–1721.
http://dx.doi.org/10.1038%2Fsj.bjc.6601330

CD95, TRAIL-R1 (tumor necrosis factor-related apoptosis inducing ligand-receptor 1) and TRAIL-R2 are members of the TNF-receptor family of transmembrane proteins that are capable of inducing apoptosis (Wiley et al, 1995Pitti et al, 1996Pan et al, 1997Peter et al, 1998). Following ligand binding, the receptors oligomerize and the pro-apoptotic molecules TRADD, FADD and FLICE/caspase-8 are recruited to their intracellular death domain forming the ‘death-inducing signaling complex’ (DISC) (Krammer, 1999). The subsequent events leading to apoptosis depend on the specific cell type being challenged. In type I cells the bulk induction of caspase-8 at the DISC leads to the direct activation of the effector caspase 3. In type II cells only little amounts of caspase-8 are activated at the DISC requiring the pro-apoptotic mitochondrial amplification loop for efficient caspase-3 activation (Scaffidi et al, 1998).
In Vivo Imaging of Chemotherapy-Induced Apoptosis in Human Cancers
T Belhocine, N Steinmetz, A Green, P Rigo
Ibid 525-529. http://dx.doi.org:/10.1196/annals.1299.097

Rationale. Induction of apoptosis in sensitive tumor cells is the main mechanism of action of chemotherapy agents in human cancers. Also, the assessment of drug-induced apoptosis soon after chemotherapy may be an early predictor of treatment efficacy. Patients and Methods. A phase I/II study was prospectively conducted in 15 patients presenting with proven lung cancers (n= 10), breast cancers (n= 2), and lymphomas (n= 3) to assess the value of the 99mTc-radiolabeled recombinant human (rh) Annexin V for imaging apoptosis immediately after completion of the first course of chemotherapy. Early Annexin V findings post-chemotherapy (day+1, day+2) were also compared to the tumor status at 6 to 12 weeks post-treatment.
Results. All lung and lymphoma patients with an increased tracer uptake post-treatment (n= 8) had either partial or complete tumor response. Five patients with no tracer uptake had progressive disease. However, two breast cancers had a response to treatment, although no significant tracer uptake was observed. Tumor response and survival time were significantly correlated with the 99mTc-labeled Annexin V uptake. No serious events related to tracer administration were noted. Conclusion. Preliminary results of this pilot study demonstrate the feasibility of the 99mTc-labeled Annexin V uptake for the in vivo imaging of apoptosis after one course of chemotherapy. If confirmed on larger series, these promising results may open new perspectives in the management of oncology patients.

In vivo photoacoustic imaging of chemotherapy-induced apoptosis in squamous cell carcinoma using a near-infrared caspase-9 probe.
Yang Q1Cui HCai SYang XForrest ML.
J Biomed Opt. 2011 Nov; 16(11):116026.
http://dx.doi.org:/10.1117/1.3650240

Anti-cancer drugs typically exert their pharmacological effect on tumors by inducing apoptosis, or programmed cell death, within the cancer cells. However, no tools exist in the clinic for detecting apoptosis in real time. Microscopic examination of surgical biopsies and secondary responses, such as morphological changes, are used to verify efficacy of a treatment. Here, we developed a novel near-infrared dye-based imaging probe to directly detect apoptosis with high specificity in cancer cells by utilizing a noninvasive photoacoustic imaging (PAI) technique. Nude mice bearing head and neck tumors received cisplatin chemotherapy (10 mg/kg) and were imaged by PAI after tail vein injection of the contrast agent. In vivo PAI indicated a strong apoptotic response to chemotherapy on the peripheral margins of tumors, whereas untreated controls showed no contrast enhancement by PAI. The apoptotic status of the mouse tumor tissue was verified by immunohistochemical techniques staining for cleaved caspase-3 p11 subunit. The results demonstrated the potential of this imaging probe to guide the evaluation of chemotherapy treatment.

Noninvasive imaging techniques are necessary for early cancer detection and evaluation of the chemotherapeutic effect on tumors. Current diagnostic imaging techniques generally include γ-scintigraphy, magnetic resonance imaging, computed tomography, and ultrasonography; however, these techniques only give morphological information on the tumor. These techniques do not report the biochemical response of the tumor to treatment and physical changes in the tumor in response to treatment may take days to weeks to fully manifest. Positron emission topography and SPECT can indirectly detect tumor response to treatment due to changes in metabolic activity and blood perfusion, respectively. However, no clinical imaging technique can directly detect the biochemical response, e.g., apoptosis, of tumors to treatment. Since apoptosis often occurs within in the first 18 to 36 h after treatment, direct imaging of apoptosis would rapidly indicate if there is a response in the tumor to chemotherapy.

Photoacoustic imaging (PAI) overcomes the spatial and resolution limitations of conventional imaging techniques at a relatively low cost,12 and it has shown its potential to monitor the growth of melanoma brain tumors3 and melanoma metastasis in sentinel lymph nodes.4 However, ascribed to the fact that PAI utilizes the optical absorption of tissues for contrast, it cannot differentiate normal from cancerous cells unless the cells are overexpressing chromomeric marker (e.g., melanomas) or labeled by reporter moieties as contrast agent to enhance the contrast between normal and pathological tissues. In this case, application of a contrast agent such as fluorochromes is expected to facilitate both the visualization of head and neck squamous cell carcinoma (HNSCC) cancer cells and their response to treatment in vivo by PAI.

We have synthesized a near-infrared fluorescent imaging probe – IR780-linker-Val-Ala-Glu(OMe)-FMK by conjugating a fluorochrome (IR780) to Z-Val-Ala-Glu (OMe), a cell permeable caspase inhibitor. The activation of caspase family of cysteine proteases has been recognized as a critical event of apoptosis, which is a physiological process of type I programmed cell death. Typically anti-cancer agents act on cancer cells to induce apoptosis, so apoptosis is a rapid and definite indicator of tumor response. For this reason, apoptosis is used in screening drug candidates in cell culture. The fluoromethyl ketone of the tripeptides valine, alanine, and O-methyle-glutamic acid [Val-Ala-Glu(OMe)-FMK] can specifically and irreversibly bind to the cysteine residue at the active site of caspase-9.5 Our preliminary in vitro cell-imaging test with prostate cancer DU 145 cells demonstrated the sensitivity of this imaging probe for cell apoptosis.6 In this study, we evaluated the application of IR780-linker-Val-Ala-Glu(OMe)-FMK for PAI to detect procaspase-9 activation caused by anticancer drug treatment in living nude mice bearing HNSCC tumors.

Increase in PA amplitude within the HNSCC tumor after intravenous injection of imaging agent

Increase in PA amplitude within the HNSCC tumor after intravenous injection of imaging agent

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3221716/bin/JBOPFO-000016-116026_1-g002.jpg

Fourteen micron (thickness) sections of the tumor tissue were stained with a goat primary polyclonal antibody for cleaved caspase-3 p11 subunit (Asp-175-Ser-176) and a donkey anti-goat secondary antibody with a fluorescein isothiocyanate (FITC) fluorophore (Santa Cruz Biotechnology Inc., Santa Cruz, California). Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI).

Maximum amplitude projection images obtained from the PAI of the HNSCC tumor region shown in Fig. ​(Fig.2).2 were converted to grayscale images. The grayscale images at various time points were linearly aligned using the scale-invariant feature transform function of Fiji/ImageJA software (ver. 20110307, http://pacific.mpi-cbg.de/wiki/index.php/Fiji) (Fig. ​(Fig.3).3). Quantification of PA signal intensity within the tumor region was performed in triplet for each image by measuring the mean gray value (units: gray/pixel) of the circled tumor region. The extent of signal enhancement was calculated by normalizing the tumor signal against a background reading taken immediately before injection of the imaging agent (Fig. ​(Fig.2).2)

Apoptosis in the tumor tissues was independently verified by immunohistochemical staining for caspase 3, a downstream indicator of apoptosome-activated caspase-mediated apoptosis that would not cross-react with the caspase-9 PA probe. Figure ​(Figure4a) 4a represents a control section stained with the secondary antibody alone (autofluorescence of the tissue without apparent staining); while, Fig. ​(Fig.4b).4b shows the immunostaining of the caspase-3 p11 subunit (green) and the DAPI staining of cell nuclei. The intense green fluorescence in these sections suggests the wide spread apoptosis of cells in the tumor tissues after intravenous administration of high-dose cisplatin. In addition, cells on the peripheral of the tumor stained more strongly for caspase 3 (green fluorescence) compared to cells at the tumor interior. This was consistent with the PA imaging of apoptosis that showed strong apoptosis at the tumor peripheral, suggesting chemotherapeutics had penetrated the outer layers of the tumor and induced apoptosis.

Immunostaining for apoptosis in tumor

Immunostaining for apoptosis in tumor

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3221716/bin/JBOPFO-000016-116026_1-g004.jpg

Immunostaining for apoptosis in tumor. (a) Representative control section stained with secondary antibody alone and (b) tissue section of the HNSCC tumor stained for caspase-3 p11 subunit after cisplatin treatment.

I now consider mechanisms of metastasis as currently viewed.

Metastasis mechanisms.
Geiger TR1Peeper DS.
Biochim Biophys Acta. 2009 Dec; 1796(2):293-308.  http://dx.doi.org:/10.1016/j.bbcan.2009.07.006.

Metastasis, the spread of malignant cells from a primary tumor to distant sites, poses the biggest problem to cancer treatment and is the main cause of death of cancer patients. It occurs in a series of discrete steps, which have been modeled into a “metastatic cascade”. In this review, we comprehensively describe the molecular and cellular mechanisms underlying the different steps, including Epithelial-Mesenchymal Transition (EMT), invasion, anoikis, angiogenesis, transport through vessels and outgrowth of secondary tumors. Furthermore, we implement recent findings that have broadened and challenged the classical view on the metastatic cascade, for example the establishment of a “premetastatic niche”, the requirement of stem cell-like properties, the role of the tumor stroma and paracrine interactions of the tumor with cells in distant anatomical sites. A better understanding of the molecular processes underlying metastasis will conceivably present us with novel targets for therapeutic intervention.

Axis of evil: molecular mechanisms of cancer metastasis Thomas Bogenrieder1 and Meenhard Herlyn1
Oncogene (2003) 22, 6524–6536.
http://dx.doi.org:/doi:10.1038/sj.onc.1206757

Although the genetic basis of tumorigenesis may vary greatly between different cancer types, the cellular and molecular steps required for metastasis are similar for all cancer cells. Not surprisingly, the molecular mechanisms that propel invasive growth and metastasis are also found in embryonic development, and to a less perpetual extent, in adult tissue repair processes. It is increasingly apparent that the stromal microenvironment, in which neoplastic cells develop, profoundly influences many steps of cancer progression, including the ability of tumor cells to metastasize. In carcinomas, the influences of the microenvironment are mediated, in large part, by bidirectional interactions (adhesion, survival, proteolysis, migration, immune escape mechanisms lymph-/angiogenesis, and homing on target organs) between epithelial tumor cells and neighboring stromal cells, such as fibroblasts as well as endothelial and immune cells. In this review, we summarize recent advances in understanding the molecular mechanisms that govern this frequently lethal metastatic progression along an axis from primary tumor to regional lymph nodes to distant organ sites. Affected proteins include growth factor signaling molecules, chemokines, cell–cell adhesion molecules (cadherins, integrins) as well as extracellular proteases (matrix metalloproteinases). We then discuss promising new therapeutic approaches targeting the microenvironment. We note, however, that there is still too little knowledge of how the many events are coordinated and integrated by the cancer cell, with conspiratorial help by the stromal component of the host. Before drug development can proceed with a legitimate chance of success, significant gaps in basic knowledge need to be filled.

Metastases to regional lymph nodes are detected at diagnosis and surgery in approximately one-third of breast, colorectal, uterine cervix, and oral cavity and pharynx cancer patients, and one-quarter of esophageal, lung pancreas, gastric and bladder cancer patients (Greenlee et al., 2001). The high mortality rates associated with cancer are caused by the metastatic spread of tumor cells from the site of their origin. In fact, metastases are the cause of 90% of cancer deaths (Hanahan and Weinberg, 2000). The prognosis for a patient who is diagnosed with advanced invasive or metastatic disease remains little better than it was decades ago (Sporn, 1997). Tumor cells invade either the blood or lymphatic vessels to access the general circulation and then establish themselves in other (visceral) tissues. Ultimately, they become surgically unresectable, with pharmacological or radiological long-term control being uncommon (Stacker et al., 2002).

Although the genetic basis of tumorigenesis may vary greatly between different cancer types, the cellular and molecular steps required for metastasis are generally similar for all solid tumor cells (Woodhouse et al., 1997Liotta and Kohn, 2003). Not surprisingly, the molecular mechanisms that propel invasive growth and metastasis are also found in embryonic development, and, however to a less perpetual/chronic/aggressive/quantitatively different extent, in adult tissue maintenance (e.g. involving stem cell differentiation) and repair processes (‘tumors are wounds that do not heal’) (Dvorak, 1986). We now view cancer as a complex tissue resulting from disrupted organ homeostasis, rather than focusing on the cancer cell, and the genes within it, alone (Hanahan and Weinberg, 2000;Bissell and Radisky, 2001Bogenrieder and Herlyn, 2002Wiseman and Werb, 2002). Normal tissue homeostasis is maintained between epithelial cells and their microenvironment, such as fibroblasts, endothelial and immunocompetent cells, and the extracellular matrix (ECM). Similarly, during malignant transformation and progression, there are (however deregulated) reciprocal and conspirational interactions between the neoplastic cells and the adjacent stromal cells (Hsu et al., 2002). A series of recent investigations have shown that aberrations in the stroma can both precede and stimulate the development of cancers (reviewed in Bissell and Radisky, 2001Wiseman and Werb, 2002).

The process of metastasis involves an intricate interplay between altered cell adhesion, survival, proteolysis, migration, lymph-/angiogenesis (see articles in this issue by R Kerbel and P Campochiaro, pp. NNN–NNN), immune escape mechanisms, and homing on target organs (Table 1). However, there is still very little knowledge of how these events are coordinated by the cancer cell, with conspirational help by the stromal component (microenvironment) of the host (Clark, 1991Hsu et al., 2002). This process is usually said to be ‘uncontrolled’. As we shall see, however, it is by no means purely stochastic, but rather a finely tuned molecular machinery with active tumor cell–host collaboration. Thus, all explanations of ‘success’ of the metastatic axis contain a strong element of determinism. Whereas the early steps in the metastastic campaign are completed very efficiently, metastasis is an inefficient process in its later steps, especially the regulation of cancer cell growth at the secondary sites (Luzzi et al., 1998Cameron et al., 2000Chambers et al., 2002). Given that spread of the tumor to distant organs is usually lethal, more intense studies of these molecular mechanisms assume general importance to develop more effective anticancer strategies. In the following discussion of specific molecular mechanisms, we have often chosen to draw mainly from examples that pertain to melanoma progression, although similar processes are most likely also operating during oncogenesis of a wide range of cancers.

The classical metastatic cascade encompasses intravasation by tumor cells, their circulation in lymph and blood vascular systems, arrest in distant organs, extravasation, and growth into metastatic foci (Herlyn and Malkowicz, 1991;Woodhouse et al., 1997). Ann Chambers et al. (2001),(2002) have demonstrated in murine models that the limiting factor for metastasis formation is growth after extravasation (Figure 1a). Recently, this extravasation model has been challenged by Ruth Muschel and co-workers (Al-Mehdi et al., 2000Wong et al., 2002), who showed that tumor cells can readily proliferate after arrest in blood vessels, suggesting that extravasation is not a prerequisite for metastatic growth (Figure 1b). In a separate series of experiments, Mary Hendrix and co-workers described that tumor cells can even have endothelial cell-like functions and form channels that allow fluid flow (Maniotis et al., 1999Folberg et al., 2000) (Figure 1c). The group has identified some of the players, such as EphA2 and VE-cadherin, on aggressive melanoma cells that are shared with endothelial cells and that are likely involved in ‘vasculogenic mimicry’. Vasculogenic mimicry is the ability of aggressive cancer cells to form de novo vessel-like networks in vitro in the absence of endothelial cells or fibroblasts, concomitant with their expression of vascular-associated cellular marker (Sood et al., 2001,2002). Tumor cell plasticity is demonstrated by the ability of tumor cells to adopt a variety of phenotypes, including an endothelial phenotype (Sood et al., 2001, 2002). These exciting new findings underscore the plasticity of malignant cells from advanced tumor progression stages, and they require from tumor biologists a more dynamic view of the metastatic cascade. If the biological phenotype of metastasis must be portrayed flexibly, then we need a new ‘yardstick,’ a normal cell, to better characterize and understand the many faces of metastasis. We need to understand how the malignant cells exert cooperation from the normal cells. Our central hypothesis is that both normal and malignant cells utilize the same molecules for invasion, but that differences in downstream signaling events allow the tumor cells to dominate over normal cells in the microenvironment. This ‘dominant plasticity’ model of cancer metastasis takes into account the flexible response of malignant cells to microenvironmental pressures while maintaining dominance over the normal parenchymal and stromal cells.

Models of metastasis

Models of metastasis

http://www.nature.com/onc/journal/v22/n42/images/1206757f1.jpg

Models of metastasis. (a) According to Chambers and co-workers, only a very small population of injected cells (2%) form micrometastases, although over 87% are arrested in the liver. Furthermore, not all of the micrometastases persist, and the progressively growing metastases that kill the mice arise only from a small subset (0.02%) of the injected cells. (b) Muschel and co-workers recently proposed a new model for pulmonary metastasis in which endothelium-attached tumor cells that survived the initial apoptotic stimuli proliferate intravascularly. Thus, a principal tenet of this new model is that the extravasation of tumor cells is not a prerequisite for metastatic colony formation and that the initial proliferation takes place within the blood vessels. (c) The unique ability of aggressive tumor cells to generate patterned networks, similar to the patterned networks during embryonic vasculogenesis, and concomitantly to express vascular markers associated with endothelial cells, their precursors and other vascular cells has been termed ‘vasculogenic mimicry’ by Hendrix and co-workers

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Hypoxia Inducible Factor 1 (HIF-1)

Writer and Curator: Larry H Bernstein, MD, FCAP

7.9  Hypoxia Inducible Factor 1 (HIF-1)

7.9.1 Hypoxia and mitochondrial oxidative metabolism

7.9.2 Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

7.9.3 Hypoxia-Inducible Factors in Physiology and Medicine

7.9.4 Hypoxia-inducible factor 1. Regulator of mitochondrial metabolism and mediator of ischemic preconditioning

7.9.5 Regulation of cancer cell metabolism by hypoxia-inducible factor 1

7.9.6 Coming up for air. HIF-1 and mitochondrial oxygen consumption

7.9.7 HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption

7.9.8 HIF-1. upstream and downstream of cancer metabolism

7.9.9 In Vivo HIF-Mediated Reductive Carboxylation

7.9.10 Evaluation of HIF-1 inhibitors as anticancer agents

 

 

7.9.1 Hypoxia and mitochondrial oxidative metabolism

Solaini G1Baracca ALenaz GSgarbi G.
Biochim Biophys Acta. 2010 Jun-Jul; 1797(6-7):1171-7
http://dx.doi.org/10.1016/j.bbabio.2010.02.011

It is now clear that mitochondrial defects are associated with a large variety of clinical phenotypes. This is the result of the mitochondria’s central role in energy production, reactive oxygen species homeostasis, and cell death. These processes are interdependent and may occur under various stressing conditions, among which low oxygen levels (hypoxia) are certainly prominent. Cells exposed to hypoxia respond acutely with endogenous metabolites and proteins promptly regulating metabolic pathways, but if low oxygen levels are prolonged, cells activate adapting mechanisms, the master switch being the hypoxia-inducible factor 1 (HIF-1). Activation of this factor is strictly bound to the mitochondrial function, which in turn is related with the oxygen level. Therefore in hypoxia, mitochondria act as [O2] sensors, convey signals to HIF-1directly or indirectly, and contribute to the cell redox potential, ion homeostasis, and energy production. Although over the last two decades cellular responses to low oxygen tension have been studied extensively, mechanisms underlying these functions are still indefinite. Here we review current knowledge of the mitochondrial role in hypoxia, focusing mainly on their role in cellular energy and reactive oxygen species homeostasis in relation with HIF-1 stabilization. In addition, we address the involvement of HIF-1 and the inhibitor protein of F1F0 ATPase in the hypoxia-induced mitochondrial autophagy.

Over the last two decades a defective mitochondrial function associated with hypoxia has been invoked in many diverse complex disorders, such as type 2 diabetes [1] and [2], Alzheimer’s disease [3] and [4], cardiac ischemia/reperfusion injury [5] and [6], tissue inflammation [7], and cancer [8][9][10],[11] and [12].

The [O2] in air-saturated aqueous buffer at 37 °C is approx. 200 μM [13]; however, mitochondria in vivo are exposed to a considerably lower [O2] that varies with tissue and physiological state. Under physiological conditions, most human resting cells experience some 5% oxygen tension, however the [O2] gradient occurring between the extracellular environment and mitochondria, where oxygen is consumed by cytochrome c oxidase, results in a significantly lower [O2] exposition of mitochondria. Below this oxygen level, most mammalian tissues are exposed to hypoxic conditions  [14]. These may arise in normal development, or as a consequence of pathophysiological conditions where there is a reduced oxygen supply due to a respiratory insufficiency or to a defective vasculature. Such conditions include inflammatory diseases, diabetes, ischemic disorders (cerebral or cardiovascular), and solid tumors. Mitochondria consume the greatest amount (some 85–90%) of oxygen in cells to allow oxidative phosphorylation (OXPHOS), which is the primary metabolic pathway for ATP production. Therefore hypoxia will hamper this metabolic pathway, and if the oxygen level is very low, insufficient ATP availability might result in cell death [15].

When cells are exposed to an atmosphere with reduced oxygen concentration, cells readily “respond” by inducing adaptive reactions for their survival through the AMP-activated protein kinase (AMPK) pathway (see for a recent review [16]) which inter alia increases glycolysis driven by enhanced catalytic efficiency of some enzymes, including phosphofructokinase-1 and pyruvate kinase (of note, this oxidative flux is thermodynamically allowed due to both reduced phosphorylation potential [ATP]/([ADP][Pi]) and the physiological redox state of the cell). However, this is particularly efficient only in the short term, therefore cells respond to prolonged hypoxia also by stimulation of hypoxia-inducible factors (HIFs: HIF-1 being the mostly studied), which are heterodimeric transcription factors composed of α and β subunits, first described by Semenza and Wang [17]. These HIFs in the presence of hypoxic oxygen levels are activated through a complex mechanism in which the oxygen tension is critical (see below). Afterwards HIFs bind to hypoxia-responsive elements, activating the transcription of more than two hundred genes that allow cells to adapt to the hypoxic environment [18] and [19].

Several excellent reviews appeared in the last few years describing the array of changes induced by oxygen deficiency in both isolated cells and animal tissues. In in vivo models, a coordinated regulation of tissue perfusion through vasoactive molecules such as nitric oxide and the action of carotid bodies rapidly respond to changes in oxygen demand [20][21][22][23] and [24]. Within isolated cells, hypoxia induces significant metabolic changes due to both variation of metabolites level and activation/inhibition of enzymes and transporters; the most important intracellular effects induced by different pathways are expertly described elsewhere (for recent reviews, see [25][26] and [27]). It is reasonable to suppose that the type of cells and both the severity and duration of hypoxia may determine which pathways are activated/depressed and their timing of onset [3][6][10][12][23] and [28]. These pathways will eventually lead to preferential translation of key proteins required for adaptation and survival to hypoxic stress. Although in the past two decades, the discovery of HIF-1 by Gregg Semenza et al. provided a molecular platform to investigate the mechanism underlying responses to oxygen deprivation, the molecular and cellular biology of hypoxia has still to be completely elucidated. This review summarizes recent experimental data concerned with mitochondrial structure and function adaptation to hypoxia and evaluates it in light of the main structural and functional parameters defining the mitochondrial bioenergetics. Since mitochondria contain an inhibitor protein, IF1, whose action on the F1F0 ATPase has been considered for decades of critical importance in hypoxia/ischemia, particular notice will be dedicated to analyze molecular aspects of IF1 regulation of the enzyme and its possible role in the metabolic changes induced by low oxygen levels in cells.

Mechanism(s) of HIF-1 activation

HIF-1 consists of an oxygen-sensitive HIF-1α subunit that heterodimerizes with the HIF-1β subunit to bind DNA. In high O2 tension, HIF-1α is oxidized (hydroxylated) by prolyl hydroxylases (PHDs) using α-ketoglutarate derived from the tricarboxylic acid (TCA) cycle. The hydroxylated HIF-1α subunit interacts with the von Hippel–Lindau protein, a critical member of an E3 ubiquitin ligase complex that polyubiquitylates HIF. This is then catabolized by proteasomes, such that HIF-1α is continuously synthesized and degraded under normoxic conditions [18]. Under hypoxia, HIF-1α hydroxylation does not occur, thereby stabilizing HIF-1 (Fig. 1). The active HIF-1 complex in turn binds to a core hypoxia response element in a wide array of genes involved in a diversity of biological processes, and directly transactivates glycolytic enzyme genes [29]. Notably, O2 concentration, multiple mitochondrial products, including the TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, hence the cellular responses to O2 depletion [30] and [31]. Incidentally, impaired TCA cycle flux, particularly if it is caused by succinate dehydrogenase dysfunction, results in decreased or loss of energy production from both the electron-transport chain and the Krebs cycle, and also in overproduction of free radicals [32]. This leads to severe early-onset neurodegeneration or, as it occurs in individuals carrying mutations in the non-catalytic subunits of the same enzyme, to tumors such as phaeochromocytoma and paraganglioma. However, impairment of the TCA cycle may be relevant also for the metabolic changes occurring in mitochondria exposed to hypoxia, since accumulation of succinate has been reported to inhibit PHDs [33]. It has to be noticed that some authors believe reactive oxygen species (ROS) to be essential to activate HIF-1 [34], but others challenge this idea [35], therefore the role of mitochondrial ROS in the regulation of HIF-1 under hypoxia is still controversial [36]. Moreover, the contribution of functional mitochondria to HIF-1 regulation has also been questioned by others [37][38] and [39].

http://ars.els-cdn.com/content/image/1-s2.0-S0005272810000575-gr1.jpg

Major mitochondrial changes in hypoxia

Major mitochondrial changes in hypoxia

Fig. 1. Major mitochondrial changes in hypoxia. Hypoxia could decrease electron-transport rate determining Δψm reduction, increased ROS generation, and enhanced NO synthase. One (or more) of these factors likely contributes to HIF stabilization, that in turn induces metabolic adaptation of both hypoxic cells and mitophagy. The decreased Δψm could also induce an active binding of IF1, which might change mitochondrial morphology and/or dynamics, and inhibit mitophagy. Solid lines indicate well established hypoxic changes in cells, whilst dotted lines indicate changes not yet stated. Inset, relationships between extracellular O2concentration and oxygen tension.

Oxygen is a major determinant of cell metabolism and gene expression, and as cellular O2 levels decrease, either during isolated hypoxia or ischemia-associated hypoxia, metabolism and gene expression profiles in the cells are significantly altered. Low oxygen reduces OXPHOS and Krebs cycle rates, and participates in the generation of nitric oxide (NO), which also contributes to decrease respiration rate [23] and [40]. However, oxygen is also central in the generation of reactive oxygen species, which can participate in cell signaling processes or can induce irreversible cellular damage and death [41].

As specified above, cells adapt to oxygen reduction by inducing active HIF, whose major effect on cells energy homeostasis is the inactivation of anabolism, activation of anaerobic glycolysis, and inhibition of the mitochondrial aerobic metabolism: the TCA cycle, and OXPHOS. Since OXPHOS supplies the majority of ATP required for cellular processes, low oxygen tension will severely reduce cell energy availability. This occurs through several mechanisms: first, reduced oxygen tension decreases the respiration rate, due first to nonsaturating substrate for cytochrome c oxidase (COX), secondarily, to allosteric modulation of COX[42]. As a consequence, the phosphorylation potential decreases, with enhancement of the glycolysis rate primarily due to allosteric increase of phosphofructokinase activity; glycolysis however is poorly efficient and produces lactate in proportion of 0.5 mol/mol ATP, which eventually drops cellular pH if cells are not well perfused, as it occurs under defective vasculature or ischemic conditions  [6]. Besides this “spontaneous” (thermodynamically-driven) shift from aerobic to anaerobic metabolism which is mediated by the kinetic changes of most enzymes, the HIF-1 factor activates transcription of genes encoding glucose transporters and glycolytic enzymes to further increase flux of reducing equivalents from glucose to lactate[43] and [44]. Second, HIF-1 coordinates two different actions on the mitochondrial phase of glucose oxidation: it activates transcription of the PDK1 gene encoding a kinase that phosphorylates and inactivates pyruvate dehydrogenase, thereby shunting away pyruvate from the mitochondria by preventing its oxidative decarboxylation to acetyl-CoA [45] and [46]. Moreover, HIF-1 induces a switch in the composition of cytochrome c oxidase from COX4-1 to COX4-2 isoform, which enhances the specific activity of the enzyme. As a result, both respiration rate and ATP level of hypoxic cells carrying the COX4-2 isoform of cytochrome c oxidase were found significantly increased with respect to the same cells carrying the COX4-1 isoform [47]. Incidentally, HIF-1 can also increase the expression of carbonic anhydrase 9, which catalyses the reversible hydration of CO2 to HCO3 and H+, therefore contributing to pH regulation.

Effects of hypoxia on mitochondrial structure and dynamics

Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. The fusion/fission machineries are modulated in response to changes in the metabolic conditions of the cell, therefore one should expect that hypoxia affect mitochondrial dynamics. Oxygen availability to cells decreases glucose oxidation, whereas oxygen shortage consumes glucose faster in an attempt to produce ATP via the less efficient anaerobic glycolysis to lactate (Pasteur effect). Under these conditions, mitochondria are not fueled with substrates (acetyl-CoA and O2), inducing major changes of structure, function, and dynamics (for a recent review see [48]). Concerning structure and dynamics, one of the first correlates that emerge is that impairment of mitochondrial fusion leads to mitochondrial depolarization, loss of mtDNA that may be accompanied by altered respiration rate, and impaired distribution of the mitochondria within cells [49][50] and [51]. Indeed, exposure of cortical neurons to moderate hypoxic conditions for several hours, significantly altered mitochondrial morphology, decreased mitochondrial size and reduced mitochondrial mean velocity. Since these effects were either prevented by exposing the neurons to inhibitors of nitric oxide synthase or mimicked by NO donors in normoxia, the involvement of an NO-mediated pathway was suggested [52]. Mitochondrial motility was also found inhibited and controlled locally by the [ADP]/[ATP] ratio [53]. Interestingly, the author used an original approach in which mitochondria were visualized using tetramethylrhodamineethylester and their movements were followed by applying single-particle tracking.

Of notice in this chapter is that enzymes controlling mitochondrial morphology regulators provide a platform through which cellular signals are transduced within the cell in order to affect mitochondrial function [54]. Accordingly, one might expect that besides other mitochondrial factors [30] and [55] playing roles in HIF stabilization, also mitochondrial morphology might reasonably be associated with HIF stabilization. In order to better define the mechanisms involved in the morphology changes of mitochondria and in their dynamics when cells experience hypoxic conditions, these pioneering studies should be corroborated by and extended to observations on other types of cells focusing also on single proteins involved in both mitochondrial fusion/fission and motion.

Effects of hypoxia on the respiratory chain complexes

O2 is the terminal acceptor of electrons from cytochrome c oxidase (Complex IV), which has a very high affinity for it, being the oxygen concentration for half-maximal respiratory rate at pH 7.4 approximately 0.7 µM [56]. Measurements of mitochondrial oxidative phosphorylation indicated that it is not dependent on oxygen concentration up to at least 20 µM at pH 7.0 and the oxygen dependence becomes markedly greater as the pH is more alkaline [56]. Similarly, Moncada et al. [57] found that the rate of O2 consumption remained constant until [O2] fell below 15 µM. Accordingly, most reports in the literature consider hypoxic conditions occurring in cells at 5–0.5% O2, a range corresponding to 46–4.6 µM O2 in the cells culture medium (see Fig. 1 inset). Since between the extracellular environment and mitochondria an oxygen pressure gradient is established [58], the O2 concentration experienced by Complex IV falls in the range affecting its kinetics, as reported above.

Under these conditions, a number of changes on the OXPHOS machinery components, mostly mediated by HIF-1 have been found. Thus, Semenza et al. [59] and others thereafter [46] reported that activation of HIF-1α induces pyruvate dehydrogenase kinase, which inhibits pyruvate dehydrogenase, suggesting that respiration is decreased by substrate limitation. Besides, other HIF-1 dependent mechanisms capable to affect respiration rate have been reported. First, the subunit composition of COX is altered in hypoxic cells by increased degradation of the COX4-1 subunit, which optimizes COX activity under aerobic conditions, and increased expression of the COX4-2 subunit, which optimizes COX activity under hypoxic conditions [29]. On the other hand, direct assay of respiration rate in cells exposed to hypoxia resulted in a significant reduction of respiration [60]. According with the evidence of Zhang et al., the respiration rate decrease has to be ascribed to mitochondrial autophagy, due to HIF-1-mediated expression of BNIP3. This interpretation is in line with preliminary results obtained in our laboratory where the assay of the citrate synthase activity of cells exposed to different oxygen tensions was performed. Fig. 2 shows the citrate synthase activity, which is taken as an index of the mitochondrial mass [11], with respect to oxygen tension: [O2] and mitochondrial mass are directly linked.

Citrate synthase activity

Citrate synthase activity

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Fig. 2. Citrate synthase activity. Human primary fibroblasts, obtained from skin biopsies of 5 healthy donors, were seeded at a density of 8,000 cells/cm2 in high glucose Dulbecco’s Modified Eagle Medium, DMEM (25 mM glucose, 110 mg/l pyruvate, and 4 mM glutamine) supplemented with 15% Foetal Bovine Serum (FBS). 18 h later, cell culture dishes were washed once with Hank’s Balanced Salt Solution (HBSS) and the medium was replaced with DMEM containing 5 mM glucose, 110 mg/l pyruvate, and 4 mM glutamine supplemented with 15% FBS. Cell culture dishes were then placed into an INVIVO2 humidified hypoxia workstation (Ruskinn Technologies, Bridgend, UK) for 72 h changing the medium at 48 h, and oxygen partial pressure (tension) conditions were: 20%, 4%, 2%, 1% and 0.5%. Cells were subsequently collected within the workstation with trypsin-EDTA (0.25%), washed with PBS and resuspended in a buffer containing 10 mM Tris/HCl, 0.1 M KCl, 5 mM KH2PO4, 1 mM EGTA, 3 mM EDTA, and 2 mM MgCl2 pH 7.4 (all the solutions were preconditioned to the appropriate oxygen tension condition). The citrate synthase activity was assayed essentially by incubating 40 µg of cells with 0.02% Triton X-100, and monitoring the reaction by measuring spectrophotometrically the rate of free coenzyme A released, as described in [90]. Enzymatic activity was expressed as nmol/min/mg of protein. Three independent experiments were carried out and assays were performed in either duplicate or triplicate.

However, the observations of Semenza et al. must be seen in relation with data reported by Moncada et al.[57] and confirmed by others [61] in which it is clearly shown that when cells (various cell lines) experience hypoxic conditions, nitric oxide synthases (NOSs) are activated, therefore NO is released. As already mentioned above, NO is a strong competitor of O2 for cytochrome c oxidase, whose apparent Km results increased, hence reduction of mitochondrial cytochromes and all the other redox centres of the respiratory chain occurs. In addition, very recent data indicate a potential de-activation of Complex I when oxygen is lacking, as it occurs in prolonged hypoxia [62]. According to Hagen et al. [63] the NO-dependent inhibition of cytochrome c oxidase should allow “saved” O2 to redistribute within the cell to be used by other enzymes, including PHDs which inactivate HIF. Therefore, unless NO inhibition of cytochrome c oxidase occurs only when [O2] is very low, inhibition of mitochondrial oxygen consumption creates the paradox of a situation in which the cell may fail to register hypoxia. It has been tempted to solve this paradox, but to date only hypotheses have been proposed [23] and [26]. Interestingly, recent observations on yeast cells exposed to hypoxia revealed abnormal protein carbonylation and protein tyrosine nitration that were ascribed to increased mitochondrially generated superoxide radicals and NO, two species typically produced at low oxygen levels, that combine to form ONOO [64]. Based on these studies a possible explanation has been proposed for the above paradox.

Finally, it has to be noticed that the mitochondrial respiratory deficiency observed in cardiomyocytes of dogs in which experimental heart failure had been induced lies in the supermolecular assembly rather than in the individual components of the electron-transport chain [65]. This observation is particularly intriguing since loss of respirasomes is thought to facilitate ROS generation in mitochondria [66], therefore supercomplexes disassembly might explain the paradox of reduced [O2] and the enhanced ROS found in hypoxic cells. Specifically, hypoxia could reduce mitochondrial fusion by impairing mitochondrial membrane potential, which in turn could induce supercomplexes disassembly, increasing ROS production[11].

Complex III and ROS production

It has been estimated that, under normoxic physiological conditions, 1–2% of electron flow through the mitochondrial respiratory chain gives rise to ROS [67] and [68]. It is now recognized that the major sites of ROS production are within Complexes I and III, being prevalent the contribution of Complex I [69] (Fig. 3). It might be expected that hypoxia would decrease ROS production, due to the low level of O2 and to the diminished mitochondrial respiration [6] and [46], but ROS level is paradoxically increased. Indeed, about a decade ago, Chandel et al. [70] provided good evidence that mitochondrial reactive oxygen species trigger hypoxia-induced transcription, and a few years later the same group [71] showed that ROS generated at Complex III of the mitochondrial respiratory chain stabilize HIF-1α during hypoxia (Fig. 1 and Fig. 3). Although others have proposed mechanisms indicating a key role of mitochondria in HIF-1α regulation during hypoxia (for reviews see [64] and [72]), the contribution of mitochondria to HIF-1 regulation has been questioned by others [35][36] and [37]. Results of Gong and Agani [35] for instance show that inhibition of electron-transport Complexes I, III, and IV, as well as inhibition of mitochondrial F0F1 ATPase, prevents HIF-1α expression and that mitochondrial reactive oxygen species are not involved in HIF-1α regulation during hypoxia. Concurrently, Tuttle et al. [73], by means of a non invasive, spectroscopic approach, could find no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize HIF-1α under moderate hypoxia. The same authors found the levels of HIF-1α comparable in both normal and ρ0 cells (i.e. cells lacking mitochondrial DNA). On the contrary, experiments carried out on genetic models consisting of either cells lacking cytochrome c or ρ0 cells both could evidence the essential role of mitochondrial respiration to stabilize HIF-1α [74]. Thus, cytochrome c null cells, being incapable to respire, exposed to moderate hypoxia (1.5% O2) prevented oxidation of ubiquinol and generation of the ubisemiquinone radical, thus eliminating superoxide formation at Complex III [71]. Concurrently, ρ0 cells lacking electron transport, exposed 4 h to moderate hypoxia failed to stabilize HIF-1α, suggesting the essential role of the respiratory chain for the cellular sensing of low O2 levels. In addition, recent evidence obtained on genetic manipulated cells (i.e. cytochrome b deficient cybrids) showed increased ROS levels and stabilized HIF-1α protein during hypoxia [75]. Moreover, RNA interference of the Complex III subunit Rieske iron sulfur protein in the cytochrome b deficient cells, abolished ROS generation at the Qo site of Complex III, preventing HIF-1α stabilization. These observations, substantiated by experiments with MitoQ, an efficient mitochondria-targeted antioxidant, strongly support the involvement of mitochondrial ROS in regulating HIF-1α. Nonetheless, collectively, the available data do not allow to definitely state the precise role of mitochondrial ROS in regulating HIF-1α, but the pathway stabilizing HIF-1α appears undoubtedly mitochondria-dependent [30].

Overview of mitochondrial electron and proton flux in hypoxia

Overview of mitochondrial electron and proton flux in hypoxia

Overview of mitochondrial electron and proton flux in hypoxia

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Fig. 3. Overview of mitochondrial electron and proton flux in hypoxia. Electrons released from reduced cofactors (NADH and FADH2) under normoxia flow through the redox centres of the respiratory chain (r.c.) to molecular oxygen (blue dotted line), to which a proton flux from the mitochondrial matrix to the intermembrane space is coupled (blue arrows). Protons then flow back to the matrix through the F0 sector of the ATP synthase complex, driving ATP synthesis. ATP is carried to the cell cytosol by the adenine nucleotide translocator (blue arrows). Under moderate to severe hypoxia, electrons escape the r.c. redox centres and reduce molecular oxygen to the superoxide anion radical before reaching the cytochrome c (red arrow). Under these conditions, to maintain an appropriate Δψm, ATP produced by cytosolic glycolysis enters the mitochondria where it is hydrolyzed by the F1F0ATPase with extrusion of protons from the mitochondrial matrix (red arrows).

Hypoxia and ATP synthase

The F1F0 ATPase (ATP synthase) is the enzyme responsible of catalysing ADP phosphorylation as the last step of OXPHOS. It is a rotary motor using the proton motive force across the mitochondrial inner membrane to drive the synthesis of ATP [76]. It is a reversible enzyme with ATP synthesis or hydrolysis taking place in the F1 sector at the matrix side of the membrane, chemical catalysis being coupled to H+transport through the transmembrane F0 sector.

Under normoxia the enzyme synthesizes ATP, but when mitochondria experience hypoxic conditions the mitochondrial membrane potential (Δψm) decreases below its endogenous steady-state level (some 140 mV, negative inside the matrix [77]) and the F1F0 ATPase may work in the reversal mode: it hydrolyses ATP (produced by anaerobic glycolysis) and uses the energy released to pump protons from the mitochondrial matrix to the intermembrane space, concurring with the adenine nucleotide translocator (i.e. in hypoxia it exchanges cytosolic ATP4− for matrix ADP3−) to maintain the physiological Δψm ( Fig. 3). Since under conditions of limited oxygen availability the decline in cytoplasmic high energy phosphates is mainly due to hydrolysis by the ATP synthase working in reverse [6] and [78], the enzyme must be strictly regulated in order to avoid ATP dissipation. This is achieved by a natural protein, the H+ψm-dependent IF1, that binds to the catalytic F1 sector at low pH and low Δψm (such as it occurs in hypoxia/ischemia) [79]. IF1 binding to the ATP synthase results in a rapid and reversible inhibition of the enzyme [80], which could reach about 50% of maximal activity (for recent reviews see [6] and [81]).

Besides this widely studied effect, IF1 appears to be associated with ROS production and mitochondrial autophagy (mitophagy). This is a mechanism involving the catabolic degradation of macromolecules and organelles via the lysosomal pathway that contributes to housekeeping and regenerate metabolites. Autophagic degradation is involved in the regulation of the ageing process and in several human diseases, such as myocardial ischemia/reperfusion [82], Alzheimer’s Disease, Huntington diseases, and inflammatory diseases (for recent reviews see [83] and [84], and, as mentioned above, it promotes cell survival by reducing ROS and mtDNA damage under hypoxic conditions.

Campanella et al. [81] reported that, in HeLa cells under normoxic conditions, basal autophagic activity varies in relation to the expression levels of IF1. Accordingly, cells overexpressing IF1 result in ROS production similar to controls, conversely cells in which IF1 expression is suppressed show an enhanced ROS production. In parallel, the latter cells show activation of the mitophagy pathway (Fig. 1), therefore suggesting that variations in IF1 expression level may play a significant role in defining two particularly important parameters in the context of the current review: rates of ROS generation and mitophagy. Thus, the hypoxia-induced enhanced expression level of IF1[81] should be associated with a decrease of both ROS production and autophagy, which is in apparent conflict with the hypoxia-induced ROS increase and with the HIF-1-dependent mitochondrial autophagy shown by Zhang et al. [60] as an adaptive metabolic response to hypoxia. However, in the experiments of Zhang et al. the cells were exposed to hypoxia for 48 h, whereas the F1F0-ATPase inhibitor exerts a prompt action on the enzyme and to our knowledge, it has never been reported whether its action persists during prolonged hypoxic expositions. Pertinent with this problem is the very recent observation that IEX-1 (immediate early response gene X-1), a stress-inducible gene that suppresses production of ROS and protects cells from apoptosis [85], targets the mitochondrial F1F0-ATPase inhibitor for degradation, reducing ROS by decreasing Δψm. It has to be noticed that the experiments described were carried out under normal oxygen availability, but it does not seem reasonable to rule out IEX-1 from playing a role under stress conditions as those induced by hypoxia in cells, therefore this issue might deserve an investigation also at low oxygen levels.

In conclusion, data are still emerging regarding the regulation of mitochondrial function by the F1F0 ATPase within hypoxic responses in different cellular and physiological contexts. Given the broad pathophysiological role of hypoxic cellular modulation, an understanding of the subtle tuning among different effectors of the ATP synthase is desirable to eventually target future therapeutics most effectively. Our laboratory is actually involved in carrying out investigations to clarify this context.

Conclusions and perspectives

The mitochondria are important cellular platforms that both propagate and initiate intracellular signals that lead to overall cellular and metabolic responses. During the last decades, a significant amount of relevant data has been obtained on the identification of mechanisms of cellular adaptation to hypoxia. In hypoxic cells there is an enhanced transcription and synthesis of several glycolytic pathway enzymes/transporters and reduction of synthesis of proteins involved in mitochondrial catabolism. Although well defined kinetic parameters of reactions in hypoxia are lacking, it is usually assumed that these transcriptional changes lead to metabolic flux modification. The required biochemical experimentation has been scarcely addressed until now and only in few of the molecular and cellular biology studies the transporter and enzyme kinetic parameters and flux rate have been determined, leaving some uncertainties.

Central to mitochondrial function and ROS generation is an electrochemical proton gradient across the mitochondrial inner membrane that is established by the proton pumping activity of the respiratory chain, and that is strictly linked to the F1F0-ATPase function. Evaluation of the mitochondrial membrane potential in hypoxia has only been studied using semiquantitative methods based on measurements of the fluorescence intensity of probes taken up by cells experiencing normal or hypoxic conditions. However, this approach is intrinsically incorrect due to the different capability that molecular oxygen has to quench fluorescence [86] and [87] and to the uncertain concentration the probe attains within mitochondria, whose mass may be reduced by a half in hypoxia [60]. In addition, the uncertainty about measurement of mitochondrial superoxide radical and H2O2 formation in vivo [88] hampers studies on the role of mitochondrial ROS in hypoxic oxidative damage, redox signaling, and HIF-1 stabilization.

The duration and severity of hypoxic stress differentially activate the responses discussed throughout and lead to substantial phenotypic variations amongst tissues and cell models, which are not consistently and definitely known. Certainly, understanding whether a hierarchy among hypoxia response mechanisms exists and which are the precise timing and conditions of each mechanism to activate, will improve our knowledge of the biochemical mechanisms underlying hypoxia in cells, which eventually may contribute to define therapeutic targets in hypoxia-associated diseases. To this aim it might be worth investigating the hypoxia-induced structural organization of both the respiratory chain enzymes in supramolecular complexes and the assembly of the ATP synthase to form oligomers affecting ROS production [65] and inner mitochondrial membrane structure [89], respectively.

7.9.2 Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

DR WisePS WardJES ShayJR CrossJJ Gruber, UM Sachdeva, et al.
Proc Nat Acad Sci Oct 27, 2011; 108(49):19611–19616
http://dx.doi.org:/10.1073/pnas.1117773108

Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived α-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived α-ketoglutarate in hypoxia is associated with a concomitant increased synthesis of 2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells are unable to proliferate. The reductive carboxylation of glutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutamine-derived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions.

Citrate plays a critical role at the center of cancer cell metabolism. It provides the cell with a source of carbon for fatty acid and cholesterol synthesis (1). The breakdown of citrate by ATP-citrate lyase is a primary source of acetyl-CoA for protein acetylation (2). Metabolism of cytosolic citrate by aconitase and IDH1 can also provide the cell with a source of NADPH for redox regulation and anabolic synthesis. Mammalian cells depend on the catabolism of glucose and glutamine to fuel proliferation (3). In cancer cells cultured at atmospheric oxygen tension (21% O2), glucose and glutamine have both been shown to contribute to the cellular citrate pool, with glutamine providing the major source of the four-carbon molecule oxaloacetate and glucose providing the major source of the two-carbon molecule acetyl-CoA (45). The condensation of oxaloacetate and acetyl-CoA via citrate synthase generates the 6 carbon citrate molecule. However, both the conversion of glucose-derived pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH) and the conversion of glutamine to oxaloacetate through the TCA cycle depend on NAD+, which can be compromised under hypoxic conditions. This raises the question of how cells that can proliferate in hypoxia continue to synthesize the citrate required for macromolecular synthesis.

This question is particularly important given that many cancers and stem/progenitor cells can continue proliferating in the setting of limited oxygen availability (67). Louis Pasteur first highlighted the impact of hypoxia on nutrient metabolism based on his observation that hypoxic yeast cells preferred to convert glucose into lactic acid rather than burning it in an oxidative fashion. The molecular basis for this shift in mammalian cells has been linked to the activity of the transcription factor HIF1 (810). Stabilization of the labile HIF1α subunit occurs in hypoxia. It can also occur in normoxia through several mechanisms including loss of the von Hippel-Lindau tumor suppressor (VHL), a common occurrence in renal carcinoma (11). Although hypoxia and/or HIF1α stabilization is a common feature of multiple cancers, to date the source of citrate in the setting of hypoxia or HIF activation has not been determined.

Here, we study the sources of hypoxic citrate synthesis in a glioblastoma cell line that proliferates in profound hypoxia (0.5% O2). Glucose uptake and conversion to lactic acid increased in hypoxia. However, glucose conversion into citrate dramatically declined. Glutamine consumption remained constant in hypoxia, and hypoxic cells were addicted to the use of glutamine in hypoxia as a source of α-ketoglutarate. Glutamine provided the major carbon source for citrate synthesis during hypoxia. However, the TCA cycle-dependent conversion of glutamine into citric acid was significantly suppressed. In contrast, there was a relative increase in glutamine-dependent citrate production in hypoxia that resulted from carboxylation of α-ketoglutarate. This reductive synthesis required the presence of mitochondrial isocitrate dehydrogenase 2 (IDH2). In confirmation of the reverse flux through IDH2, the increased reductive metabolism of glutamine-derived α-ketoglutarate in hypoxia was associated with increased synthesis of 2HG. Finally, constitutive HIF1α-expressing cells also demonstrated significant reductive-carboxylation-dependent synthesis of citrate in normoxia and a relative defect in the oxidative conversion of glutamine into citrate. Collectively, the data demonstrate that mitochondrial glutamine metabolism can be rerouted through IDH2-dependent citrate synthesis in support of hypoxic cell growth.

Some Cancer Cells Can Proliferate at 0.5% O2 Despite a Sharp Decline in Glucose-Dependent Citrate Synthesis.

At 21% O2, cancer cells have been shown to synthesize citrate by condensing glucose-derived acetyl-CoA with glutamine-derived oxaloacetate through the activity of the canonical TCA cycle enzyme citrate synthase (4). In contrast, less is known regarding the synthesis of citrate by cells that can continue proliferating in hypoxia. The glioblastoma cell line SF188 is able to proliferate at 0.5% O2 (Fig. 1A), a level of hypoxia that is sufficient to stabilize HIF1α (Fig. 1B) and predicted to limit respiration (1213). Consistent with previous observations in hypoxic cells, we found that SF188 cells demonstrated increased lactate production when incubated in hypoxia (Fig. 1C), and the ratio of lactate produced to glucose consumed increased demonstrating an increase in the rate of anaerobic glycolysis. When glucose-derived carbon in the form of pyruvate is converted to lactate, it is diverted away from subsequent metabolism that can contribute to citrate production. However, we observed that SF188 cells incubated in hypoxia maintain their intracellular citrate to ∼75% of the level maintained under normoxia (Fig. 1D). This prompted an investigation of how proliferating cells maintain citrate production under hypoxia.

SF188 glioblastoma cells proliferate at 0.5% O2 despite a profound reduction in glucose-dependent citrate synthesis.

SF188 glioblastoma cells proliferate at 0.5% O2 despite a profound reduction in glucose-dependent citrate synthesis.

http://www.pnas.org/content/108/49/19611/F1.medium.gif

Fig. 1. SF188 glioblastoma cells proliferate at 0.5% O2 despite a profound reduction in glucose-dependent citrate synthesis. (A) SF188 cells were plated in complete medium equilibrated with 21% O2 (Normoxia) or 0.5% O2 (Hypoxia), total viable cells were counted 24 h and 48 h later (Day 1 and Day 2), and population doublings were calculated. Data are the mean ± SEM of four independent experiments. (B) Western blot demonstrates stabilized HIF1α protein in cells cultured in hypoxia compared with normoxia. (C) Cells were grown in normoxia or hypoxia for 24 h, after which culture medium was collected. Medium glucose and lactate levels were measured and compared with the levels in fresh medium. (D) Cells were cultured for 24 h as in C. Intracellular metabolism was then quenched with 80% MeOH prechilled to −80 °C that was spiked with a 13C-labeled citrate as an internal standard. Metabolites were then extracted, and intracellular citrate levels were analyzed with GC-MS and normalized to cell number. Data for C and D are the mean ± SEM of three independent experiments. (E) Model depicting the pathway for cit+2 production from [U-13C]glucose. Glucose uniformly 13C-labeled will generate pyruvate+3. Pyruvate+3 can be oxidatively decarboxylated by PDH to produce acetyl-CoA+2, which can condense with unlabeled oxaloacetate to produce cit+2. (F) Cells were cultured for 24 h as in C and D, followed by an additional 4 h of culture in glucose-deficient medium supplemented with 10 mM [U-13C]glucose. Intracellular metabolites were then extracted, and 13C-enrichment in cellular citrate was analyzed by GC-MS and normalized to the total citrate pool size. Data are the mean ± SD of three independent cultures from a representative of two independent experiments. *P < 0.05, ***P < 0.001.

Increased glucose uptake and glycolytic metabolism are critical elements of the metabolic response to hypoxia. To evaluate the contributions made by glucose to the citrate pool under normoxia or hypoxia, SF188 cells incubated in normoxia or hypoxia were cultured in medium containing 10 mM [U-13C]glucose. Following a 4-h labeling period, cellular metabolites were extracted and analyzed for isotopic enrichment by gas chromatography-mass spectrometry (GC-MS). In normoxia, the major 13C-enriched citrate species found was citrate enriched with two 13C atoms (cit+2), which can arise from the NAD+-dependent decarboxylation of pyruvate+3 to acetyl-CoA+2 by PDH, followed by the condensation of acetyl-CoA+2 with unenriched oxaloacetate (Fig. 1 E and F). Compared with the accumulation of cit+2, we observed minimal accumulation of cit+3 and cit+5 under normoxia. Cit+3 arises from pyruvate carboxylase (PC)-dependent conversion of pyruvate+3 to oxaloacetate+3, followed by the condensation of oxaloacetate+3 with unenriched acetyl-CoA. Cit+5 arises when PC-generated oxaloacetate+3 condenses with PDH-generated acetyl-CoA+2. The lack of cit+3 and cit+5 accumulation is consistent with PC activity not playing a major role in citrate production in normoxic SF188 cells, as reported (4).

In hypoxic cells, the major citrate species observed was unenriched. Cit+2, cit+3, and cit+5 all constituted minor fractions of the total citrate pool, consistent with glucose carbon not being incorporated into citrate through either PDH or PC-mediated metabolism under hypoxic conditions (Fig. 1F). These data demonstrate that in contrast to normoxic cells, where a large percentage of citrate production depends on glucose-derived carbon, hypoxic cells significantly reduce their rate of citrate production from glucose.

Glutamine Carbon Metabolism Is Required for Viability in Hypoxia.

In addition to glucose, we have previously reported that glutamine can contribute to citrate production during cell growth under normoxic conditions (4). Surprisingly, under hypoxic conditions, we observed that SF188 cells retained their high rate of glutamine consumption (Fig. 2A). Moreover, hypoxic cells cultured in glutamine-deficient medium displayed a significant loss of viability (Fig. 2B). In normoxia, the requirement for glutamine to maintain viability of SF188 cells can be satisfied by α-ketoglutarate, the downstream metabolite of glutamine that is devoid of nitrogenous groups (14). α-ketoglutarate cannot fulfill glutamine’s roles as a nitrogen source for nonessential amino acid synthesis or as an amide donor for nucleotide or hexosamine synthesis, but can be metabolized through the oxidative TCA cycle to regenerate oxaloacetate, and subsequently condense with glucose-derived acetyl-CoA to produce citrate. To test whether the restoration of carbon from glutamine metabolism in the form of α-ketoglutarate could rescue the viability defect of glutamine-starved SF188 cells even under hypoxia, SF188 cells incubated in hypoxia were cultured in glutamine-deficient medium supplemented with a cell-penetrant form of α-ketoglutarate (dimethyl α-ketoglutarate). The addition of dimethyl α-ketoglutarate rescued the defect in cell viability observed upon glutamine withdrawal (Fig. 2B). These data demonstrate that, even under hypoxic conditions, when the ability of glutamine to replenish oxaloacetate through oxidative TCA cycle metabolism is diminished, SF188 cells retain their requirement for glutamine as the carbon backbone for α-ketoglutarate. This result raised the possibility that glutamine could be the carbon source for citrate production through an alternative, nonoxidative, pathway in hypoxia.

Glutamine carbon is required for hypoxic cell viability

Glutamine carbon is required for hypoxic cell viability

Glutamine carbon is required for hypoxic cell viability

http://www.pnas.org/content/108/49/19611/F2.medium.gif

Fig. 2. Glutamine carbon is required for hypoxic cell viability and contributes to increased citrate production through reductive carboxylation relative to oxidative metabolism in hypoxia. (A) SF188 cells were cultured for 24 h in complete medium equilibrated with either 21% O2 (Normoxia) or 0.5% O2(Hypoxia). Culture medium was then removed from cells and analyzed for glutamine levels which were compared with the glutamine levels in fresh medium. Data are the mean ± SEM of three independent experiments. (B) The requirement for glutamine to maintain hypoxic cell viability can be satisfied by α-ketoglutarate. Cells were cultured in complete medium equilibrated with 0.5% O2 for 24 h, followed by an additional 48 h at 0.5% O2 in either complete medium (+Gln), glutamine-deficient medium (−Gln), or glutamine-deficient medium supplemented with 7 mM dimethyl α-ketoglutarate (−Gln +αKG). All medium was preconditioned in 0.5% O2. Cell viability was determined by trypan blue dye exclusion. Data are the mean and range from two independent experiments. (C) Model depicting the pathways for cit+4 and cit+5 production from [U-13C]glutamine (glutamine+5). Glutamine+5 is catabolized to α-ketoglutarate+5, which can then contribute to citrate production by two divergent pathways. Oxidative metabolism produces oxaloacetate+4, which can condense with unlabeled acetyl-CoA to produce cit+4. Alternatively, reductive carboxylation produces isocitrate+5, which can isomerize to cit+5. (D) Glutamine contributes to citrate production through increased reductive carboxylation relative to oxidative metabolism in hypoxic proliferating cancer cells. Cells were cultured for 24 h as in A, followed by 4 h of culture in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. 13C enrichment in cellular citrate was quantitated with GC-MS. Data are the mean ± SD of three independent cultures from a representative of three independent experiments. **P < 0.01.

Cells Proliferating in Hypoxia Maintain Levels of Additional Metabolites Through Reductive Carboxylation.

Previous work has documented that, in normoxic conditions, SF188 cells use glutamine as the primary anaplerotic substrate, maintaining the pool sizes of TCA cycle intermediates through oxidative metabolism (4). Surprisingly, we found that, when incubated in hypoxia, SF188 cells largely maintained their levels of aspartate (in equilibrium with oxaloacetate), malate, and fumarate (Fig. 3A). To distinguish how glutamine carbon contributes to these metabolites in normoxia and hypoxia, SF188 cells incubated in normoxia or hypoxia were cultured in medium containing 4 mM [U-13C]glutamine. After a 4-h labeling period, metabolites were extracted and the intracellular pools of aspartate, malate, and fumarate were analyzed by GC-MS.

In normoxia, the majority of the enriched intracellular asparatate, malate, and fumarate were the +4 species, which arise through oxidative metabolism of glutamine-derived α-ketoglutarate (Fig. 3 B and C). The +3 species, which can be derived from the citrate generated by the reductive carboxylation of glutamine-derived α-ketoglutarate, constituted a significantly lower percentage of the total aspartate, malate, and fumarate pools. By contrast, in hypoxia, the +3 species constituted a larger percentage of the total aspartate, malate, and fumarate pools than they did in normoxia. These data demonstrate that, in addition to citrate, hypoxic cells preferentially synthesize oxaloacetate, malate, and fumarate through the pathway of reductive carboxylation rather than the oxidative TCA cycle.

IDH2 Is Critical in Hypoxia for Reductive Metabolism of Glutamine and for Cell Proliferation.

We hypothesized that the relative increase in reductive carboxylation we observed in hypoxia could arise from the suppression of α-ketoglutarate oxidation through the TCA cycle. Consistent with this, we found that α-ketoglutarate levels increased in SF188 cells following 24 h in hypoxia (Fig. 4A). Surprisingly, we also found that levels of the closely related metabolite 2-hydroxyglutarate (2HG) increased in hypoxia, concomitant with the increase in α-ketoglutarate under these conditions. 2HG can arise from the noncarboxylating reduction of α-ketoglutarate (Fig. 4B). Recent work has found that specific cancer-associated mutations in the active sites of either IDH1 or IDH2 lead to a 10- to 100-fold enhancement in this activity facilitating 2HG production (1517), but SF188 cells lack IDH1/2 mutations. However, 2HG levels are also substantially elevated in the inborn error of metabolism 2HG aciduria, and the majority of patients with this disease lack IDH1/2 mutations. As 2HG has been demonstrated to arise in these patients from mitochondrial α-ketoglutarate (18), we hypothesized that both the increased reductive carboxylation of glutamine-derived α-ketoglutarate to citrate and the increased 2HG accumulation we observed in hypoxia could arise from increased reductive metabolism by wild-type IDH2 in the mitochondria.

Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2

Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2

Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2

http://www.pnas.org/content/108/49/19611/F4.medium.gif

Fig. 4. Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2. (A) α-ketoglutarate and 2HG increase in hypoxia. SF188 cells were cultured in complete medium equilibrated with either 21% O2 (Normoxia) or 0.5% O2 (Hypoxia) for 24 h. Intracellular metabolites were then extracted, cell extracts spiked with a 13C-labeled citrate as an internal standard, and intracellular α-ketoglutarate and 2HG levels were analyzed with GC-MS. Data shown are the mean ± SEM of three independent experiments. (B) Model for reductive metabolism from glutamine-derived α-ketoglutarate. Glutamine+5 is catabolized to α-ketoglutarate+5. Carboxylation of α-ketoglutarate+5 followed by reduction of the carboxylated intermediate (reductive carboxylation) will produce isocitrate+5, which can then isomerize to cit+5. In contrast, reductive activity on α-ketoglutarate+5 that is uncoupled from carboxylation will produce 2HG+5. (C) IDH2 is required for reductive metabolism of glutamine-derived α-ketoglutarate in hypoxia. SF188 cells transfected with a siRNA against IDH2 (siIDH2) or nontargeting negative control (siCTRL) were cultured for 2 d in complete medium equilibrated with 0.5% O2. (Upper) Cells were then cultured at 0.5% O2 for an additional 4 h in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. 13C enrichment in intracellular citrate and 2HG was determined and normalized to the relevant metabolite total pool size. (Lower) Cells transfected and cultured in parallel at 0.5% O2 were counted by hemacytometer (excluding nonviable cells with trypan blue staining) or harvested for protein to assess IDH2 expression by Western blot. Data shown for GC-MS and cell counts are the mean ± SD of three independent cultures from a representative experiment. **P < 0.01, ***P < 0.001.

In an experiment to test this hypothesis, SF188 cells were transfected with either siRNA directed against mitochondrial IDH2 (siIDH2) or nontargeting control, incubated in hypoxia for 2 d, and then cultured for another 4 h in hypoxia in media containing 4 mM [U-13C]glutamine. After the labeling period, metabolites were extracted and analyzed by GC-MS (Fig. 4C). Hypoxic SF188 cells transfected with siIDH2 displayed a decreased contribution of cit+5 to the total citrate pool, supporting an important role for IDH2 in the reductive carboxylation of glutamine-derived α-ketoglutarate in hypoxic conditions. The contribution of cit+4 to the total citrate pool did not decrease with siIDH2 treatment, consistent with IDH2 knockdown specifically affecting the pathway of reductive carboxylation and not other fundamental TCA cycle-regulating processes. In confirmation of reverse flux occurring through IDH2, the contribution of 2HG+5 to the total 2HG pool decreased in siIDH2-treated cells. Supporting the importance of citrate production by IDH2-mediated reductive carboxylation for hypoxic cell proliferation, siIDH2-transfected SF188 cells displayed a defect in cellular accumulation in hypoxia. Decreased expression of IDH2 protein following siIDH2 transfection was confirmed by Western blot. Collectively, these data point to the importance of mitochondrial IDH2 for the increase in reductive carboxylation flux of glutamine-derived α-ketoglutarate to maintain citrate levels in hypoxia, and to the importance of this reductive pathway for hypoxic cell proliferation.

Reprogramming of Metabolism by HIF1 in the Absence of Hypoxia Is Sufficient to Induce Increased Citrate Synthesis by Reductive Carboxylation Relative to Oxidative Metabolism.

The relative increase in the reductive metabolism of glutamine-derived α-ketoglutarate at 0.5% O2 may be explained by the decreased ability to carry out oxidative NAD+-dependent reactions as respiration is inhibited (1213). However, a shift to preferential reductive glutamine metabolism could also result from the active reprogramming of cellular metabolism by HIF1 (810), which inhibits the generation of mitochondrial acetyl-CoA necessary for the synthesis of citrate by oxidative glucose and glutamine metabolism (Fig. 5A). To better understand the role of HIF1 in reductive glutamine metabolism, we used VHL-deficient RCC4 cells, which display constitutive expression of HIF1α under normoxia (Fig. 5B). RCC4 cells expressing either a nontargeting control shRNA (shCTRL) or an shRNA directed at HIF1α (shHIF1α) were incubated in normoxia and cultured in medium with 4 mM [U-13C]glutamine. Following a 4-h labeling period, metabolites were extracted and the cellular citrate pool was analyzed by GC-MS. In shCTRL cells, which have constitutive HIF1α expression despite incubation in normoxia, the majority of the total citrate pool was constituted by the cit+5 species, with low levels of all other species including cit+4 (Fig. 5C). By contrast, in HIF1α-deficient cells the contribution of cit+5 to the total citrate pool was greatly decreased, whereas the contribution of cit+4 to the total citrate pool increased and was the most abundant citrate species. These data demonstrate that the relative enhancement of the reductive carboxylation pathway for citrate synthesis can be recapitulated by constitutive HIF1 activation in normoxia.

Reprogramming of metabolism by HIF1 in the absence of hypoxia

Reprogramming of metabolism by HIF1 in the absence of hypoxia

http://www.pnas.org/content/108/49/19611/F5.medium.gif

Reprogramming of metabolism by HIF1 in the absence of hypoxia is sufficient to induce reductive carboxylation of glutamine-derived α-ketoglutarate.

Fig. 5. Reprogramming of metabolism by HIF1 in the absence of hypoxia is sufficient to induce reductive carboxylation of glutamine-derived α-ketoglutarate. (A) Model depicting how HIF1 signaling’s inhibition of pyruvate dehydrogenase (PDH) activity and promotion of lactate dehydrogenase-A (LDH-A) activity can block the generation of mitochondrial acetyl-CoA from glucose-derived pyruvate, thereby favoring citrate synthesis from reductive carboxylation of glutamine-derived α-ketoglutarate. (B) Western blot demonstrating HIF1α protein in RCC4 VHL−/− cells in normoxia with a nontargeting shRNA (shCTRL), and the decrease in HIF1α protein in RCC4 VHL−/− cells stably expressing HIF1α shRNA (shHIF1α). (C) HIF1-induced reprogramming of glutamine metabolism. Cells from B at 21% O2 were cultured for 4 h in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. Intracellular metabolites were then extracted, and 13C enrichment in cellular citrate was determined by GC-MS. Data shown are the mean ± SD of three independent cultures from a representative of three independent experiments. ***P < 0.001.

Compared with glucose metabolism, much less is known regarding how glutamine metabolism is altered under hypoxia. It has also remained unclear how hypoxic cells can maintain the citrate production necessary for macromolecular biosynthesis. In this report, we demonstrate that in contrast to cells at 21% O2, where citrate is predominantly synthesized through oxidative metabolism of both glucose and glutamine, reductive carboxylation of glutamine carbon becomes the major pathway of citrate synthesis in cells that can effectively proliferate at 0.5% O2. Moreover, we show that in these hypoxic cells, reductive carboxylation of glutamine-derived α-ketoglutarate is dependent on mitochondrial IDH2. Although others have previously suggested the existence of reductive carboxylation in cancer cells (1920), these studies failed to demonstrate the intracellular localization or specific IDH isoform responsible for the reductive carboxylation flux. Recently, we identified IDH2 as an isoform that contributes to reductive carboxylation in cancer cells incubated at 21% O2 (16), but remaining unclear were the physiological importance and regulation of this pathway relative to oxidative metabolism, as well as the conditions where this reductive pathway might be advantageous for proliferating cells.

Here we report that IDH2-mediated reductive carboxylation of glutamine-derived α-ketoglutarate to citrate is an important feature of cells proliferating in hypoxia. Moreover, the reliance on reductive glutamine metabolism can be recapitulated in normoxia by constitutive HIF1 activation in cells with loss of VHL. The mitochondrial NADPH/NADP+ ratio required to fuel the reductive reaction through IDH2 can arise from the increased NADH/NAD+ ratio existing in the mitochondria under hypoxic conditions (2122), with the transfer of electrons from NADH to NADP+ to generate NADPH occurring through the activity of the mitochondrial transhydrogenase (23). Our data do not exclude a complementary role for cytosolic IDH1 in impacting reductive glutamine metabolism, potentially through its oxidative function in an IDH2/IDH1 shuttle that transfers high energy electrons in the form of NADPH from mitochondria to cytosol (1624).

In further support of the increased mitochondrial reductive glutamine metabolism that we observe in hypoxia, we report here that incubation in hypoxia can lead to elevated 2HG levels in cells lacking IDH1/2 mutations. 2HG production from glutamine-derived α-ketoglutarate significantly decreased with knockdown of IDH2, supporting the conclusion that 2HG is produced in hypoxia by enhanced reverse flux of α-ketoglutarate through IDH2 in a truncated, noncarboxylating reductive reaction. However, other mechanisms may also contribute to 2HG elevation in hypoxia. These include diminished oxidative activity and/or enhanced reductive activity of the 2HG dehydrogenase, a mitochondrial enzyme that normally functions to oxidize 2HG back to α-ketoglutarate (25). The level of 2HG elevation we observe in hypoxic cells is associated with a concomitant increase in α-ketoglutarate, and is modest relative to that observed in cancers with IDH1/2 gain-of-function mutations. Nonetheless, 2HG elevation resulting from hypoxia in cells with wild-type IDH1/2 may hold promise as a cellular or serum biomarker for tissues undergoing chronic hypoxia and/or excessive glutamine metabolism.

The IDH2-dependent reductive carboxylation pathway that we propose in this report allows for continued citrate production from glutamine carbon when hypoxia and/or HIF1 activation prevents glucose carbon from contributing to citrate synthesis. Moreover, as opposed to continued oxidative TCA cycle functioning in hypoxia which can increase reactive oxygen species (ROS), reductive carboxylation of α-ketoglutarate in the mitochondria may serve as an electron sink that decreases the generation of ROS. HIF1 activity is not limited to the setting of hypoxia, as a common feature of several cancers is the normoxic stabilization of HIF1α through loss of the VHL tumor suppressor or other mechanisms. We demonstrate here that altered glutamine metabolism through a mitochondrial reductive pathway is a central aspect of hypoxic proliferating cell metabolism and HIF1-induced metabolic reprogramming. These findings are relevant for the understanding of numerous constitutive HIF1-expressing malignancies, as well as for populations, such as stem progenitor cells, which frequently proliferate in hypoxic conditions.

7.9.3 Hypoxia-Inducible Factors in Physiology and Medicine

Gregg L. Semenza
Cell. 2012 Feb 3; 148(3): 399–408.
http://dx.doi.org/10.1016%2Fj.cell.2012.01.021

Oxygen homeostasis represents an organizing principle for understanding metazoan evolution, development, physiology, and pathobiology. The hypoxia-inducible factors (HIFs) are transcriptional activators that function as master regulators of oxygen homeostasis in all metazoan species. Rapid progress is being made in elucidating homeostatic roles of HIFs in many physiological systems, determining pathological consequences of HIF dysregulation in chronic diseases, and investigating potential targeting of HIFs for therapeutic purposes. Oxygen homeostasis represents an organizing principle for understanding metazoan evolution, development, physiology, and pathobiology. The hypoxia-inducible factors (HIFs) are transcriptional activators that function as master regulators of oxygen homeostasis in all metazoan species. Rapid progress is being made in elucidating homeostatic roles of HIFs in many physiological systems, determining pathological consequences of HIF dysregulation in chronic diseases, and investigating potential targeting of HIFs for therapeutic purposes.

 

Oxygen is central to biology because of its utilization in the process of respiration. O2 serves as the final electron acceptor in oxidative phosphorylation, which carries with it the risk of generating reactive oxygen species (ROS) that react with cellular macromolecules and alter their biochemical or physical properties, resulting in cell dysfunction or death. As a consequence, metazoan organisms have evolved elaborate cellular metabolic and systemic physiological systems that are designed to maintain oxygen homeostasis. This review will focus on the role of hypoxia-inducible factors (HIFs) as master regulators of oxygen homeostasis and, in particular, on recent advances in understanding their roles in physiology and medicine. Due to space limitations and the remarkably pleiotropic effects of HIFs, the description of such roles will be illustrative rather than comprehensive.

O2 and Evolution, Part 1

Accumulation of O2 in Earth’s atmosphere starting ~2.5 billion years ago led to evolution of the extraordinarily efficient system of oxidative phosphorylation that transfers chemical energy stored in carbon bonds of organic molecules to the high-energy phosphate bond in ATP, which is used to power physicochemical reactions in living cells. Energy produced by mitochondrial respiration is sufficient to power the development and maintenance of multicellular organisms, which could not be sustained by energy produced by glycolysis alone (Lane and Martin, 2010). The modest dimensions of primitive metazoan species were such that O2 could diffuse from the atmosphere to all of the organism’s thousand cells, as is the case for the worm Caenorhabditis elegans. To escape the constraints placed on organismal growth by diffusion, systems designed to conduct air to cells deep within the body evolved and were sufficient for O2delivery to organisms with hundreds of thousands of cells, such as the fly Drosophila melanogaster. The final leap in body scale occurred in vertebrates and was associated with the evolution of complex respiratory, circulatory, and nervous systems designed to efficiently capture and distribute O2 to hundreds of millions of millions of cells in the case of the adult Homo sapiens.

Hypoxia-Inducible Factors

Hypoxia-inducible factor 1 (HIF-1) is expressed by all extant metazoan species analyzed (Loenarz et al., 2011). HIF-1 consists of HIF-1α and HIF-1β subunits, which each contain basic helix-loop-helix-PAS (bHLH-PAS) domains (Wang et al., 1995) that mediate heterodimerization and DNA binding (Jiang et al., 1996a). HIF-1β heterodimerizes with other bHLH-PAS proteins and is present in excess, such that HIF-1α protein levels determine HIF-1 transcriptional activity (Semenza et al., 1996).

Under well-oxygenated conditions, HIF-1α is bound by the von Hippel-Lindau (VHL) protein, which recruits an ubiquitin ligase that targets HIF-1α for proteasomal degradation (Kaelin and Ratcliffe, 2008). VHL binding is dependent upon hydroxylation of a specific proline residue in HIF-1α by the prolyl hydroxylase PHD2, which uses O2 as a substrate such that its activity is inhibited under hypoxic conditions (Epstein et al., 2001). In the reaction, one oxygen atom is inserted into the prolyl residue and the other atom is inserted into the co-substrate α-ketoglutarate, splitting it into CO2 and succinate (Kaelin and Ratcliffe, 2008). Factor inhibiting HIF-1 (FIH-1) represses HIF-1α transactivation function (Mahon et al., 2001) by hydroxylating an asparaginyl residue, using O2 and α-ketoglutarate as substrates, thereby blocking the association of HIF-1α with the p300 coactivator protein (Lando et al., 2002). Dimethyloxalylglycine (DMOG), a competitive antagonist of α-ketoglutarate, inhibits the hydroxylases and induces HIF-1-dependent transcription (Epstein et al., 2001). HIF-1 activity is also induced by iron chelators (such as desferrioxamine) and cobalt chloride, which inhibit hydroxylases by displacing Fe(II) from the catalytic center (Epstein et al., 2001).

Studies in cultured cells (Jiang et al., 1996b) and isolated, perfused, and ventilated lung preparations (Yu et al., 1998) revealed an exponential increase in HIF-1α levels at O2 concentrations less than 6% (~40 mm Hg), which is not explained by known biochemical properties of the hydroxylases. In most adult tissues, O2concentrations are in the range of 3-5% and any decrease occurs along the steep portion of the dose-response curve, allowing a graded response to hypoxia. Analyses of cultured human cells have revealed that expression of hundreds of genes was increased in response to hypoxia in a HIF-1-dependent manner (as determined by RNA interference) with direct binding of HIF-1 to the gene (as determined by chromatin immunoprecipitation [ChIP] assays); in addition, the expression of hundreds of genes was decreased in response to hypoxia in a HIF-1-dependent manner but binding of HIF-1 to these genes was not detected (Mole et al., 2009), indicating that HIF-dependent repression occurs via indirect mechanisms, which include HIF-1-dependent expression of transcriptional repressors (Yun et al., 2002) and microRNAs (Kulshreshtha et al., 2007). ChIP-seq studies have revealed that only 40% of HIF-1 binding sites are located within 2.5 kb of the transcription start site (Schödel et al., 2011).

In vertebrates, HIF-2α is a HIF-1α paralog that is also regulated by prolyl and asparaginyl hydroxylation and dimerizes with HIF-1β, but is expressed in a cell-restricted manner and plays important roles in erythropoiesis, vascularization, and pulmonary development, as described below. In D. melanogaster, the gene encoding the HIF-1α ortholog is designated similar and its paralog is designated trachealess because inactivating mutations result in defective development of the tracheal tubes (Wilk et al., 1996). In contrast, C. elegans has only a single HIF-1α homolog (Epstein et al., 2001). Thus, in both invertebrates and vertebrates, evolution of specialized systems for O2 delivery was associated with the appearance of a HIF-1α paralog.

O2 and Metabolism

The regulation of metabolism is a principal and primordial function of HIF-1. Under hypoxic conditions, HIF-1 mediates a transition from oxidative to glycolytic metabolism through its regulation of: PDK1, encoding pyruvate dehydrogenase (PDH) kinase 1, which phosphorylates and inactivates PDH, thereby inhibiting the conversion of pyruvate to acetyl coenzyme A for entry into the tricarboxylic acid cycle (Kim et al., 2006Papandreou et al., 2006); LDHA, encoding lactate dehydrogenase A, which converts pyruvate to lactate (Semenza et al. 1996); and BNIP3 (Zhang et al. 2008) and BNIP3L (Bellot et al., 2009), which mediate selective mitochondrial autophagy (Figure 1). HIF-1 also mediates a subunit switch in cytochrome coxidase that improves the efficiency of electron transfer under hypoxic conditions (Fukuda et al., 2007). An analogous subunit switch is also observed in Saccharomyces cerevisiae, although it is mediated by a completely different mechanism (yeast lack HIF-1), suggesting that it may represent a fundamental response of eukaryotic cells to hypoxia.

Regulation of Glucose Metabolism nihms-350382-f0001

Regulation of Glucose Metabolism nihms-350382-f0001

Regulation of Glucose Metabolism

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Figure 1
Regulation of Glucose Metabolism

It is conventional wisdom that cells switch to glycolysis when O2 becomes limiting for mitochondrial ATP production. Yet, HIF-1α-null mouse embryo fibroblasts, which do not down-regulate respiration under hypoxic conditions, have higher ATP levels at 1% O2 than wild-type cells at 20% O2, demonstrating that under these conditions O2 is not limiting for ATP production (Zhang et al., 2008). However, the HIF-1α-null cells die under prolonged hypoxic conditions due to ROS toxicity (Kim et al. 2006Zhang et al., 2008). These studies have led to a paradigm shift with regard to our understanding of the regulation of cellular metabolism (Semenza, 2011): the purpose of this switch is to prevent excess mitochondrial generation of ROS that would otherwise occur due to the reduced efficiency of electron transfer under hypoxic conditions (Chandel et al., 1998). This may be particularly important in stem cells, in which avoidance of DNA damage is critical (Suda et al., 2011).

Role of HIFs in Development

Much of mammalian embryogenesis occurs at O2 concentrations of 1-5% and O2 functions as a morphogen (through HIFs) in many developmental systems (Dunwoodie, 2009). Mice that are homozygous for a null allele at the locus encoding HIF-1α die by embryonic day 10.5 with cardiac malformations, vascular defects, and impaired erythropoiesis, indicating that all three components of the circulatory system are dependent upon HIF-1 for normal development (Iyer et al., 1998Yoon et al., 2011). Depending on the genetic background, mice lacking HIF-2α: die by embryonic day 12.5 with vascular defects (Peng et al., 2000) or bradycardia due to deficient catecholamine production (Tian et al., 1998); die as neonates due to impaired lung maturation (Compernolle et al., 2002); or die several months after birth due to ROS-mediated multi-organ failure (Scortegagna et al., 2003). Thus, while vertebrate evolution was associated with concomitant appearance of the circulatory system and HIF-2α, both HIF-1 and HIF-2 have important roles in circulatory system development. Conditional knockout of HIF-1α in specific cell types has demonstrated important roles in chondrogenesis (Schipani et al., 2001), adipogenesis (Yun et al., 2002), B-lymphocyte development (Kojima et al., 2002), osteogenesis (Wang et al., 2007), hematopoiesis (Takubo et al., 2010), T-lymphocyte differentiation (Dang et al., 2011), and innate immunity (Zinkernagel et al., 2007). While knockout mouse experiments point to the adverse effects of HIF-1 loss-of-function on development, it is also possible that increased HIF-1 activity, induced by hypoxia in embryonic tissues as a result of abnormalities in placental blood flow, may also dysregulate development and result in congenital malformations. For example, HIF-1α has been shown to interact with, and stimulate the transcriptional activity of, Notch, which plays a key role in many developmental pathways (Gustafsson et al., 2005).

Translational Prospects

Drug discovery programs have been initiated at many pharmaceutical and biotech companies to develop prolyl hydroxylase inhibitors (PHIs) that, as described above for DMOG, induce HIF activity for treatment of disorders in which HIF mediates protective physiological responses. Local and/or short term induction of HIF activity by PHIs, gene therapy, or other means are likely to be useful novel therapies for many of the diseases described above. In the case of ischemic cardiovascular disease, local therapy is needed to provide homing signals for the recruitment of BMDACs. Chronic systemic use of PHIs must be approached with great caution: individuals with genetic mutations that constitutively activate the HIF pathway (described below) have increased incidence of cardiovascular disease and mortality (Yoon et al., 2011). On the other hand, the profound inhibition of HIF activity and vascular responses to ischemia that are associated with aging suggest that systemic replacement therapy might be contemplated as a preventive measure for subjects in whom impaired HIF responses to hypoxia can be documented. In C. elegans, VHL loss-of-function increases lifespan in a HIF-1-dependent manner (Mehta et al., 2009), providing further evidence for a mutually antagonistic relationship between HIF-1 and aging.

Cancer

Cancers contain hypoxic regions as a result of high rates of cell proliferation coupled with the formation of vasculature that is structurally and functionally abnormal. Increased HIF-1α and/or HIF-2α levels in diagnostic tumor biopsies are associated with increased risk of mortality in cancers of the bladder, brain, breast, colon, cervix, endometrium, head/neck, lung, ovary, pancreas, prostate, rectum, and stomach; these results are complemented by experimental studies, which demonstrate that genetic manipulations that increase HIF-1α expression result in increased tumor growth, whereas loss of HIF activity results in decreased tumor growth (Semenza, 2010). HIFs are also activated by genetic alterations, most notably, VHL loss of function in clear cell renal carcinoma (Majmunder et al., 2010). HIFs activate transcription of genes that play key roles in critical aspects of cancer biology, including stem cell maintenance (Wang et al., 2011), cell immortalization, epithelial-mesenchymal transition (Mak et al., 2010), genetic instability (Huang et al., 2007), vascularization (Liao and Johnson, 2007), glucose metabolism (Luo et al., 2011), pH regulation (Swietach et al., 2007), immune evasion (Lukashev et al., 2007), invasion and metastasis (Chan and Giaccia, 2007), and radiation resistance (Moeller et al., 2007). Given the extensive validation of HIF-1 as a potential therapeutic target, drugs that inhibit HIF-1 have been identified and shown to have anti-cancer effects in xenograft models (Table 1Semenza, 2010).

Table 1  Drugs that Inhibit HIF-1

Process Inhibited Drug Class Prototype
HIF-1 α synthesis Cardiac glycosidemTOR inhibitorMicrotubule targeting agent

Topoisomerase I inhibitor

DigoxinRapamycin2-Methoxyestradiol

Topotecan

HIF-1 α protein stability HDAC inhibitorHSP90 inhibitorCalcineurin inhibitor

Guanylate cyclase activator

LAQ82417-AAGCyclosporine

YC-1

Heterodimerization Antimicrobial agent Acriflavine
DNA binding AnthracyclineQuinoxaline antibiotic DoxorubicinEchinomycin
Transactivation Proteasome inhibitorAntifungal agent BortezomibAmphotericin B
Signal transduction BCR-ABL inhibitorCyclooxygenase inhibitorEGFR inhibitor

HER2 inhibitor

ImatinibIbuprofenErlotinib, Gefitinib

Trastuzumab

Over 100 women die every day of breast cancer in the U.S. The mean PO2 is 10 mm Hg in breast cancer as compared to > 60 mm Hg in normal breast tissue and cancers with PO2 < 10 mm Hg are associated with increased risk of metastasis and patient mortality (Vaupel et al., 2004). Increased HIF-1α protein levels, as identified by immunohistochemical analysis of tumor biopsies, are associated with increased risk of metastasis and/or patient mortality in unselected breast cancer patients and in lymph node-positive, lymph node-negative, HER2+, or estrogen receptor+ subpopulations (Semenza, 2011). Metastasis is responsible for > 90% of breast cancer mortality. The requirement for HIF-1 in breast cancer metastasis has been demonstrated for both autochthonous tumors in transgenic mice (Liao et al., 2007) and orthotopic transplants in immunodeficient mice (Zhang et al., 2011Wong et al., 2011). Primary tumors direct the recruitment of bone marrow-derived cells to the lungs and other sites of metastasis (Kaplan et al., 2005). In breast cancer, hypoxia induces the expression of lysyl oxidase (LOX), a secreted protein that remodels collagen at sites of metastatic niche formation (Erler et al., 2009). In addition to LOX, breast cancers also express LOX-like proteins 2 and 4. LOX, LOXL2, and LOXL4 are all HIF-1-regulated genes and HIF-1 inhibition blocks metastatic niche formation regardless of which LOX/LOXL protein is expressed, whereas available LOX inhibitors are not effective against all LOXL proteins (Wong et al., 2011), again illustrating the role of HIF-1 as a master regulator that controls the expression of multiple genes involved in a single (patho)physiological process.

Translational Prospects

Small molecule inhibitors of HIF activity that have anti-cancer effects in mouse models have been identified (Table 1). Inhibition of HIF impairs both vascular and metabolic adaptations to hypoxia, which may decrease O2 delivery and increase O2 utilization. These drugs are likely to be useful (as components of multidrug regimens) in the treatment of a subset of cancer patients in whom high HIF activity is driving progression. As with all novel cancer therapeutics, successful translation will require the development of methods for identifying the appropriate patient cohort. Effects of combination drug therapy also need to be considered. VEGF receptor tyrosine kinase inhibitors, which induce tumor hypoxia by blocking vascularization, have been reported to increase metastasis in mouse models (Ebos et al., 2009), which may be mediated by HIF-1; if so, combined use of HIF-1 inhibitors with these drugs may prevent unintended counter-therapeutic effects.

HIF inhibitors may also be useful in the treatment of other diseases in which dysregulated HIF activity is pathogenic. Proof of principle has been established in mouse models of ocular neovascularization, a major cause of blindness in the developed world, in which systemic or intraocular injection of the HIF-1 inhibitor digoxin is therapeutic (Yoshida et al., 2010). Systemic administration of HIF inhibitors for cancer therapy would be contraindicated in patients who also have ischemic cardiovascular disease, in which HIF activity is protective. The analysis of SNPs at the HIF1A locus described above suggests that the population may include HIF hypo-responders, who are at increased risk of severe ischemic cardiovascular disease. It is also possible that HIF hyper-responders, such as individuals with hereditary erythrocytosis, are at increased risk of particularly aggressive cancer.

O2 and Evolution, Part 2

When lowlanders sojourn to high altitude, hypobaric hypoxia induces erythropoiesis, which is a relatively ineffective response because the problem is not insufficient red cells, but rather insufficient ambient O2. Chronic erythrocytosis increases the risk of heart attack, stroke, and fetal loss during pregnancy. Many high-altitude Tibetans maintain the same hemoglobin concentration as lowlanders and yet, despite severe hypoxemia, they also maintain aerobic metabolism. The basis for this remarkable evolutionary adaptation appears to have involved the selection of genetic variants at multiple loci encoding components of the oxygen sensing system, particularly HIF-2α (Beall et al., 2010Simonson et al., 2010Yi et al., 2010). Given that hereditary erythrocytosis is associated with modest HIF-2α gain-of-function, the Tibetan genotype associated with absence of an erythrocytotic response to hypoxia may encode reduced HIF-2α activity along with other alterations that increase metabolic efficiency. Delineating the molecular mechanisms underlying these metabolic adaptations may lead to novel therapies for ischemic disorders, illustrating the importance of oxygen homeostasis as a nexus where evolution, biology, and medicine converge.

7.9.4 Hypoxia-inducible factor 1. Regulator of mitochondrial metabolism and mediator of ischemic preconditioning

Semenza GL1.
Biochim Biophys Acta. 2011 Jul; 1813(7):1263-8.
http://dx.doi.org/10.1016%2Fj.bbamcr.2010.08.006

Hypoxia-inducible factor 1 (HIF-1) mediates adaptive responses to reduced oxygen availability by regulating gene expression. A critical cell-autonomous adaptive response to chronic hypoxia controlled by HIF-1 is reduced mitochondrial mass and/or metabolism. Exposure of HIF-1-deficient fibroblasts to chronic hypoxia results in cell death due to excessive levels of reactive oxygen species (ROS). HIF-1 reduces ROS production under hypoxic conditions by multiple mechanisms including: a subunit switch in cytochrome c oxidase from the COX4-1 to COX4-2 regulatory subunit that increases the efficiency of complex IV; induction of pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; induction of BNIP3, which triggers mitochondrial selective autophagy; and induction of microRNA-210, which blocks assembly of Fe/S clusters that are required for oxidative phosphorylation. HIF-1 is also required for ischemic preconditioning and this effect may be due in part to its induction of CD73, the enzyme that produces adenosine. HIF-1-dependent regulation of mitochondrial metabolism may also contribute to the protective effects of ischemic preconditioning.

The story of life on Earth is a tale of oxygen production and utilization. Approximately 3 billion years ago, primitive single-celled organisms evolved the capacity for photosynthesis, a biochemical process in which photons of solar energy are captured by chlorophyll and used to power the reaction of CO2 and H2O to form glucose and O2. The subsequent rise in the atmospheric O2 concentration over the next billion years set the stage for the ascendance of organisms with the capacity for respiration, a process that consumes glucose and O2 and generates CO2, H2O, and energy in the form of ATP. Some of these single-celled organisms eventually took up residence within the cytoplasm of other cells and devoted all of their effort to energy production as mitochondria. Compared to the conversion of glucose to lactate by glycolysis, the complete oxidation of glucose by respiration provided such a large increase in energy production that it made possible the evolution of multicellular organisms. Among metazoan organisms, the progressive increase in body size during evolution was accompanied by progressively more complex anatomic structures that function to ensure the adequate delivery of O2 to all cells, ultimately resulting in the sophisticated circulatory and respiratory systems of vertebrates.

All metazoan cells can sense and respond to reduced O2 availability (hypoxia). Adaptive responses to hypoxia can be cell autonomous, such as the alterations in mitochondrial metabolism that are described below, or non-cell-autonomous, such as changes in tissue vascularization (reviewed in ref. 1). Primary responses to hypoxia need to be distinguished from secondary responses to sequelae of hypoxia, such as the adaptive responses to ATP depletion that are mediated by AMP kinase (reviewed in ref 2). In contrast, recent data suggest that O2 and redox homeostasis are inextricably linked and that changes in oxygenation are inevitably associated with changes in the levels of reactive oxygen species (ROS), as will be discussed below.

HIF-1 Regulates Oxygen Homeostasis in All Metazoan Species

A key regulator of the developmental and physiological networks required for the maintenance of O2homeostasis is hypoxia-inducible factor 1 (HIF-1). HIF-1 is a heterodimeric transcription factor that is composed of an O2-regulated HIF-1α subunit and a constitutively expressed HIF-1β subunit [3,4]. HIF-1 regulates the expression of hundreds of genes through several major mechanisms. First, HIF-1 binds directly to hypoxia response elements, which are cis-acting DNA sequences located within target genes [5]. The binding of HIF-1 results in the recruitment of co-activator proteins that activate gene transcription (Fig. 1A). Only rarely does HIF-1 binding result in transcriptional repression [6]. Instead, HIF-1 represses gene expression by indirect mechanisms, which are described below. Second, among the genes activated by HIF-1 are many that encode transcription factors [7], which when synthesized can bind to and regulate (either positively or negatively) secondary batteries of target genes (Fig. 1B). Third, another group of HIF-1 target genes encode members of the Jumonji domain family of histone demethylases [8,9], which regulate gene expression by modifying chromatin structure (Fig. 1C). Fourth, HIF-1 can activate the transcription of genes encoding microRNAs [10], which bind to specific mRNA molecules and either block their translation or mediate their degradation (Fig. 1D). Fifth, the isolated HIF-1α subunit can bind to other transcription factors [11,12] and inhibit (Fig. 1E) or potentiate (Fig. 1F) their activity.

Mechanisms by which HIF-1 regulates gene expression. nihms232046f1

Mechanisms by which HIF-1 regulates gene expression. nihms232046f1

Mechanisms by which HIF-1 regulates gene expression.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010308/bin/nihms232046f1.gif

Fig. 1 Mechanisms by which HIF-1 regulates gene expression. (A) Top: HIF-1 binds directly to target genes at a cis-acting hypoxia response element (HRE) and recruits coactivator proteins such as p300 to increase gene transcription.

HIF-1α and HIF-1β are present in all metazoan species, including the simple roundworm Caenorhabitis elegans [13], which consists of ~103 cells and has no specialized systems for O2 delivery. The fruit flyDrosophila melanogaster evolved tracheal tubes, which conduct air into the interior of the body from which it diffuses to surrounding cells. In vertebrates, the development of the circulatory and respiratory systems was accompanied by the appearance of HIF-2α, which is also O2-regulated and heterodimerizes with HIF-1β [14] but is only expressed in a restricted number of cell types [15], whereas HIF-1α and HIF-1β are expressed in all human and mouse tissues [16]. In Drosophila, the ubiquitiously expressed HIF-1α ortholog is designatedSimilar [17] and the paralogous gene that is expressed specifically in tracheal tubes is designated Trachealess[18].

HIF-1 Activity is Regulated by Oxygen

In the presence of O2, HIF-1α and HIF-2α are subjected to hydroxylation by prolyl-4-hydroxylase domain proteins (PHDs) that use O2 and α-ketoglutarate as substrates and generate CO2 and succinate as by-products [19]. Prolyl hydroxylation is required for binding of the von Hipple-Lindau protein, which recruits a ubiquitin-protein ligase that targets HIF-1α and HIF-2α for proteasomal degradation (Fig. 2). Under hypoxic conditions, the rate of hydroxylation declines and the non-hydroxylated proteins accumulate. HIF-1α transactivation domain function is also O2-regulated [20,21]. Factor inhibiting HIF-1 (FIH-1) represses transactivation domain function [22] by hydroxylating asparagine residue 803 in HIF-1α, thereby blocking the binding of the co-activators p300 and CBP [23].

Negative regulation of HIF-1 activity by oxygen nihms232046f2

Negative regulation of HIF-1 activity by oxygen nihms232046f2

Negative regulation of HIF-1 activity by oxygen

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010308/bin/nihms232046f2.gif

Fig. 2 Negative regulation of HIF-1 activity by oxygen. Top: In the presence of O2: prolyl hydroxylation of HIF-1a leads to binding of the von Hippel-Lindau protein (VHL), which recruits a ubiquitin protein-ligase that targets HIF-1a for proteasomal degradation;

When cells are acutely exposed to hypoxic conditions, the generation of ROS at complex III of the mitochondrial electron transport chain (ETC) increases and is required for the induction of HIF-1α protein levels [24]. More than a decade after these observations were first made, the precise mechanism by which hypoxia increases ROS generation and by which ROS induces HIF-1α accumulation remain unknown. However, the prolyl and asparaginyl hydroxylases contain Fe2+ in their active site and oxidation to Fe3+would block their catalytic activity. Since O2 is a substrate for the hydroxylation reaction, anoxia also results in a loss of enzyme activity. However, the concentration at which O2 becomes limiting for prolyl or asparaginyl hydroxylase activity in vivo is not known.

HIF-1 Regulates the Balance Between Oxidative and Glycolytic Metabolism

All metazoan organisms depend on mitochondrial respiration as the primary mechanism for generating sufficient amounts of ATP to maintain cellular and systemic homeostasis. Respiration, in turn, is dependent on an adequate supply of O2 to serve as the final electron acceptor in the ETC. In this process, electrons are transferred from complex I (or complex II) to complex III, then to complex IV, and finally to O2, which is reduced to water. This orderly transfer of electrons generates a proton gradient across the inner mitochondrial membrane that is used to drive the synthesis of ATP. At each step of this process, some electrons combine with O2 prematurely, resulting in the production of superoxide anion, which is reduced to hydrogen peroxide through the activity of mitochondrial superoxide dismutase. The efficiency of electron transport appears to be optimized to the physiological range of O2 concentrations, such that ATP is produced without the production of excess superoxide, hydrogen peroxide, and other ROS at levels that would result in the increased oxidation of cellular macromolecules and subsequent cellular dysfunction or death. In contrast, when O2levels are acutely increased or decreased, an imbalance between O2 and electron flow occurs, which results in increased ROS production.

MEFs require HIF-1 activity to make two critical metabolic adaptations to chronic hypoxia. First, HIF-1 activates the gene encoding pyruvate dehydrogenase (PDH) kinase 1 (PDK1), which phosphorylates and inactivates the catalytic subunit of PDH, the enzyme that converts pyruvate to acetyl coenzyme A (AcCoA) for entry into the mitochondrial tricarboxylic acid (TCA) cycle [25]. Second, HIF-1 activates the gene encoding BNIP3, a member of the Bcl-2 family of mitochondrial proteins, which triggers selective mitochondrial autophagy [26]. Interference with the induction of either of these proteins in hypoxic cells results in increased ROS production and increased cell death. Overexpression of either PDK1 or BNIP3 rescues HIF-1α-null MEFs. By shunting pyruvate away from the mitochondria, PDK1 decreases flux through the ETC and thereby counteracts the reduced efficiency of electron transport under hypoxic conditions, which would otherwise increase ROS production. PDK1 functions cooperatively with the product of another HIF-1 target gene, LDHA [27], which converts pyruvate to lactate, thereby further reducing available substrate for the PDH reaction.

PDK1 effectively reduces flux through the TCA cycle and thereby reduces flux through the ETC in cells that primarily utilize glucose as a substrate for oxidative phosphorylation. However, PDK1 is predicted to have little effect on ROS generation in cells that utilize fatty acid oxidation as their source of AcCoA. Hence another strategy to reduce ROS generation under hypoxic conditions is selective mitochondrial autophagy [26]. MEFs reduce their mitochondrial mass and O2 consumption by >50% after only two days at 1% O2. BNIP3 competes with Beclin-1 for binding to Bcl-2, thereby freeing Beclin-1 to activate autophagy. Using short hairpin RNAs to knockdown expression of BNIP3, Beclin-1, or Atg5 (another component of the autophagy machinery) phenocopied HIF-1α-null cells by preventing hypoxia-induced reductions in mitochondrial mass and O2 consumption as a result of failure to induce autophagy [26]. HIF-1-regulated expression of BNIP3L also contributes to hypoxia-induced autophagy [28]. Remarkably, mice heterozygous for the HIF-1α KO allele have a significantly increased ratio of mitochondrial:nuclear DNA in their lungs (even though this is the organ that is exposed to the highest O2 concentrations), indicating that HIF-1 regulates mitochondrial mass under physiological conditions in vivo [26]. In contrast to the selective mitochondrial autophagy that is induced in response to hypoxia as described above, autophagy (of unspecified cellular components) induced by anoxia does not require HIF-1, BNIP3, or BNIP3L, but is instead regulated by AMP kinase [29].

The multiplicity of HIF-1-mediated mechanisms identified so far by which cells regulate mitochondrial metabolism in response to changes in cellular O2 concentration (Fig. 3) suggests that this is a critical adaptive response to hypoxia. The fundamental nature of this physiological response is underscored by the fact that yeast also switch COX4 subunits in an O2-dependent manner but do so by an entirely different molecular mechanism [33], since yeast do not have a HIF-1α homologue. Thus, it appears that by convergent evolution both unicellular and multicellular eukaryotes possess mechanisms by which they modulate mitochondrial metabolism to maintain redox homeostasis despite changes in O2 availability. Indeed, it is the balance between energy, oxygen, and redox homeostasis that represents the key to life with oxygen.

Regulation of mitochondrial metabolism by HIF-1  nihms232046f3

Regulation of mitochondrial metabolism by HIF-1 nihms232046f3

Regulation of mitochondrial metabolism by HIF-1α

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010308/bin/nihms232046f3.gif

Fig. 3 Regulation of mitochondrial metabolism by HIF-1α. Acute hypoxia leads to increased mitochondrial generation of reactive oxygen species (ROS). Decreased O2 and increased ROS levels lead to decreased HIF-1α hydroxylation (see Fig. 2) and increased HIF-1-dependent 

 

7.9.5 Regulation of cancer cell metabolism by hypoxia-inducible factor 1

Semenza GL1.
Semin Cancer Biol. 2009 Feb; 19(1):12-6.

The Warburg Effect: The Re-discovery of the Importance of Aerobic Glycolysis in Tumor Cells
http://dx.doi.org:/10.1016/j.semcancer.2008.11.009

The induction of hypoxia-inducible factor 1 (HIF-1) activity, either as a result of intratumoral hypoxia or loss-of-function mutations in the VHL gene, leads to a dramatic reprogramming of cancer cell metabolism involving increased glucose transport into the cell, increased conversion of glucose to pyruvate, and a concomitant decrease in mitochondrial metabolism and mitochondrial mass. Blocking these adaptive metabolic responses to hypoxia leads to cell death due to toxic levels of reactive oxygen species. Targeting HIF-1 or metabolic enzymes encoded by HIF-1 target genes may represent a novel therapeutic approach to cancer.

http://ars.els-cdn.com/content/image/1-s2.0-S1044579X08001065-gr1.sml

http://ars.els-cdn.com/content/image/1-s2.0-S1044579X08001065-gr2.sml

7.9.6 Coming up for air. HIF-1 and mitochondrial oxygen consumption

Simon MC1.
Cell Metab. 2006 Mar;3(3):150-1.
http://dx.doi.org/10.1016/j.cmet.2006.02.007

Hypoxic cells induce glycolytic enzymes; this HIF-1-mediated metabolic adaptation increases glucose flux to pyruvate and produces glycolytic ATP. Two papers in this issue of Cell Metabolism (Kim et al., 2006; Papandreou et al., 2006) demonstrate that HIF-1 also influences mitochondrial function, suppressing both the TCA cycle and respiration by inducing pyruvate dehydrogenase kinase 1 (PDK1). PDK1 regulation in hypoxic cells promotes cell survival.

Comment on

Oxygen deprivation (hypoxia) occurs in tissues when O2 supply via the cardiovascular system fails to meet the demand of O2-consuming cells. Hypoxia occurs naturally in physiological settings (e.g., embryonic development and exercising muscle), as well as in pathophysiological conditions (e.g., myocardial infarction, inflammation, and solid tumor formation). For over a century, it has been appreciated that O2-deprived cells exhibit increased conversion of glucose to lactate (the “Pasteur effect”). Activation of the Pasteur effect during hypoxia in mammalian cells is facilitated by HIF-1, which mediates the upregulation of glycolytic enzymes that support an increase in glycolytic ATP production as mitochondria become starved for O2, the substrate for oxidative phosphorylation (Seagroves et al., 2001). Thus, mitochondrial respiration passively decreases due to O2 depletion in hypoxic tissues. However, reports by Kim et al. (2006) and Papandreou et al. (2006) in this issue of Cell Metabolism demonstrate that this critical metabolic adaptation is more complex and includes an active suppression of mitochondrial pyruvate catabolism and O2consumption by HIF-1.

Mitochondrial oxidative phosphorylation is regulated by multiple mechanisms, including substrate availability. Major substrates include O2 (the terminal electron acceptor) and pyruvate (the primary carbon source). Pyruvate, as the end product of glycolysis, is converted to acetyl-CoA by the pyruvate dehydrogenase enzymatic complex and enters the tricarboxylic acid (TCA) cycle. Pyruvate conversion into acetyl-CoA is irreversible; this therefore represents an important regulatory point in cellular energy metabolism. Pyruvate dehydrogenase kinase (PDK) inhibits pyruvate dehydrogenase activity by phosphorylating its E1 subunit (Sugden and Holness, 2003). In the manuscripts by Kim et al. (2006) and Papandreou et al. (2006), the authors find that PDK1 is a HIF-1 target gene that actively regulates mitochondrial respiration by limiting pyruvate entry into the TCA cycle. By excluding pyruvate from mitochondrial metabolism, hypoxic cells accumulate pyruvate, which is then converted into lactate via lactate dehydrogenase (LDH), another HIF-1-regulated enzyme. Lactate in turn is released into the extracellular space, regenerating NAD+ for continued glycolysis by O2-starved cells (see Figure 1). This HIF-1-dependent block to mitochondrial O2 consumption promotes cell survival, especially when O2 deprivation is severe and prolonged.

multiple-hypoxia-induced-cellular-metabolic-changes-are-regulated-by-hif-1

multiple-hypoxia-induced-cellular-metabolic-changes-are-regulated-by-hif-1

http://ars.els-cdn.com/content/image/1-s2.0-S1550413106000672-gr1.jpg

Figure 1. Multiple hypoxia-induced cellular metabolic changes are regulated by HIF-1

By stimulating the expression of glucose transporters and glycolytic enzymes, HIF-1 promotes glycolysis to generate increased levels of pyruvate. In addition, HIF-1 promotes pyruvate reduction to lactate by activating lactate dehydrogenase (LDH). Pyruvate reduction to lactate regenerates NAD+, which permits continued glycolysis and ATP production by hypoxic cells. Furthermore, HIF-1 induces pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase and blocks conversion of pyruvate to acetyl CoA, resulting in decreased flux through the tricarboxylic acid (TCA) cycle. Decreased TCA cycle activity results in attenuation of oxidative phosphorylation and excessive mitochondrial reactive oxygen species (ROS) production. Because hypoxic cells already exhibit increased ROS, which have been shown to promote HIF-1 accumulation, the induction of PDK1 prevents the persistence of potentially harmful ROS levels.

Papandreou et al. demonstrate that hypoxic regulation of PDK has important implications for antitumor therapies. Recent interest has focused on cytotoxins that target hypoxic cells in tumor microenvironments, such as the drug tirapazamine (TPZ). Because intracellular O2 concentrations are decreased by mitochondrial O2 consumption, HIF-1 could protect tumor cells from TPZ-mediated cell death by maintaining intracellular O2 levels. Indeed, Papandreou et al. show that HIF-1-deficient cells grown at 2% O2 exhibit increased sensitivity to TPZ relative to wild-type cells, presumably due to higher rates of mitochondrial O2 consumption. HIF-1 inhibition in hypoxic tumor cells should have multiple therapeutic benefits, but the use of HIF-1 inhibitors in conjunction with other treatments has to be carefully evaluated for the most effective combination and sequence of drug delivery. One result of HIF-1 inhibition would be a relative decrease in intracellular O2 levels, making hypoxic cytotoxins such as TPZ more potent antitumor agents. Because PDK expression has been detected in multiple human tumor samples and appears to be induced by hypoxia (Koukourakis et al., 2005), small molecule inhibitors of HIF-1 combined with TPZ represent an attractive therapeutic approach for future clinical studies.

Hypoxic regulation of PDK1 has other important implications for cell survival during O2 depletion. Because the TCA cycle is coupled to electron transport, Kim et al. suggest that induction of the pyruvate dehydrogenase complex by PDK1 attenuates not only mitochondrial respiration but also the production of mitochondrial reactive oxygen species (ROS) in hypoxic cells. ROS are a byproduct of electron transfer to O2, and cells cultured at 1 to 5% O2 generate increased mitochondrial ROS relative to those cultured at 21% O2 (Chandel et al., 1998 and Guzy et al., 2005). In fact, hypoxia-induced mitochondrial ROS have also been shown to be necessary for the stabilization of HIF-1 in hypoxic cells (Brunelle et al., 2005Guzy et al., 2005 and Mansfield et al., 2005). However, the persistence of ROS could ultimately be lethal to tissues during chronic O2 deprivation, and PDK1 induction by HIF-1 should promote cell viability during long-term hypoxia. Kim et al. present evidence that HIF-1-deficient cells exhibit increased apoptosis after 72 hr of culture at 0.5% O2 compared to wild-type cells and that cell survival is rescued by enforced expression of exogenous PDK1. Furthermore, PDK1 reduces ROS production by the HIF-1 null cells. These findings support a novel prosurvival dimension of cellular hypoxic adaptation where PDK1 inhibits the TCA cycle, mitochondrial respiration, and chronic ROS production.

The HIF-1-mediated block to mitochondrial O2 consumption via PDK1 regulation also has implications for O2-sensing pathways by hypoxic cells. One school of thought suggests that perturbing mitochondrial O2consumption increases intracellular O2 concentrations and suppresses HIF-1 induction by promoting the activity of HIF prolyl hydroxylases, the O2-dependent enzymes that regulate HIF-1 stability (Hagen et al., 2003 and Doege et al., 2005). This model suggests that mitochondria function as “O2 sinks.” Although Papandreou et al. demonstrate that increased mitochondrial respiration due to PDK1 depletion results in decreased intracellular O2 levels (based on pimonidazole staining), these changes failed to reduce HIF-1 levels in hypoxic cells. Another model for hypoxic activation of HIF-1 describes a critical role for mitochondrial ROS in prolyl hydroxylase inhibition and HIF-1 stabilization in O2-starved cells (Brunelle et al., 2005Guzy et al., 2005 and Mansfield et al., 2005) (see Figure 1). The mitochondrial “O2 sink” hypothesis can account for some observations in the literature but fails to explain the inhibition of HIF-1 stabilization by ROS scavengers (Chandel et al., 1998Brunelle et al., 2005Guzy et al., 2005 and Sanjuán-Pla et al., 2005). While the relationship between HIF-1 stability, mitochondrial metabolism, ROS, and intracellular O2 redistribution will continue to be debated for some time, these most recent findings shed new light on findings by Louis Pasteur over a century ago.

Selected reading

Brunelle et al., 2005

J.K. Brunelle, E.L. Bell, N.M. Quesada, K. Vercauteren, V. Tiranti, M. Zeviani, R.C. Scarpulla, N.S. Chandel

Cell Metab., 1 (2005), pp. 409–414

Article  PDF (324 K) View Record in Scopus Citing articles (357)

Chandel et al., 1998

N.S. Chandel, E. Maltepe, E. Goldwasser, C.E. Mathieu, M.C. Simon, P.T. Schumacker

Proc. Natl. Acad. Sci. USA, 95 (1998), pp. 11715–11720

View Record in Scopus Full Text via CrossRef Citing articles (973)

Doege et al., 2005Doege, S. Heine, I. Jensen, W. Jelkmann, E. Metzen

Blood, 106 (2005), pp. 2311–2317

View Record in Scopus Full Text via CrossRef Citing articles (84)

Guzy et al., 2005

R.D. Guzy, B. Hoyos, E. Robin, H. Chen, L. Liu, K.D. Mansfield, M.C. Simon, U. Hammerling, P.T. Schumacker

Cell Metab., 1 (2005), pp. 401–408

Article  PDF (510 K) View Record in Scopus Citing articles (593)

Hagen et al., 2003

Hagen, C.T. Taylor, F. Lam, S. Moncada

Science, 302 (2003), pp. 1975–1978

View Record in Scopus Full Text via CrossRef Citing articles (450)

7.9.7 HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption

Papandreou I1Cairns RAFontana LLim ALDenko NC.
Cell Metab. 2006 Mar; 3(3):187-97.
http://dx.doi.org/10.1016/j.cmet.2006.01.012

The HIF-1 transcription factor drives hypoxic gene expression changes that are thought to be adaptive for cells exposed to a reduced-oxygen environment. For example, HIF-1 induces the expression of glycolytic genes. It is presumed that increased glycolysis is necessary to produce energy when low oxygen will not support oxidative phosphorylation at the mitochondria. However, we find that while HIF-1 stimulates glycolysis, it also actively represses mitochondrial function and oxygen consumption by inducing pyruvate dehydrogenase kinase 1 (PDK1). PDK1 phosphorylates and inhibits pyruvate dehydrogenase from using pyruvate to fuel the mitochondrial TCA cycle. This causes a drop in mitochondrial oxygen consumption and results in a relative increase in intracellular oxygen tension. We show by genetic means that HIF-1-dependent block to oxygen utilization results in increased oxygen availability, decreased cell death when total oxygen is limiting, and reduced cell death in response to the hypoxic cytotoxin tirapazamine.

Comment in

Tissue hypoxia results when supply of oxygen from the bloodstream does not meet demand from the cells in the tissue. Such a supply-demand mismatch can occur in physiologic conditions such as the exercising muscle, in the pathologic condition such as the ischemic heart, or in the tumor microenvironment (Hockel and Vaupel, 2001 and Semenza, 2004). In either the physiologic circumstance or pathologic conditions, there is a molecular response from the cell in which a program of gene expression changes is initiated by the hypoxia-inducible factor-1 (HIF-1) transcription factor. This program of gene expression changes is thought to help the cells adapt to the stressful environment. For example, HIF-1-dependent expression of erythropoietin and angiogenic compounds results in increased blood vessel formation for delivery of a richer supply of oxygenated blood to the hypoxic tissue. Additionally, HIF-1 induction of glycolytic enzymes allows for production of energy when the mitochondria are starved of oxygen as a substrate for oxidative phosphorylation. We now find that this metabolic adaptation is more complex, with HIF-1 not only regulating the supply of oxygen from the bloodstream, but also actively regulating the oxygen demand of the tissue by reducing the activity of the major cellular consumer of oxygen, the mitochondria.

Perhaps the best-studied example of chronic hypoxia is the hypoxia associated with the tumor microenvironment (Brown and Giaccia, 1998). The tumor suffers from poor oxygen supply through a chaotic jumble of blood vessels that are unable to adequately perfuse the tumor cells. The oxygen tension within the tumor is also a function of the demand within the tissue, with oxygen consumption influencing the extent of tumor hypoxia (Gulledge and Dewhirst, 1996 and Papandreou et al., 2005b). The net result is that a large fraction of the tumor cells are hypoxic. Oxygen tensions within the tumor range from near normal at the capillary wall, to near zero in the perinecrotic regions. This perfusion-limited hypoxia is a potent microenvironmental stress during tumor evolution (Graeber et al., 1996 and Hockel and Vaupel, 2001) and an important variable capable of predicting for poor patient outcome. (Brizel et al., 1996Cairns and Hill, 2004Hockel et al., 1996 and Nordsmark and Overgaard, 2004).

The HIF-1 transcription factor was first identified based on its ability to activate the erythropoetin gene in response to hypoxia (Wang and Semenza, 1993). Since then, it is has been shown to be activated by hypoxia in many cells and tissues, where it can induce hypoxia-responsive target genes such as VEGF and Glut1 (Airley et al., 2001 and Kimura et al., 2004). The connection between HIF-regulation and human cancer was directly linked when it was discovered that the VHL tumor suppressor gene was part of the molecular complex responsible for the oxic degradation of HIF-1α (Maxwell et al., 1999). In normoxia, a family of prolyl hydroxylase enzymes uses molecular oxygen as a substrate and modifies HIF-1α and HIF2α by hydroxylation of prolines 564 and 402 (Bruick and McKnight, 2001 and Epstein et al., 2001). VHL then recognizes the modified HIF-α proteins, acts as an E3-type of ubiquitin ligase, and along with elongins B and C is responsible for the polyubiquitination of HIF-αs and their proteosomal degradation (Bruick and McKnight, 2001Chan et al., 2002Ivan et al., 2001 and Jaakkola et al., 2001). Mutations in VHL lead to constitutive HIF-1 gene expression, and predispose humans to cancer. The ability to recognize modified HIF-αs is at least partly responsible for VHL activity as a tumor suppressor, as introduction of nondegradable HIF-2α is capable of overcoming the growth–inhibitory activity of wild-type (wt) VHL in renal cancer cells (Kondo et al., 2003).

Mitochondrial function can be regulated by PDK1 expression. Mitochondrial oxidative phosphorylation (OXPHOS) is regulated by several mechanisms, including substrate availability (Brown, 1992). The major substrates for OXPHOS are oxygen, which is the terminal electron acceptor, and pyruvate, which is the primary carbon source. Pyruvate is the end product of glycolysis and is converted to acetyl-CoA through the activity of the pyruvate dehydrogenase complex of enzymes. The acetyl-CoA then directly enters the TCA cycle at citrate synthase where it is combined with oxaloacetate to generate citrate. In metazoans, the conversion of pyruvate to acetyl-CoA is irreversible and therefore represents a critical regulatory point in cellular energy metabolism. Pyruvate dehydrogenase is regulated by three known mechanisms: it is inhibited by acetyl-CoA and NADH, it is stimulated by reduced energy in the cell, and it is inhibited by regulatory phosphorylation of its E1 subunit by pyruvate dehydrogenase kinase (PDK) (Holness and Sugden, 2003 and Sugden and Holness, 2003). There are four members of the PDK family in vertebrates, each with specific tissue distributions (Roche et al., 2001). PDK expression has been observed in human tumor biopsies (Koukourakis et al., 2005), and we have reported that PDK3 is hypoxia-inducible in some cell types (Denko et al., 2003). In this manuscript, we find that PDK1 is also a hypoxia-responsive protein that actively regulates the function of the mitochondria under hypoxic conditions by reducing pyruvate entry into the TCA cycle. By excluding pyruvate from mitochondrial consumption, PDK1 induction may increase the conversion of pyruvate to lactate, which is in turn shunted to the extracellular space, regenerating NAD for continued glycolysis.

Identification of HIF-dependent mitochondrial proteins through genomic and bioinformatics approaches

In order to help elucidate the role of HIF-1α in regulating metabolism, we undertook a genomic search for genes that were regulated by HIF-1 in tumor cells exposed to hypoxia in vitro. We used genetically matched human RCC4 cells that had lost VHL during tumorigenesis and displayed constitutive HIF-1 activity, and a cell line engineered to re-express VHL to establish hypoxia-dependent HIF activation. These cells were treated with 18 hr of stringent hypoxia (<0.01% oxygen), and microarray analysis performed. Using a strict 2.5-fold elevation as our cutoff, we identified 173 genes that were regulated by hypoxia and/or VHL status (Table S1 in the Supplemental Data available with this article online). We used the pattern of expression in these experiments to identify putative HIF-regulated genes—ones that were constitutively elevated in the parent RCC4s independent of hypoxia, downregulated in the RCC4VHL cells under normoxia, and elevated in response to hypoxia. Of the 173 hypoxia and VHL-regulated genes, 74 fit the putative HIF-1 target pattern. The open reading frames of these genes were run through a pair of bioinformatics engines in order to predict subcellular localization, and 10 proteins scored as mitochondrial on at least one engine. The genes, fold induction, and mitochondrial scores are listed in Table 1.

HIF-1 downregulates mitochondrial oxygen consumption

Having identified several putative HIF-1 responsive gene products that had the potential to regulate mitochondrial function, we then directly measured mitochondrial oxygen consumption in cells exposed to long-term hypoxia. While other groups have studied mitochondrial function under acute hypoxia (Chandel et al., 1997), this is one of the first descriptions of mitochondrial function after long-term hypoxia where there have been extensive hypoxia-induced gene expression changes. Figure 1A is an example of the primary oxygen trace from a Clark electrode showing a drop in oxygen concentration in cell suspensions of primary fibroblasts taken from normoxic and hypoxic cultures. The slope of the curve is a direct measure of the total cellular oxygen consumption rate. Exposure of either primary human or immortalized mouse fibroblasts to 24 hr of hypoxia resulted in a reduction of this rate by approximately 50% (Figures 1A and 1B). In these experiments, the oxygen consumption can be stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide 3-chloro phenylhydrazone) and was completely inhibited by 2 mM potassium cyanide. We determined that the change in total cellular oxygen consumption was due to changes in mitochondrial activity by the use of the cell-permeable poison of mitochondrial complex 3, Antimycin A. Figure 1C shows that the difference in the normoxic and hypoxic oxygen consumption in murine fibroblasts is entirely due to the Antimycin-sensitive mitochondrial consumption. The kinetics with which mitochondrial function slows in hypoxic tumor cells also suggests that it is due to gene expression changes because it takes over 6 hr to achieve maximal reduction, and the reversal of this repression requires at least another 6 hr of reoxygenation (Figure 1D). These effects are not likely due to proliferation or toxicity of the treatments as these conditions are not growth inhibitory or toxic to the cells (Papandreou et al., 2005a).

Since we had predicted from the gene expression data that the mitochondrial oxygen consumption changes were due to HIF-1-mediated expression changes, we tested several genetically matched systems to determine what role HIF-1 played in the process (Figure 2). We first tested the cell lines that had been used for microarray analysis and found that the parental RCC4 cells had reduced mitochondrial oxygen consumption when compared to the VHL-reintroduced cells. Oxygen consumption in the parental cells was insensitive to hypoxia, while it was reduced by hypoxia in the wild-type VHL-transfected cell lines. Interestingly, stable introduction of a tumor-derived mutant VHL (Y98H) that cannot degrade HIF was also unable to restore oxygen consumption. These results indicate that increased expression of HIF-1 is sufficient to reduce oxygen consumption (Figure 2A). We also investigated whether HIF-1 induction was required for the observed reduction in oxygen consumption in hypoxia using two genetically matched systems. We measured normoxic and hypoxic oxygen consumption in murine fibroblasts derived from wild-type or HIF-1α null embryos (Figure 2B) and from human RKO tumor cells and RKO cells constitutively expressing ShRNAs directed against the HIF-1α gene (Figures 2C and 4C). Neither of the HIF-deficient cell systems was able to reduce oxygen consumption in response to hypoxia. These data from the HIF-overexpressing RCC cells and the HIF-deficient cells indicate that HIF-1 is both necessary and sufficient for reducing mitochondrial oxygen consumption in hypoxia.

HIF-dependent mitochondrial changes are functional, not structural

Because addition of CCCP could increase oxygen consumption even in the hypoxia-treated cells, we hypothesized that the hypoxic inhibition was a regulated activity, not a structural change in the mitochondria in response to hypoxic stress. We confirmed this interpretation by examining several additional mitochondrial characteristics in hypoxic cells such as mitochondrial morphology, quantity, and membrane potential. We examined morphology by visual inspection of both the transiently transfected mitochondrially localized DsRed protein and the endogenous mitochondrial protein cytochrome C. Both markers were indistinguishable in the parental RCC4 and the RCC4VHL cells (Figure 3A). Likewise, we measured the mitochondrial membrane potential with the functional dye rhodamine 123 and found that it was identical in the matched RCC4 cells and the matched HIF wt and knockout (KO) cells when cultured in normoxia or hypoxia (Figure 3B). Finally, we determined that the quantity of mitochondria per cell was not altered in response to HIF or hypoxia by showing that the amount of the mitochondrial marker protein HSP60 was identical in the RCC4 and HIF cell lines (Figure 3C)

PDK1 is a HIF-1 inducible target protein

After examination of the list of putative HIF-regulated mitochondrial target genes, we hypothesized that PDK1 could mediate the functional changes that we observed in hypoxia. We therefore investigated PDK1 protein expression in response to HIF and hypoxia in the genetically matched cell systems. Figure 4A shows that in the RCC4 cells PDK1 and the HIF-target gene BNip3 (Greijer et al., 2005 and Papandreou et al., 2005a) were both induced by hypoxia in a VHL-dependent manner, with the expression of PDK1 inversely matching the oxygen consumption measured in Figure 1 above. Likewise, the HIF wt MEFs show oxygen-dependent induction of PDK1 and BNip3, while the HIF KO MEFs did not show any expression of either of these proteins under any oxygen conditions (Figure 4B). Finally, the parental RKO cells were able to induce PDK1 and the HIF target gene BNip3L in response to hypoxia, while the HIF-depleted ShRNA RKO cells could not induce either protein (Figure 4C). Therefore, in all three cell types, the HIF-1-dependent regulation of oxygen consumption seen in Figure 2, corresponds to the HIF-1-dependent induction of PDK1 seen in Figure 4.

In order to determine if PDK1 was a direct HIF-1 target gene, we analyzed the genomic sequence flanking the 5′ end of the gene for possible HIF-1 binding sites based on the consensus core HRE element (A/G)CGTG (Caro, 2001). Several such sites exist within the first 400 bases upstream, so we generated reporter constructs by fusing the genomic sequence from −400 to +30 of the start site of transcription to the firefly luciferase gene. In transfection experiments, the chimeric construct showed significant induction by either cotransfection with a constitutively active HIF proline mutant (P402A/P564G) (Chan et al., 2002) or exposure of the transfected cells to 0.5% oxygen (Figure 4D). Most noteworthy, when the reporter gene was transfected into the HIF-1α null cells, it did not show induction when the cells were cultured in hypoxia, but it did show induction when cotransfected with expression HIF-1α plasmid. We then generated deletions down to the first 36 bases upstream of transcription and found that even this short sequence was responsive to HIF-1 (Figure 4D). Analysis of this small fragment showed only one consensus HRE site located in an inverted orientation in the 5′ untranslated region. We synthesized and cloned a mutant promoter fragment in which the core element ACGTG was replaced with AAAAG, and this construct lost over 90% of its hypoxic induction. These experiments suggest that it is this HRE within the proximal 5′ UTR that HIF-1 uses to transactivate the endogenous PDK1 gene in response to hypoxia.

PDK1 is responsible for the HIF-dependent mitochondrial oxygen consumption changes

In order to directly test if PDK1 was the HIF-1 target gene responsible for the hypoxic reduction in mitochondrial oxygen consumption, we generated RKO cell lines with either knockdown or overexpression of PDK1 and measured the oxygen consumption in these derivatives. The PDK1 ShRNA stable knockdown line was generated as a pool of clones cotransfected with pSUPER ShPDK1 and pTK-hygro resistance gene. After selection for growth in hygromycin, the cells were tested by Western blot for the level of PDK1 protein expression. We found that normoxic PDK1 is reduced by 75%, however, there was measurable expression of PDK1 in these cells in response to hypoxia (Figure 5A). When we measured the corresponding oxygen consumption in these cells, we found a change commensurate with the level of PDK1. The knockdown cells show elevated baseline oxygen consumption, and partial reduction in this activity in response to hypoxia. Therefore, reduction of PDK1 expression by genetic means increased mitochondrial oxygen consumption in both normoxic and hypoxic conditions. Interestingly, these cells still induced HIF-1α (Figure 5A) and HIF-1 target genes such as BNip3L in response to hypoxia (data not shown), suggesting that altered PDK1 levels do not alter HIF-1α function.

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PDK1 expression directly regulates cellular oxygen consumption rate

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Figure 5. PDK1 expression directly regulates cellular oxygen consumption rate

  1. A)Western blot of RKO cell and ShRNAPDK1RKO cell lysates after exposure to 24 hr of normoxia or 0.5% O2. Blots were probed for HIF 1α, PDK1, and tubulin as a loading control.
  2. B)Oxygen consumption rate in RKO and ShRNAPDK1RKO cells after exposure to 24 hr of normoxia or 0.5% O2.
  3. C)Western blot of RKOiresGUS cell and RKOiresPDK1 cell lysates after exposure to 24 hr of normoxia or 0.5% O2. Blots were probed for HIF 1α, PDK1, and tubulin as a loading control.
  4. D)Oxygen consumption rate in RKOiresGUS and RKOiresPDK1 cells after exposure to 24 hr of normoxia or 0.5% O2.
  5. E)Model describing the interconnected effects of HIF-1 target gene activation on hypoxic cell metabolism. Reduced oxygen conditions causes HIF-1 to coordinately induce the enzymes shown in boxes. HIF-1 activation results in increased glucose transporter expression to increase intracellular glucose flux, induction of glycolytic enzymes increases the conversion of glucose to pyruvate generating energy and NADH, induction of PDK1 decreases mitochondrial utilization of pyruvate and oxygen, and induction of LDH increases the removal of excess pyruvate as lactate and also regenerates NAD+ for increased glycolysis.

For all graphs, the error bars represent the standard error of the mean.

We also determined if overexpression of PDK1 could lead to reduced mitochondrial oxygen consumption. A separate culture of RKO cells was transfected with a PDK1-IRES-puro expression plasmid and selected for resistance to puromycin. The pool of puromycin resistant cells was tested for PDK1 expression by Western blot. These cells showed a modest increase in PDK1 expression under control conditions when compared to the cells transfected with GUS-IRES-puro, with an additional increase in PDK1 protein in response to hypoxia (Figure 5C). The corresponding oxygen consumption measurements showed that the mitochondria is very sensitive to changes in the levels of PDK1, as even this slight increase was able to significantly reduce oxygen consumption in the normoxic PDK1-puro cultures. Further increase in PDK1 levels with hypoxia further reduced oxygen consumption in both cultures (Figure 5D). The model describing the relationship between hypoxia, HIF-1, PDK1, and intermediate metabolism is described inFigure 5E.

Altering oxygen consumption alters intracellular oxygen tension and sensitivity to hypoxia-dependent cell killing

The intracellular concentration of oxygen is a net result of the rate at which oxygen diffuses into the cell and the rate at which it is consumed. We hypothesized that the rate at which oxygen was consumed within the cell would significantly affect its steady-state intracellular concentrations. We tested this hypothesis in vitro using the hypoxic marker drug pimonidazole (Bennewith and Durand, 2004). We plated high density cultures of HIF wild-type and HIF knockout cells and placed these cultures in normoxic, 2% oxygen, and anoxic incubators for overnight treatment. The overnight treatment gives the cells time to adapt to the hypoxic conditions and establish altered oxygen consumption profiles. Pimonidozole was then added for the last 4 hr of the growth of the culture. Pimonidazole binding was detected after fixation of the cells using an FITC labeled anti-pimonidazole antibody and it was quantitated by flow cytometry. The quantity of the bound drug is a direct indication of the oxygen concentration within the cell (Bennewith and Durand, 2004). The histograms in Figure 6A show that the HIF-1 knockout and wild-type cells show similar staining in the cells grown in 0% oxygen. However, the cells treated with 2% oxygen show the consequence of the genetic removal of HIF-1. The HIF-proficient cells showed relatively less pimonidazole binding at 2% when compared to the 0% culture, while the HIF-deficient cells showed identical binding between the cells at 2% and those at 0%. We interpret these results to mean that the HIF-deficient cells have greater oxygen consumption, and this has lowered the intracellular oxygenation from the ambient 2% to close to zero intracellularly. The HIF-proficient cells reduced their oxygen consumption rate so that the rate of diffusion into the cell is greater than the rate of consumption.

Figure 6. HIF-dependent decrease in oxygen consumption raises intracellular oxygen concentration, protects when oxygen is limiting, and decreases sensitivity to tirapazamine in vitro

  1. A)Pimonidazole was used to determine the intracellular oxygen concentration of cells in culture. HIF wt and HIF KO MEFs were grown at high density and exposed to 2% O2or anoxia for 24 hr in glass dishes. For the last 4 hr of treatment, cells were exposed to 60 μg/ml pimonidazole. Pimonidazole binding was quantitated by flow cytometry after binding of an FITC conjugated anti-pimo mAb. Results are representative of two independent experiments.
  2. B)HIF1α reduces oxygen consumption and protects cells when total oxygen is limited. HIF wt and HIF KO cells were plated at high density and sealed in aluminum jigs at <0.02% oxygen. At the indicated times, cells were harvested, and dead cells were quantitated by trypan blue exclusion. Note both cell lines are equally sensitive to anoxia-induced apoptosis, so the death of the HIF null cells indicates that the increased oxygen consumption removed any residual oxygen in the jig and resulted in anoxia-induced death.
  3. C)PDK1 is responsible for HIF-1’s adaptive response when oxygen is limiting. A similar jig experiment was performed to measure survival in the parental RKO, the RKO ShRNAHIF1α, and the RKOShPDK1 cells. Cell death by trypan blue uptake was measured 48 hr after the jigs were sealed.
  4. D)HIF status alters sensitivity to TPZ in vitro. HIF wt and HIF KO MEFs were grown at high density in glass dishes and exposed to 21%, 2%, and <0.01% O2conditions for 18 hr in the presence of varying concentrations of Tirapazamine. After exposure, cells were harvested and replated under normoxia to determine clonogenic viability. Survival is calculated relative to the plating efficiency of cells exposed to 0 μM TPZ for each oxygen concentration.
  5. E)Cell density alters sensitivity to TPZ. HIF wt and HIF KO MEFs were grown at varying cell densities in glass dishes and exposed to 2% O2in the presence of 10 μM TPZ for 18 hr. After the exposure, survival was determined as described in (C).

For all graphs, the error bars represent the standard error of the mean.

HIF-induced PDK1 can reduce the total amount of oxygen consumed per cell. The reduction in the amount of oxygen consumed could be significant if there is a finite amount of oxygen available, as would be the case in the hours following a blood vessel occlusion. The tissue that is fed by the vessel would benefit from being economical with the oxygen that is present. We experimentally modeled such an event using aluminum jigs that could be sealed with defined amounts of cells and oxygen present (Siim et al., 1996). We placed 10 × 106 wild-type or HIF null cells in the sealed jig at 0.02% oxygen, waited for the cells to consume the remaining oxygen, and measured cell viability. We have previously shown that these two cell types are resistant to mild hypoxia and equally sensitive to anoxia-induced apoptosis (Papandreou et al., 2005a). Therefore, any death in this experiment would be the result of the cells consuming the small amount of remaining oxygen and dying in response to anoxia. We found that in sealed jigs, the wild-type cells are more able to adapt to the limited oxygen supply by reducing consumption. The HIF null cells continued to consume oxygen, reached anoxic levels, and started to lose viability within 36 hr (Figure 6B). This is a secondary adaptive effect of HIF1. We confirmed that PDK1 was responsible for this difference by performing a similar experiment using the parental RKO cells, the RKOShRNAHIF1α and the RKOShRNAPDK1 cells. We found similar results in which both the cells with HIF1α knockdown and PDK1 knockdown were sensitive to the long-term effects of being sealed in a jig with a defined amount of oxygen (Figure 6c). Note that the RKOShPDK1 cells are even more sensitive than the RKOShHIF1α cells, presumably because they have higher basal oxygen consumption rates (Figure 5B).

Because HIF-1 can help cells adapt to hypoxia and maintain some intracellular oxygen level, it may also protect tumor cells from killing by the hypoxic cytotoxin tirapazamine (TPZ). TPZ toxicity is very oxygen dependent, especially at oxygen levels between 1%–4% (Koch, 1993). We therefore tested the relative sensitivity of the HIF wt and HIF KO cells to TPZ killing in high density cultures (Figure 6D). We exposed the cells to the indicated concentrations of drug and oxygen concentrations overnight. The cells were then harvested and replated to determine reproductive viability by colony formation. Both cell types were equally resistant to TPZ at 21% oxygen, while both cell types are equally sensitive to TPZ in anoxic conditions where intracellular oxygen levels are equivalent (Figure 6A). The identical sensitivity of both cell types in anoxia indicates that both cell types are equally competent in repairing the TPZ-induced DNA damage that is presumed to be responsible for its toxicity. However, in 2% oxygen cultures, the HIF null cells displayed a significantly greater sensitivity to the drug than the wild-type cells. This suggests that the increased oxygen consumption rate in the HIF-deficient cells is sufficient to lower the intracellular oxygen concentration relative to that in the HIF-proficient cells. The lower oxygen level is significant enough to dramatically sensitize these cells to killing by TPZ.

If the increased sensitivity to TPZ in the HIF ko cells is determined by intracellular oxygen consumption differences, then this effect should also be cell-density dependent. We showed that this is indeed the case in Figure 6E where oxygen and TPZ concentrations were held constant, and increased cell density lead to increased TPZ toxicity. The effect was much more pronounced in the HIF KO cells, although the HIF wt cells showed some increased toxicity in the highest density cultures, consistent with the fact they were still consuming some oxygen, even with HIF present (Figure 1). The in vitro TPZ survival data is therefore consistent with our hypothesis that control of oxygen consumption can regulate intracellular oxygen concentration, and suggests that increased oxygen consumption could sensitize cells to hypoxia-dependent therapy.

Discussion

The findings presented here show that HIF-1 is actively responsible for regulating energy production in hypoxic cells by an additional, previously unrecognized mechanism. It has been shown that HIF-1 induces the enzymes responsible for glycolysis when it was presumed that low oxygen did not support efficient oxidative phosphorylation (Iyer et al., 1998 and Seagroves et al., 2001). The use of glucose to generate ATP is capable of satisfying the energy requirements of a cell if glucose is in excess (Papandreou et al., 2005a). We now find that at the same time that glycolysis is increasing, mitochondrial respiration is decreasing. However, the decreased respiration is not because there is not enough oxygen present to act as a substrate for oxidative phosphorylation, but because the flow of pyruvate into the TCA cycle has been reduced by the activity of pyruvate dehydrogenase kinase. Other reports have suggested that oxygen utilization is shifted in cells exposed to hypoxia, but these reports have focused on other regulators such as nitric oxide synthase (Hagen et al., 2003). NO can reduce oxygen consumption through direct inhibition of cytochrome oxidase, but this effect seems to be more significant at physiologic oxygen concentrations, not at severe levels seen in the tumor (Palacios-Callender et al., 2004).

7.9.8 HIF-1. upstream and downstream of cancer metabolism

Semenza GL1.
Curr Opin Genet Dev. 2010 Feb; 20(1):51-6
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Hypoxia-inducible factor 1 (HIF-1) plays a key role in the reprogramming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which take up glucose and convert it to lactate; pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; and BNIP3, which triggers selective mitochondrial autophagy. The shift from oxidative to glycolytic metabolism allows maintenance of redox homeostasis and cell survival under conditions of prolonged hypoxia. Many metabolic abnormalities in cancer cells increase HIF-1 activity. As a result, a feed-forward mechanism can be activated that drives HIF-1 activation and may promote tumor progression. Hypoxia-inducible factor 1 (HIF-1) plays a key role in the reprogramming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which take up glucose and convert it to lactate; pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; and BNIP3, which triggers selective mitochondrial autophagy. The shift from oxidative to glycolytic metabolism allows maintenance of redox homeostasis and cell survival under conditions of prolonged hypoxia. Many metabolic abnormalities in cancer cells increase HIF-1 activity. As a result, a feed-forward mechanism can be activated that drives HIF-1 activation and may promote tumor progression.

Metastatic cancer is characterized by reprogramming of cellular metabolism leading to increased uptake of glucose for use as both an anabolic and catabolic substrate. Increased glucose uptake is such a reliable feature that it is utilized clinically to detect metastases by positron emission tomography using 18F-fluorodeoxyglucose (FDG-PET) with a sensitivity of ~90% [1]. As with all aspects of cancer biology, the details of metabolic reprogramming differ widely among individual tumors. However, the role of specific signaling pathways and transcription factors in this process is now understood in considerable detail. This review will focus on the involvement of hypoxia-inducible factor 1 (HIF-1) in both mediating metabolic reprogramming and responding to metabolic alterations. The placement of HIF-1 both upstream and downstream of cancer metabolism results in a feed-forward mechanism that may play a major role in the development of the invasive, metastatic, and lethal cancer phenotype.

O2 concentrations are significantly reduced in many human cancers compared to the surrounding normal tissue. The median PO2 in breast cancers is ~10 mm Hg, as compared to ~65 mm Hg in normal breast tissue [2]. Reduced O2 availability induces HIF-1, which regulates the transcription of hundreds of genes [3*,4*] that encode proteins involved in every aspect of cancer biology, including: cell immortalization and stem cell maintenance; genetic instability; glucose and energy metabolism; vascularization; autocrine growth factor signaling; invasion and metastasis; immune evasion; and resistance to chemotherapy and radiation therapy [5].

HIF-1 is a transcription factor that consists of an O2-regulated HIF-1α and a constitutively expressed HIF-1β subunit [6]. In well-oxygenated cells, HIF-1α is hydroxylated on proline residue 402 (Pro-402) and/or Pro-564 by prolyl hydroxylase domain protein 2 (PHD2), which uses O2 and α-ketoglutarate as substrates in a reaction that generates CO2 and succinate as byproducts [7]. Prolyl-hydroxylated HIF-1α is bound by the von Hippel-Lindau tumor suppressor protein (VHL), which recruits an E3-ubiquitin ligase that targets HIF-1α for proteasomal degradation (Figure 1A). Asparagine 803 in the transactivation domain is hydroxylated in well-oxygenated cells by factor inhibiting HIF-1 (FIH-1), which blocks the binding of the coactivators p300 and CBP [7]. Under hypoxic conditions, the prolyl and asparaginyl hydroxylation reactions are inhibited by substrate (O2) deprivation and/or the mitochondrial generation of reactive oxygen species (ROS), which may oxidize Fe(II) present in the catalytic center of the hydroxylases [8].

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HIF-1 and metabolism

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Figure 1 HIF-1 and metabolism. (A) Regulation of HIF-1α protein synthesis and stability and HIF-1-dependent metabolic reprogramming. The rate of translation of HIF-1α mRNA into protein in cancer cells is dependent upon the activity of the mammalian 

The finding that acute changes in PO2 increase mitochondrial ROS production suggests that cellular respiration is optimized at physiological PO2 to limit ROS generation and that any deviation in PO2 — up or down — results in increased ROS generation. If hypoxia persists, induction of HIF-1 leads to adaptive mechanisms to reduce ROS and re-establish homeostasis, as described below. Prolyl and asparaginyl hydroxylation provide a molecular mechanism by which changes in cellular oxygenation can be transduced to the nucleus as changes in HIF-1 activity. This review will focus on recent advances in our understanding of the role of HIF-1 in controlling glucose and energy metabolism, but it should be appreciated that any increase in HIF-1 activity that leads to changes in cell metabolism will also affect many other critical aspects of cancer biology [5] that will not be addressed here.

HIF-1 target genes involved in glucose and energy metabolism

HIF-1 activates the transcription of SLC2A1 and SLC2A3, which encode the glucose transporters GLUT1 and GLUT3, respectively, as well as HK1 and HK2, which encode hexokinase, the first enzyme of the Embden-Meyerhoff (glycolytic) pathway [9]. Once taken up by GLUT and phosphorylated by HK, FDG cannot be metabolized further; thus, FDG-PET signal is determined by FDG delivery to tissue (i.e. perfusion) and GLUT/HK expression/activity. Unlike FDG, glucose is further metabolized to pyruvate by the action of the glycolytic enzymes, which are all encoded by HIF-1 target genes (Figure 1A). Glycolytic intermediates are also utilized for nucleotide and lipid synthesis [10]. Lactate dehydrogenase A (LDHA), which converts pyruvate to lactate, and monocarboxylate transporter 4 (MCT4), which transports lactate out of the cell (Figure 1B), are also regulated by HIF-1 [9,11]. Remarkably, lactate produced by hypoxic cancer cells can be taken up by non-hypoxic cells and used as a respiratory substrate [12**].

Pyruvate represents a critical metabolic control point, as it can be converted to acetyl coenzyme A (AcCoA) by pyruvate dehydrogenase (PDH) for entry into the tricarboxylic acid (TCA) cycle or it can be converted to lactate by LDHA (Figure 1B). Pyruvate dehydrogenase kinase (PDK), which phosphorylates and inactivates the catalytic domain of PDH, is encoded by four genes and PDK1 is activated by HIF-1 [13,14]. (Further studies are required to determine whether PDK2PDK3, or PDK4 is regulated by HIF-1.) As a result of PDK1 activation, pyruvate is actively shunted away from the mitochondria, which reduces flux through the TCA cycle, thereby reducing delivery of NADH and FADH2 to the electron transport chain. This is a critical adaptive response to hypoxia, because in HIF-1α–null mouse embryo fibroblasts (MEFs), PDK1 expression is not induced by hypoxia and the cells die due to excess ROS production, which can be ameliorated by forced expression of PDK1 [13]. MYC, which is activated in ~40% of human cancers, cooperates with HIF-1 to activate transcription of PDK1, thereby amplifying the hypoxic response [15]. Pharmacological inhibition of HIF-1 or PDK1 activity increases O2 consumption by cancer cells and increases the efficacy of a hypoxia-specific cytotoxin [16].

Hypoxia also induces mitochondrial autophagy in many human cancer cell lines through HIF-1-dependent expression of BNIP3 and a related BH3 domain protein, BNIP3L [19**]. Autocrine signaling through the platelet-derived growth factor receptor in cancer cells increases HIF-1 activity and thereby increases autophagy and cell survival under hypoxic conditions [21]. Autophagy may also occur in a HIF-1-independent manner in response to other physiological stimuli that are associated with hypoxic conditions, such as a decrease in the cellular ATP:AMP ratio, which activates AMP kinase signaling [22].

In clear cell renal carcinoma, VHL loss of function (LoF) results in constitutive HIF-1 activation, which is associated with impaired mitochondrial biogenesis that results from HIF-1-dependent expression of MXI1, which blocks MYC-dependent expression of PGC-1β, a coactivator that is required for mitochondrial biogenesis [23]. Inhibition of wild type MYC activity in renal cell carcinoma contrasts with the synergistic effect of HIF-1 and oncogenic MYC in activating PDK1 transcription [24].

Genetic and metabolic activators of HIF-1

Hypoxia plays a critical role in cancer progression [2,5] but not all cancer cells are hypoxic and a growing number of O2-independent mechanisms have been identified by which HIF-1 is induced [5]. Several mechanisms that are particularly relevant to cancer metabolism are described below.

Activation of mTOR

Alterations in mitochondrial metabolism

NAD+ levels

It is of interest that the NAD+-dependent deacetylase sirtuin 1 (SIRT1) was found to bind to, deacetylate, and increase transcriptional activation by HIF-2α but not HIF-1α [42**]. Another NAD+-dependent enzyme is poly(ADP-ribose) polymerase 1 (PARP1), which was recently shown to bind to HIF-1α and promote transactivation through a mechanism that required the enzymatic activity of PARP1 [43]. Thus, transactivation mediated by both HIF-1α and HIF-2α can be modulated according to NAD+ levels.

Nitric oxide

Increased expression of nitric oxide (NO) synthase isoforms and increased levels of NO have been shown to increase HIF-1α protein stability in human oral squamous cell carcinoma [44]. In prostate cancer, nuclear co-localization of endothelial NO synthase, estrogen receptor β, HIF-1α, and HIF-2α was associated with aggressive disease and the proteins were found to form chromatin complexes on the promoter of TERT gene encoding telomerase [45**]. The NOS2 gene encoding inducible NO synthase is HIF-1 regulated [5], suggesting another possible feed-forward mechanism.

7.9.9 In Vivo HIF-Mediated Reductive Carboxylation

Gameiro PA1Yang JMetelo AMPérez-Carro R, et al.
Cell Metab. 2013 Mar 5; 17(3):372-85.
http://dx.doi.org/10.1016%2Fj.cmet.2013.02.002

Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduced intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate or knocking down PDK-1 and ACLY restored citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action to maintain lipogenesis. Using [1–13C] glutamine, we demonstrated in vivo RC activity in VHL-deficient tumors growing as xenografts in mice. Lastly, HIF rendered VHL-deficient cells sensitive to glutamine deprivation in vitro, and systemic administration of glutaminase inhibitors suppressed the growth of RCC cells as mice xenografts.

Cancer cells undergo fundamental changes in their metabolism to support rapid growth, adapt to limited nutrient resources, and compete for these supplies with surrounding normal cells. One of the metabolic hallmarks of cancer is the activation of glycolysis and lactate production even in the presence of adequate oxygen. This is termed the Warburg effect, and efforts in cancer biology have revealed some of the molecular mechanisms responsible for this phenotype (Cairns et al., 2011). More recently, 13C isotopic studies have elucidated the complementary switch of glutamine metabolism that supports efficient carbon utilization for anabolism and growth (DeBerardinis and Cheng, 2010). Acetyl-CoA is a central biosynthetic precursor for lipid synthesis, being generated from glucose-derived citrate in well-oxygenated cells (Hatzivassiliou et al., 2005). Warburg-like cells, and those exposed to hypoxia, divert glucose to lactate, raising the question of how the tricarboxylic acid (TCA) cycle is supplied with acetyl-CoA to support lipogenesis. We and others demonstrated, using 13C isotopic tracers, that cells under hypoxic conditions or defective mitochondria primarily utilize glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate by isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) (Filipp et al., 2012Metallo et al., 2012;Mullen et al., 2012Wise et al., 2011).

The transcription factors hypoxia inducible factors 1α and 2α (HIF-1α, HIF-2α) have been established as master regulators of the hypoxic program and tumor phenotype (Gordan and Simon, 2007Semenza, 2010). In addition to tumor-associated hypoxia, HIF can be directly activated by cancer-associated mutations. The von Hippel-Lindau (VHL) tumor suppressor is inactivated in the majority of sporadic clear-cell renal carcinomas (RCC), with VHL-deficient RCC cells exhibiting constitutive HIF-1α and/or HIF-2α activity irrespective of oxygen availability (Kim and Kaelin, 2003). Previously, we showed that VHL-deficient cells also relied on RC for lipid synthesis even under normoxia. Moreover, metabolic profiling of two isogenic clones that differ in pVHL expression (WT8 and PRC3) suggested that reintroduction of wild-type VHL can restore glucose utilization for lipogenesis (Metallo et al., 2012). The VHL tumor suppressor protein (pVHL) has been reported to have several functions other than the well-studied targeting of HIF. Specifically, it has been reported that pVHL regulates the large subunit of RNA polymerase (Pol) II (Mikhaylova et al., 2008), p53 (Roe et al., 2006), and the Wnt signaling regulator Jade-1. VHL has also been implicated in regulation of NF-κB signaling, tubulin polymerization, cilia biogenesis, and proper assembly of extracellular fibronectin (Chitalia et al., 2008Kim and Kaelin, 2003Ohh et al., 1998Thoma et al., 2007Yang et al., 2007). Hypoxia inactivates the α-ketoglutarate-dependent HIF prolyl hydroxylases, leading to stabilization of HIF. In addition to this well-established function, oxygen tension regulates a larger family of α-ketoglutarate-dependent cellular oxygenases, leading to posttranslational modification of several substrates, among which are chromatin modifiers (Melvin and Rocha, 2012). It is therefore conceivable that the effect of hypoxia on RC that was reported previously may be mediated by signaling mechanisms independent of the disruption of the pVHL-HIF interaction. Here we (1) demonstrate that HIF is necessary and sufficient for RC, (2) provide insights into the molecular mechanisms that link HIF to RC, (3) detected RC activity in vivo in human VHL-deficient RCC cells growing as tumors in nude mice, (4) provide evidence that the reductive phenotype ofVHL-deficient cells renders them sensitive to glutamine restriction in vitro, and (5) show that inhibition of glutaminase suppresses growth of VHL-deficient cells in nude mice. These observations lay the ground for metabolism-based therapeutic strategies for targeting HIF-driven tumors (such as RCC) and possibly the hypoxic compartment of solid tumors in general.

Functional Interaction between pVHL and HIF Is Necessary to Inhibit RC

Figure 1  HIF Inactivation Is Necessary for Downregulation of Reductive Carboxylation by pVHL

We observed a concurrent regulation in glucose metabolism in the different VHL mutants. Reintroduction of wild-type or type 2C pVHL mutant, which can meditate HIF-α destruction, stimulated glucose oxidation via pyruvate dehydrogenase (PDH), as determined by the degree of 13C-labeled TCA cycle metabolites (M2 enrichment) (Figures 1D and 1E). In contrast, reintroduction of an HIF nonbinding Type 2B pVHL mutant failed to stimulate glucose oxidation, resembling the phenotype observed in VHL-deficient cells (Figures 1D and 1E). Additional evidence for the overall glucose utilization was obtained from the enrichment of M3 isotopomers using [U13-C6]glucose (Figure S1A), which shows a lower contribution of glucose-derived carbons to the TCA cycle in VHL-deficient RCC cells (via pyruvate carboxylase and/or continued TCA cycling).

To test the effect of HIF activation on the overall glutamine incorporation in the TCA cycle, we labeled an isogenic pair of VHL-deficient and VHL-reconstituted UMRC2 cells with [U-13C5]glutamine, which generates M4 fumarate, M4 malate, M4 aspartate, and M4 citrate isotopomers through glutamine oxidation. As seen in Figure S1BVHL-deficient/VHL-positive UMRC2 cells exhibit similar enrichment of M4 fumarate, M4 malate, and M4 asparate (but not citrate) showing that VHL-deficient cells upregulate reductive carboxylation without compromising oxidative metabolism from glutamine. …  Labeled carbon derived from [5-13C1]glutamine can be incorporated into fatty acids exclusively through RC, and the labeled carbon cannot be transferred to palmitate through the oxidative TCA cycle (Figure 1B, red carbons). Tracer incorporation from [5-13C1]glutamine occurs in the one carbon (C1) of acetyl-CoA, which results in labeling of palmitate at M1, M2, M3, M4, M5, M6, M7, and M8 mass isotopomers. In contrast, lipogenic acetyl-CoA molecules originating from [U-13C6]glucose are fully labeled, and the labeled palmitate is represented by M2, M4, M6, M8, M10, M12, M14, and M16 mass isotopomers.

Figure 2 HIF Inactivation Is Necessary for Downregulation of Reductive Lipogenesis by pVHL

To determine the specific contribution from glucose oxidation or glutamine reduction to lipogenic acetyl-CoA, we performed isotopomer spectral analysis (ISA) of palmitate labeling patterns. ISA indicates that wild-type pVHL or pVHL L188V mutant-reconstituted UMRC2 cells relied mainly on glucose oxidation to produce lipogenic acetyl-CoA, while UMRC2 cells reconstituted with a pVHL mutant defective in HIF inactivation (Y112N or Y98N) primarily employed RC. Upon disruption of the pVHL-HIF interaction, glutamine becomes the preferred substrate for lipogenesis, supplying 70%–80% of the lipogenic acetyl-CoA (Figure 2C). This is not a cell-line-specific phenomenon, but it applies to VHL-deficient human RCC cells in general; the same changes are observed in 786-O cells reconstituted with wild-type pVHL or mutant pVHL or infected with vector only as control (Figure S2).

HIF Is Sufficient to Induce RC (reductive carboxylation) from Glutamine in RCC Cells

As shown in Figure 3C, reintroduction of wild-type VHLinto 786-O cells suppressed RC, whereas the expression of the constitutively active HIF-2α mutant was sufficient to stimulate this reaction, restoring the M1 enrichment of TCA cycle metabolites observed in VHL-deficient 786-O cells. Expression of HIF-2α P-A also led to a concomitant decrease in glucose oxidation, corroborating the metabolic alterations observed in glutamine metabolism (Figures 3D and 3E).

Figure 3 Expression of HIF-2α Is Sufficient to Induce Reductive Carboxylation and Lipogenesis from Glutamine in RCC Cells

Expression of HIF-2α P-A in 786-O cells phenocopied the loss-of-VHL with regards to glutamine reduction for lipogenesis (Figure 3G), suggesting that HIF-2α can induce the glutamine-to-lipid pathway in RCC cells per se. Although reintroduction of wild-type VHL restored glucose oxidation in UMRC2 and UMRC3 cells (Figures S3B–S3I), HIF-2α P-A expression did not measurably affect the contribution of each substrate to the TCA cycle or lipid synthesis in these RCC cells (data not shown). UMRC2 and UMRC3 cells endogenously express both HIF-1α and HIF-2α, whereas 786-O cells exclusively express HIF-2α. There is compelling evidence suggesting, at least in RCC cells, that HIF-α isoforms have overlapping—but also distinct—functions and their roles in regulating bioenergetic processes remain an area of active investigation. Overall, HIF-1α has an antiproliferative effect, and its expression in vitro leads to rapid death of RCC cells while HIF-2α promotes tumor growth (Keith et al., 2011Raval et al., 2005).

Metabolic Flux Analysis Shows Net Reversion of the IDH Flux upon HIF Activation

To determine absolute fluxes in RCC cells, we employed 13C metabolic flux analysis (MFA) as previously described (Metallo et al., 2012). Herein, we performed MFA using a combined model of [U-13C6]glucose and [1-13C1]glutamine tracer data sets from the 786-O derived isogenic clones PRC3 (VHL−/ −)/WT8 (VHL+) cells, which show a robust metabolic regulation by reintroduction of pVHL. To this end, we first determined specific glucose/glutamine consumption and lactate/glutamate secretion rates. As expected, PRC3 exhibited increased glucose consumption and lactate production when compared to WT8 counterparts (Figure 4A). While PRC3 exhibited both higher glutamine consumption and glutamate production rates than WT8 (Figure 4A), the net carbon influx was higher in PRC3 cells (Figure 4B). Importantly, the fitted data show that the flux of citrate to α-ketoglutarate was negative in PRC3 cells (Figure 4C). This indicates that the net (forward plus reverse) flux of isocitrate dehydrogenase and aconitase (IDH + ACO) is toward citrate production. The exchange flux was also higher in PRC3 than WT8 cells, whereas the PDH flux was lower in PRC3 cells. In agreement with the tracer data, these MFA results strongly suggest that the reverse IDH + ACO fluxes surpass the forward flux in VHL-deficient cells. The estimated ATP citrate lyase (ACLY) flux was also lower in PRC3 than in WT8 cells. Furthermore, the malate dehydrogenase (MDH) flux was negative, reflecting a net conversion of oxaloacetate into malate in VHL-deficient cells (Figure 4C). This indicates an increased flux through the reductive pathway downstream of IDH, ACO, and ACLY. Additionally, some TCA cycle flux estimates downstream of α-ketoglutarate were not significantly different between PRC and WT8 (Table S1). This shows that VHL-deficient cells maintain glutamine oxidation while upregulating reductive carboxylation (Figure S1B). This finding is in agreement with the higher glutamine uptake observed in VHL-deficient cells. Table S1 shows the metabolic network and complete MFA results. …

Addition of citrate in the medium, in contrast to acetate, led to an increase in the citrate-to-α-ketoglutarate ratio (Figure 5L) and absolute citrate levels (Figure S4H) not only in VHL-deficient but alsoVHL-reconstituted cells. The ability of exogenous citrate, but not acetate, to also affect RC in VHL-reconstituted cells may be explained by compartmentalization differences or by allosteric inhibition of citrate synthase (Lehninger, 2005); that is, the ability of acetate to raise the intracellular levels of citrate may be limited in (VHL-reconstituted) cells that exhibit high endogenous levels of citrate. Whatever the mechanism, the results imply that increasing the pools of intracellular citrate has a direct biochemical effect in cells with regards to their reliance on RC. Finally, we assayed the transcript and protein levels of enzymes involved in the reductive utilization of glutamine and did not observe significant differences between VHL-deficient andVHL-reconstituted UMRC2 cells (Figures S4I and S4J), suggesting that HIF does not promote RC by direct transactivation of these enzymes. The IDH1/IDH2 equilibrium is defined as follows:

[α−ketoglutrate][NADPH][CO2]/[Isocitrate][NADP+]=K(IDH)

Figure 5 Regulation of HIF-Mediated Reductive Carboxylation by Citrate Levels

We sought to investigate whether HIF could affect the driving force of the IDH reaction by also enhancing NADPH production. We did not observe a significant alteration of the NADP+/NADPH ratio between VHL-deficient and VHL-positive cells in the cell lysate (Figure S4I). Yet, we determined the ratio of the free dinucleotides using the measured ratios of suitable oxidized (α-ketoglutarate) and reduced (isocitrate/citrate) metabolites that are linked to the NADP-dependent IDH enzymes. The determined ratios (Figure S4J) are in close agreement with the values initially reported by the Krebs lab (Veech et al., 1969) and showed that HIF-expressing UMRC2 cells exhibit a higher NADP+/NADPH ratio. Collectively, these data strongly suggest that HIF-regulated citrate levels modulate the reductive flux to maintain adequate lipogenesis.

Reductive Carboxylation from Glutamine Is Detectable In Vivo

Figure 6 Evidence for Reductive Carboxylation Activity In Vivo

Loss of VHL Renders RCC Cells Sensitive to Glutamine Deprivation

We hypothesized that VHL deficiency results in cell addiction to glutamine for proliferation. We treated the isogenic clones PRC3 (VHL-deficient cells) and WT8 (VHL-reconstituted cells) with the glutaminase inhibitor 968 (Wang et al., 2010a). VHL-deficient PRC3 cells were more sensitive to treatment with 968, compared to the VHL-reconstituted WT8 cells (Figure 7A). To confirm that this is not only a cell-line-specific phenomenon, we also cultured UMRC2 cells in the presence of 968 or diluent control and showed selective sensitivity of VHL-deficient cells (Figure 7B).

Figure 7 VHL-Deficient Cells and Tumors Are Sensitive to Glutamine Deprivation

(A–E) Cell proliferation is normalized to the corresponding cell type grown in 1 mM glutamine-containing medium. Effect of treatment with glutaminase (GLS) inhibitor 968 in PRC3/WT8 (A) and UMRC2 cells (B). Rescue of GLS inhibition with dimethyl alpha-ketoglutarate (DM-Akg; 4 mM) or acetate (4 mM) in PRC3/WT8 clonal cells (C) and polyclonal 786-O cells (D). Effect of GLS inhibitor BPTES in UMRC2 cells (E). Student’s t test compares VHL-reconstituted cells to control cells in (A), (B), and (E) and DM-Akg or acetate-rescued cells to correspondent control cells treated with 968 only in (C) and (D) (asterisk in parenthesis indicates comparison between VHL-reconstituted to control cells). Error bars represent SEM.

(F) GLS inhibitor BPTES suppresses growth of human UMRC3 RCC cells as xenografts in nu/nu mice. When the tumors reached 100mm3, injections with BPTES or vehicle control were carried out daily for 14 days (n = 12). BPTES treatment decreases tumor size and mass (see insert). Student’s t test compares control to BPTES-treated mice (F). Error bars represent SEM.

(G) Diagram showing the regulation of reductive carboxylation by HIF.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4003458/bin/nihms449661f7.jpg

In summary, our findings show that HIF is necessary and sufficient to promote RC from glutamine. By inhibiting glucose oxidation in the TCA cycle and reducing citrate levels, HIF shifts the IDH reaction toward RC to support citrate production and lipogenesis (Figure 7G). The reductive flux is active in vivo, fuels tumor growth, and can potentially be targeted pharmacologically. Understanding the significance of reductive glutamine metabolism in tumors may lead to metabolism-based therapeutic strategies.

Along with others, we reported that hypoxia and loss of VHL engage cells in reductive carboxylation (RC) from glutamine to support citrate and lipid synthesis (Filipp et al., 2012Metallo et al., 2012Wise et al., 2011). Wise et al. (2011) suggested that inactivation of HIF in VHL-deficient cells leads to reduction of RC. These observations raise the hypothesis that HIF, which is induced by hypoxia and is constitutively active inVHL-deficient cells, mediates RC. In our current work, we provide mechanistic insights that link HIF to RC. First, we demonstrate that polyclonal reconstitution of VHL in several human VHL-deficient RCC cell lines inhibits RC and restores glucose oxidation. Second, the VHL mutational analysis demonstrates that the ability of pVHL to mitigate reductive lipogenesis is mediated by HIF and is not the outcome of previously reported, HIF-independent pVHL function(s). Third, to prove our hypothesis we showed that constitutive expression of a VHL-independent HIF mutant is sufficient to phenocopy the reductive phenotype observed in VHL-deficient cells. In addition, we showed that RC is not a mere in vitro phenomenon, but it can be detected in vivo in human tumors growing as mouse xenografts. Lastly, treatment of VHL-deficient human xenografts with glutaminase inhibitors led to suppression of their growth as tumors.

7.9.10 Evaluation of HIF-1 inhibitors as anticancer agents

Semenza GL1.
Drug Discov Today. 2007 Oct; 12(19-20):853-9
http://dx.doi.org/10.1016/j.drudis.2007.08.006

Hypoxia-inducible factor 1 (HIF-1) regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, genetic instability, vascularization, metabolic reprogramming, autocrine growth factor signaling, invasion/metastasis, and treatment failure. In animal models, HIF-1 overexpression is associated with increased tumor growth, vascularization, and metastasis, whereas HIF-1 loss-of-function has the opposite effect, thus validating HIF-1 as a target. In further support of this conclusion, immunohistochemical detection of HIF-1α overexpression in biopsy sections is a prognostic factor in many cancers. A growing number of novel anticancer agents have been shown to inhibit HIF-1 through a variety of molecular mechanisms. Determining which combination of drugs to administer to any given patient remains a major obstacle to improving cancer treatment outcomes.

Aurelian Udristioiu

Aurelian

Aurelian Udristioiu

Lab Director at Emergency County Hospital Targu Jiu

Mechanisms that control T cell metabolic reprogramming are now coming to light, and many of the same oncogenes importance in cancer metabolism are also crucial to drive T cell metabolic transformations, most notably Myc, hypoxia inducible factor (HIF)1a, estrogen-related receptor (ERR) a, and the mTOR pathway.
The proto-oncogenic transcription factor, Myc, is known to promote transcription of genes for the cell cycle, as well as aerobic glycolysis and glutamine metabolism. Recently, Myc has been shown to play an essential role in inducing the expression of glycolytic and glutamine metabolism genes in the initial hours of T cell activation. In a similar fashion, the transcription factor (HIF)1a can up-regulate glycolytic genes to allow cancer cells to survive under hypoxic conditions

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