Cellular Respiration and Carcinogenesis

Cellular Respiration and Carcinogenesis

Shireesh P. Apte Rangaprasad Sarangarajan •

Editors

Cellular Respiration and Carcinogenesis

Foreword by Gregg L. Semenza

123

Editors Shireesh P. Apte Alcon Laboratories Fort Worth, TX USA [email protected]

ISBN: 978-1-934115-07-7 DOI 10.1007/978-1-59745-435-3

Rangaprasad Sarangarajan Massachusetts College of Pharmacy and Health Sciences Worcester, MA USA [email protected]

e-ISBN: 978-1-59745-435-3

Library of Congress Control Number: 2008937240 c Humana Press, a part of Springer Science+Business Media, LLC 2009  All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper springer.com

Foreword

Cancer is remarkably heterogeneous when one considers pathogenesis of the disease in affected individuals with the same diagnosis (such as “renal carcinoma”) or even when cancer in a single individual is considered over time and space. This heterogeneity is the single greatest obstacle to effective cancer therapy. As a result of disease heterogeneity, there are not too many universal truths in cancer biology. One of the most consistent findings, though, is the dramatic increase in glucose uptake by metastatic cancer cells compared with that of their normal counterparts. This is such a reliable characteristic that it is used clinically to identify occult disease by positron emission tomography after injection of [18 F]fluoro-2-deoxy-D-glucose. Increased glucose uptake reflects an increased rate of glycolysis, with conversion of glucose to lactate and decreased conversion of pyruvate to acetyl coenzyme A, the substrate for mitochondrial oxidative phosphorylation. Because of the relative inefficiency of glycolysis compared with that of oxidative phosphorylation as a means of generating ATP, flux through the glycolytic pathway must increase dramatically to maintain cellular energetics. Because it is observed in more than 95% of advanced cancers, understanding the mechanisms and consequences of this dramatic reprogramming of glucose and energy metabolism in cancer cells is an important challenge for contemporary cancer biology. The 12 chapters of this book provide the reader with state-of-the-art summaries of recent exciting progress that has been made in the field of cancer metabolism. This story starts at the beginning of the 20th century with the work of Otto Warburg, whom Hans Krebs described as a “Cell Physiologist, Biochemist, and Eccentric.” Warburg invented a device, now known as the Warburg manometer, with which he demonstrated that tumor cells consume less O2 (and produce more lactate) than do normal cells under the same ambient O2 concentrations. A century later, the struggle to understand how and why metastatic cancer cells manifest the Warburg effect is still ongoing, and 12 rounds of this heavyweight fight await the reader beyond this brief introduction. Why do cancer cells manifest altered energy metabolism? To answer this question, it is necessary to acknowledge that mitochondria, in addition to serving as the major source of ATP production, provide two other critical cellular functions. One is that they also serve as the major source of reactive oxygen species (ROS), which are generated when electrons donated to the respiratory chain are not successfully v

vi

Foreword

transferred from complex IV (cytochrome c oxidase) to O2 to form water but instead react with O2 prematurely to form superoxide anion, which is then converted to hydrogen peroxide through the action of mitochondrial superoxide dismutase. ROS are used by normal cells as signaling molecules (e.g., during cell proliferation). However, if generated in excess (amplitude or duration or both), ROS result in the oxidation of lipids, proteins, and nucleic acids leading to cell dysfunction or death, which introduces the other major role of mitochondria as arbiters of cell survival, as apoptosis is triggered through changes in mitochondrial structure and function. Thus, it appears that reduced mitochondrial respiration, which often is accompanied by reduced mitochondrial mass, occurs in cancer cells as a result of selection against excess ROS production and/or apoptosis. How do cancer cells reprogram energy metabolism? To answer this question (and to more completely answer “why?” beyond the pat generalization provided above), it is necessary to return full circle to the issue of heterogeneity. Because mitochondria play critical roles in energy production, redox balance, proliferation, and survival, many mutations that are selected for in advanced cancers impact mitochondrial function in one or more ways, for although mitochondria control cell fate, the cell controls mitochondria by virtue of the fact that most of the components of mitochondria are the products of nuclear, not mitochondrial, genes. A danger of a reductionist approach to cancer biology that focuses on changes involving key oncogenes and tumor suppressor genes is the misconception that carcinogenesis can be attributed to one or a few mutations in any given cell. As in every other aspect of cancer biology, it is the net effect of all of the mutations in nuclear and mitochondrial DNA of the cancer cell that will determine its metabolic phenotype. Another danger of a solely mutation-based approach to cancer is that it ignores the effect of the tumor microenvironment. Like normal cells, cancer cells react to changes in the concentrations of O2 , glucose, hydrogen ions, and free radicals. In fact, these changes represent the most important selective force acting on the mutant gene pool of cancer cells. As a result, not all changes in cancer metabolism are hard-wired by genetic alteration but may instead represent adaptive responses to the microenvironment. In other words, the reprogramming of energy metabolism is unique to each cancer cell. The complex considerations that are covered so superficially in this brief introduction are discussed in detail in the 12 chapters that follow. There is tremendous interest (and hope) that because altered glucose and energy metabolism appears to be a virtually universal characteristic of metastatic cancer cells (i.e., the cells that kill cancer patients), understanding the mechanisms and consequences of metabolic reprogramming may lead to novel therapeutic approaches that will more effectively fight this formidable foe. Ring the bell for Round 1 . . . Baltimore, Maryland

Gregg L. Semenza, M.D., Ph.D.

Contents

Oxidative Phosphorylation and Cancer: The Ongoing Warburg Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael Ristow and Jos´e M. Cuezva

1

The Electron Transport Chain and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . 19 Jean-Jacques Bri`ere, Paule B´enit, and Pierre Rustin Respiratory Control of Redox Signaling and Cancer . . . . . . . . . . . . . . . . . . . 33 Pauline M. Carrico, Nadine Hempel, and J. Andr´es Melendez Cellular Respiration and Dedifferentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Roberto Scatena, Patrizia Bottoni, and Bruno Giardina Cellular Adaptations to Oxidative Phosphorylation Defects in Cancer . . . . 55 Sarika Srivastava and Carlos T. Moraes Regulation of Glucose and Energy Metabolism in Cancer Cells by Hypoxia Inducible Factor 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 ´ T´ulio C´esar Ferreira and Elida Geralda Campos The Role of Glycolysis in Cellular Immortalization . . . . . . . . . . . . . . . . . . . . . 91 Hiroshi Kondoh Metabolic Modulation of Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Shireesh P. Apte and Rangaprasad Sarangarajan Mitochondrial DNA Mutations in Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Anna Czarnecka and Ewa Bartnik Cellular Respiration and Tumor Suppressor Genes . . . . . . . . . . . . . . . . . . . . . 131 Luis F. Gonzalez-Cuyar, Fabio Tavora, Iusta Caminha, George Perry, Mark A. Smith, and Rudy J. Castellani

vii

viii

Contents

Uncoupling Cellular Respiration: A Link to Cancer Cell Metabolism and Immune Privilege . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 M. Karen Newell, Elizabeth M. Villalobos-Menuey, Marilyn Burnett, and Robert E. Camley How Cancer Cells Escape Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Erica Werner Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

Contributors

Shireesh P. Apte Senior Scientist, Process Research and Support, Mail stop: R4-11 Alcon Laboratories, 6201 South Freeway, Fort Worth, Texas 76134, USA Ewa Bartnik Department of Genetics and Biotechnology, University of Warsaw; and Institute of Biochemistry and Biophysics, Polish Academy of Sciences Warsaw, Pawinskiogo 5a. 02106, Poland Paule B´enit Hˆopital Robert Debr´e, INSERM U676, Paris, France Patrizia Bottoni Dipartimento di Medicina di Laboratorio, Universit`a Cattolica, Rome, Italy Jean-Jacques Bri`ere Hˆopital Robert Debr´e, INSERM U676, Paris, France Marilyn Burnett CU Institute of Bioenergetics and Immunology, University of Colorado at Colorado Springs, Colorado Springs, Colorado Iusta Caminha Department of Pharmacology, University of Maryland, Baltimore, Maryland Robert E. Camley CU Institute of Bioenergetics and Immunology, University of Colorado at Colorado Springs, Colorado Springs, Colorado ´ Elida Geralda Campos Departamento de Biologia Celular, Universidade de Bras´ılia, Bras´ılia, DF, Brazil Pauline M. Carrico Ordway Research Institute, Albany, New York Rudy J. Castellani Department of Pathology, University of Maryland, Baltimore, Maryland ix

x

Contributors

Jos´e M. Cuezva Departamento de Biolog´ıa Molecular, Centro de Biolog´ıa Molecular “Severo Ochoa,” Centro de Investigaci´on Biom´edica en Red de Enfermedades Raras, Universidad, Aut´onoma de Madrid, Madrid, Spain Anna Czarnecka Department of Genetics and Biotechnology, University of Warsaw; Warsaw, Poland; and Postgraduate School of Molecular Medicine, Warsaw, Poland T´ulio C´esar Ferreira Departamento de Biologia Celular, Universidade de Bras´elia, Bras´elia, DF, Brazil Bruno Giardina Dipartimento di Medicina di Laboratorio, Universit`a Cattolica, Rome, Italy Luis F. Gonzalez-Cuyar Department of Pathology, University of Maryland, Baltimore, Maryland Nadine Hempel The Center for Immunology & Microbial Disease, Albany Medical College, Albany, New York Hiroshi Kondoh Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan J. Andr´es Melendez The Center for Immunology & Microbial Disease, Albany Medical College, Albany, New York Carlos T. Moraes Department of Neurology and Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, Florida M. Karen Newell CU Institute of Bioenergetics and Immunology, University of Colorado at Colorado Springs, Colorado Springs, Colorado George Perry College of Sciences, University of Texas at San Antonio, San Antonio, Texas; and Department of Pathology, Case Western Reserve University, Cleveland, Ohio Michael Ristow Department of Human Nutrition, Institute of Nutrition, University of Jena, Jena, Germany Pierre Rustin Hˆopital Robert Debr´e, Paris, France

Contributors

xi

Rangaprasad Sarangarajan Associate Professor of Pharmacology & Toxicology, School of Pharmacy (Worcester) Massachusetts College of Pharmacy and Health Sciences, 19 Foster Street, Worcester, MA 01608, Gregg L. Semenza Director, Vascular Program, Institute for Cell Engineering; Professor, Departments of Pediatrics, Medicine, Oncology, Radiation Oncology, and the McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Broadway Research Building, Suite 671, 733 North Broadway, Baltimore, MD 21205 USA Roberto Scatena Dipartimento di Medicina di Laboratorio, Universit`a Cattolica del Sacro Cuore, Rome, Italy Mark A. Smith Department of Pathology, Case Western Reserve University, Cleveland, Ohio Sarika Srivastava Department of Neurology, University of Miami Miller School of Medicine, Miami, Florida Fabio Tavora Department of Pathology, University of Maryland, Baltimore, Maryland Elizabeth M. Villalobos-Menuey CU Institute of Bioenergetics and Immunology, University of Colorado at Colorado Springs, Colorado Springs, Colorado Erica Werner Department of Cell Biology, School of Medicine, Emory University, Atlanta, Georgia

Oxidative Phosphorylation and Cancer: The Ongoing Warburg Hypothesis Michael Ristow and Jos´e M. Cuezva

Abstract More than eight decades ago, the German physiologist Otto Warburg observed that cancer cells in the presence of oxygen produced large amounts of lactate and proposed that impaired oxidative metabolism may cause cancer. This formulation, later known as the Warburg hypothesis, was investigated and debated for several decades. The development of molecular biology and the discovery of oncogenes and tumor suppressor genes in subsequent years shifted the general interest in the cancer field into directions other than metabolism and promoted the abandoning of the Warburg hypothesis or its consideration as an epiphenomenon of cell transformation. In recent years, a renaissance in the field of mitochondria has occurred in biological studies. This has happened mostly by the recognition of the key functional role that this organelle plays in the execution of cell death and, thus, for its participation in the development of a vast array of human pathologies. The cancer field was not indifferent to these changes, and the Warburg hypothesis was brought back to the scene with renewed strength. Specifically, recent findings suggest that cancer is associated with a decrease in the activity and expression of ␤-F1-ATPase, a key subunit of the mitochondrial ATP synthase. This alteration has been shown to limit oxidative phosphorylation and to trigger the induction of glycolysis to provide energy to the cell thus configuring the earlier Warburg observation in an additional hallmark of the cancer cell. Moreover, increased and decreased cellular mitochondrial activities are respectively associated with suppression and development of cancer. We suggest that reactivating mitochondrial metabolism by pharmacologic or dietary measures and/or tackling the deviant glycolysis in cancer cells may efficiently suppress malignant growth.

M. Ristow (B) Chair, Department of Human Nutrition, Institute of Nutrition, University of Jena, D-07743 Jena, Germany e-mail: [email protected] J.M. Cuezva (B) Departamento de Biolog´ıa Molecular, Centro de Biolog´ıa Molecular “Severo Ochoa”, CIBER de Entermedades Rasas, Universidad Aut´onoma de Madrid, 28049 Madrid, Spain. e-mail: [email protected] S.P. Apte, R. Sarangarajan (eds.), Cellular Respiration and Carcinogenesis, DOI 10.1007/978-1-59745-435-3 1,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

1

2

M. Ristow, J.M. Cuezva

Keywords Mitochondria · Oxidative phosphorylation · H+ -ATP synthase · Glycolysis · Cancer Nowadays, malignant disorders are a major cause of premature death. The incidence of cancers increases with age, whereas metabolic activity and resting energy expenditure resembling oxidative metabolism continuously decrease during aging of humans. Hence, an inverse relationship between mitochondrial activity and the promotion of tumor growth might possibly exist. To analyze whether this apparent link is supported by scientific evidence rather than simply being a coincidence, tremendous efforts have been undertaken in the past to elucidate this possible connection. This chapter aims to summarize published evidence on mechanistic interdependencies between cancer biology and the activity of mitochondria. Other recent reviews dealing with this subject are also available [1, 2]. In the past decades, glucose has become a major component of Western diets [3]. The most frequently used nutritive sugar, the disaccharide sucrose, contains equal amounts of the monosaccharides fructose and glucose. Glucose is actively transported into mammalian cells, and subsequently is (as well as fructose) biochemically converted into pyruvate (Fig. 1). Pyruvate either is converted into lactate, which prevents its further intracellular breakdown, or is completely oxidized to carbon dioxide and water by mitochondrial activities linked to the supply of molecular oxygen (Fig. 1). The breakdown of glucose into pyruvate is termed glycolysis. Glycolysis takes place in the cytoplasm of eukaryotic cells, and it can proceed both in the absence and presence of oxygen (Fig. 1). In order to carry on glycolysis in the former case, the carbon skeleton of pyruvate needs to be reduced to lactate to regenerate the pool of oxidized NAD+ (Fig. 1). Many prokaryotes and lower eukaryotes including Saccharomyces cerevisiae (yeast) survive in the absence of oxygen by obtaining energy from anaerobic glycolysis only [4], whereas higher eukaryotes, especially mammals, require a continuous supply of oxygen for obtaining biological energy. Glycolysis in absence or presence of oxygen provides comparably little energy equivalents per mole of glucose oxidized when compared with its terminal oxidation in the mitochondria (Fig. 1). Nevertheless, anaerobic glycolysis supplies the precursors and building blocks for the biosynthesis of cellular macromolecules, and, in yeast, as well as in certain mammalian cell types, is sufficient to maintain viability for longer periods of time. Because highly developed organisms tend to use nutritive energy equivalents more efficiently than do less developed organisms [5], mammals have significantly more efficient ways to generate energy equivalents. This is illustrated by the further oxidation of a metabolite of glycolysis (pyruvate) in mitochondria, which yields an approximately 18-fold amount of energy equivalents per mole of glucose oxidized when compared with anaerobic glycolysis (Fig. 1). Hence, the major advantage of glycolytic metabolism is to be independent from oxygen, and the major disadvantage of this process is a comparably low yield of energy equivalents (Fig. 1). For many decades, mitochondrial organelles were considered eminently important for the generation of biological energy and as the cellular site that allowed

Oxidative Phosphorylation and Cancer: The Ongoing Warburg Hypothesis HK Glucose

PFK1

PGI

G-6-P

F-6-P

3

ALD

F-1,6-P

TPI

G-3-P

G-3-P + DHAP

GAPDH ENO

PEP

PGM

2-PG

PGK

3-PG

1,3-BPG

PK

02

Pyruvate

NAD+

LDH

Lactate

Net ATP produced: 2 Glucose utilization: HIGH

AEROBIOSIS

AEROBIOSIS / ANAEROBIOSIS

NADH

pyruvate

CO2 + H2O Net ATP produced: 36 Glucose utilization: LOW

Fig. 1 Cellular ATP provision defines the rate of aerobic glycolysis. Abbreviations of glycolytic intermediates and enzymes of glycolysis are presented sequentially up to the pyruvate crossroad. The key glycolytic enzymes hexokinase (HK), phosphofructokinase 1 (PFK1), and pyruvate kinase (PK) are highlighted in bold. The reduction of pyruvate to lactate by lactate dehydrogenase (LDH) regenerates NAD+ and allows glycolysis to proceed with very low yield of energy and a high consumption of glucose. Pyruvate could be oxidized to CO2 and water only in the presence of oxygen by mitochondria. In this situation, the yield of energy obtained is high and consequently glucose consumption is low

the integration of the metabolism of macronutrients, specifically carbohydrates, fatty acids, and amino acids. Interconversion of carbohydrate derivatives (i.e., pyruvate) into fatty acids, amino acids into carbohydrate precursors, and others occur in the matrix of mitochondria, and these activities are integrated in the Krebs cycle. Concurrently, the reducing equivalents obtained from the terminal oxidation of the metabolic intermediates of the Krebs cycle in the form of NADH and FADH2 are shuttled into the complexes of the respiratory chain (Fig. 2). The respiratory chain is composed of proteins that are located in the inner mitochondrial membrane and have, as overall function, the reoxidation of NADH and FADH2 to further allow the oxidation of more metabolic substrates (Fig. 2). The ordered transfer of electrons along the carriers of the respiratory chain to molecular oxygen (Fig. 2), which is the final electron acceptor, is the process known as respiration. Electron transfer in complexes of the respiratory chain trigger the proton pumping activity of complexes CI, CIII, and CIV (Fig. 2), which extrudes protons from the matrix interior of mitochondria and finally results in the generation of the proton electrochemical gradient (⌬␮H+ ) across the inner membrane of the organelle, which is the intermediate used for the synthesis of ATP [6] (Fig. 2). The reentry of protons to the matrix interior through the proton channel of the H+ -ATP synthase (CV in Fig. 2) drives the

4

M. Ristow, J.M. Cuezva

O2·

CYTOPLASM O.M.

H+

H+

Cyt c – CI e

Q

NAD+ FADH2

CIV

e–

I.M.

CIII

– CII e

NADH

ΔμH+

H+

e– FAD

O2 H2O

CV

ATP H+ MATRIX

O2· Fig. 2 Mitochondrial activities: respiration, oxidative phosphorylation, and the production of ROS. The oxidation of metabolic substrates promotes the reduction of redox coenzymes (NAD+ and FAD) that feed the electrons (e-) to the complexes of the respiratory chain (CI to CIV) placed in the inner mitochondrial membrane (I.M.). Electron flow down the respiratory chain to molecular oxygen generates the water of respiration. Electron transfer is coupled with proton pumping from the matrix interior to the intermembrane space at CI, CIII, and CIV to generate the proton electrochemical gradient (⌬␮H+ ) across the inner membrane. ⌬␮H+ is the driving force used by the H+ -ATP synthase (CV) for the synthesis of ATP. However, at complexes I and III, some of the electrons can combine with molecular oxygen to generate the toxic superoxide radical (dashed lines). Q, ubiquinone; Cyt c, cytochrome c; O.M., outer membrane

biosynthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi) in the process known as oxidative phosphorylation (OXPHOS) (Fig. 2). Whereas the metabolism of nutrient intermediates and OXPHOS were thought to be the primary roles of mitochondria for many decades, recent findings indicate that mitochondria play additional essential cellular functions. The protist Giardia lamblia carries remnant organelles resembling mitochondria that, though they are unable to provide ATP, instead synthesize ironsulfur (Fe-S) clusters [7]. Fe-S clusters are essential parts of specific mitochondrial enzymes, namely aconitase in the Krebs cycle as well as complexes of the respiratory chain [8]. Briefly, Fe-S clusters are known to carry non-heme iron, which can both donate and accept electrons [9]. Moreover, mitochondria are central regulators and executors of cell death [10], and the acquisition of a resistant phenotype to apoptosis is a critical aspect in the biology of tumors and its therapy [11, 12]. It is now firmly established that upon the activation of cell death by various types of stimuli (genetic damage, ischemia, chemical and/or metabolic stresses), mitochondria release proteins (cyt c, AIF, Endo G, Smac/DIABLO, Omi/HtrA2) and/or other molecules such as the superoxide anion (O2 . ) that participate in signaling and

Oxidative Phosphorylation and Cancer: The Ongoing Warburg Hypothesis

5

executing cell demise. It should be noted that in most cells, reactive oxygen species (ROS) are produced by single electron transfer reactions to molecular oxygen during mitochondrial respiration (Fig. 2). In summary, mitochondria are responsible for the production of Fe-S clusters, the conversion of primary energy substrates into each other, the generation of large amounts of ATP, and the signaling and execution of cell death. The major advantage of oxidative metabolism is the comparably high energy yield, and the major disadvantage is its dependency on the availability of oxygen and the subsequent production of ROS that could damage essential cellular constituents. Unaware of the above-mentioned facts, the physiologist Otto Warburg (Fig. 3 and Fig. 4) proposed as early as 1924 that malignant growth might be caused by increased glycolysis accompanied by impaired oxygen consumption [13–16]. This hypothesis was based on his original observations that cancer tissues show a low respiratory rate compared with that of adjacent healthy (i.e., nonmalignant) tissues. After becoming the Nobel laureate for medicine and physiology in 1931 [17], his hypothesis on initiation of cancer obtained increasing attention and caused widespread debate [18–22]. Warburg died in 1970 while his hypothesis was still

Fig. 3 Otto Warburg (date unknown). (Photograph courtesy of the Archives of the Max Planck Society, Berlin, Germany.)

6

M. Ristow, J.M. Cuezva

Fig. 4 Otto Warburg in his laboratory (date unknown). (Photograph courtesy of the Archives of the Max Planck Society, Berlin, Germany.)

unconfirmed, denied, and/or neglected [23, 24] mostly because of the abandonment of metabolic studies in favor of the development of molecular biology and the discovery of oncogenes, tumor suppressor genes, and other advances in cancer research. In any case, and after both OXPHOS and respiration had been attributed to mitochondria, electron microscopy–based techniques were developed to study the morphology of these organelles in tumor tissues. In summary, mitochondria from rapidly growing cancers show less cristae and are smaller than those from betterdifferentiated cancers and normal tissues [25, 26]. Besides these structural differences in the mitochondria of the cancer cell, it has been further emphasized that some cancer tissues also have a significant reduction in the number of mitochondria per cell [25–27]. The abnormal high-aerobic glycolysis of tumors discovered by Warburg was confirmed by most researchers studying cancer cells, and a significant number of potential mechanisms have been put forward to explain the metabolic changes specific to cancer cells. These can be separated into mechanisms that impinge on cellular glucose uptake and consumption by glycolysis (Fig. 1) and those affecting the mitochondrial metabolism of pyruvate (Fig. 1 and Fig. 2). When comparing energy metabolism of tumors and healthy tissue, profound changes have been observed in virtually all cases of cancer or tissue types. Increased glycolysis has been found in cancer cells at the genomic [28], proteomic [26, 29, 30–32], and metabolic levels:

Oxidative Phosphorylation and Cancer: The Ongoing Warburg Hypothesis

7

increased glucose uptake [33, 34], as well as increased production of lactate as the end product of glycolysis [35, 36]. A modern noninvasive imaging technique called positron emission tomography (PET) is nowadays in clinical use for diagnosing and staging cancer patients [37] because it is remarkably sensitive to detect tumor tissue and its metastasis in situ based on the principles of the Warburg hypothesis [32]. By injecting the nonmetabolizable glucose analogue [18 F]fluoro-2-deoxy-d-glucose (FDG) as a tracer substance into the human body, tissues with high FDG uptake (high glucose avidity) can easily be identified from the surrounding normal tissue in images of PET scans [38]. Using this approach, we have recently showed that lung tumors with high glucose avidity have a profound alteration of the bioenergetic proteome [32]. The bioenergetic proteome (BEC index) provides a signature of the tumor that informs of the preponderance of the metabolic pathways relevant for cellular energy provision at the protein level [2, 26]. Remarkably, the analysis of the bioenergetic signature in tumor specimens and in cells in culture [32] revealed that the downregulation of the bioenergetic function of mitochondria (Fig. 1 and Fig. 2), specifically of oxidative phosphorylation, underlies the high rates of glucose uptake and consumption by aerobic glycolysis in the carcinomas (Fig. 1) [32]. These results have provided the first indications that integrate molecular and functional data to confirm Warburg’s initial observations [32]. In a recent review, Gatenby and Gillies put forward an evolutionary model to explain the apparently high glycolytic rates of cancer cells [39]. Under conditions of extensive hypoxia, which most likely occurs during hyperplastic growth, evolutionary selection favors cells with elevated glycolytic capacity [40]. Moreover, increased glycolysis causes increased lactate production and subsequent lactate-dependent acidification. Selection is in favor of those cells that exhibit increased resistance toward an unfavorable acidic environment. Therefore, a premalignant cell will be less dependent on oxygen and is still able to expand under conditions that would kill a nonmalignant cell. In support of Warburg’s hypothesis, this simple model provides an explanation on the benefit of cancer cells from an energetic trade-off (i.e., preference for nonmitochondrial energy supply). Numerous experimental findings further emphasize this assumption. Induction of glucose-specific transporters (GLUTs), and, most importantly GLUT1, was several times correlated with tumor aggressiveness [41–43]. Moreover, additional evidence links a number of established cancer genes to control of glycolytic flux. In this regard, it has been shown that the c-myc oncogene induces expression of GLUT1 and a number of additional genes involved in glycolytic flux, including glucose-6phosphate isomerase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, lactate dehydrogenase, and others (Fig. 1) [44]. Of interest, c-Myc also acts to transactivate the lactate dehydrogenase A (LDHA) gene which is necessary for c-myc–mediated transformation in fibroblasts [35]. Accordingly, expression of other oncogenes (ras, src, PDGF) causes increased glucose uptake in malignant cells [34, 35, 45]. Additionally, and acting as c-myc itself, such cells were capable of coactivating LDHA expression via proposed binding to a carbohydrate responsive element found in the LDHA promoter region [46].

8

M. Ristow, J.M. Cuezva

Another relevant mechanism to regulate glycolysis and metabolic adaptation to oxygen depletion is that mediated by the hypoxia inducible factor 1 alpha (HIF-1␣). Subsequent to its identification in 1992 [47], a significant body of evidence has emerged proposing that HIF-1␣ might be relevant for many of the effects as suggested in the initial Warburg hypothesis [48]. HIF-1␣ forms dimers with its partner HIF-1␤ to become an active transcription factor binding to promoter elements of several genes including those encoding for proteins of glucose transport [49] and activating the expression of several enzymes of the glycolytic pathway (Fig. 1) including LDHA [50, 51]. Furthermore, the active HIF-dimer promotes angiogenesis and erythropoiesis [47, 52]. HIF-1␣ is constitutively expressed in most tissues and degraded under conditions of normal oxygen pressure. In contrast, when oxygen partial pressure is decreased, degradation of HIF-1␣ is blocked causing increased transcriptional activity to regulate the respective target genes following changes in oxygen availability [53]. Accordingly, deregulated HIF-1␣ or sustained stability under conditions of normal oxygen supply has been linked to numerous types of cancer in past years [54]. Furthermore, the ubiquitin ligase responsible for HIF-1␣ inactivation, the von Hippel–Lindau tumor suppressor (VHL) protein, has been affiliated with the von Hippel–Lindau syndrome, which promotes neuronal and kidney cancers [55]. Moreover, downregulation of pyruvate dehydrogenase might lead to accumulation of pyruvate and increased lactate production, which contributes to stabilization of HIF-1␣ [56]. Hence, although hypoxia might play a key role in tumor development, it seems not to be unambiguously required for sustained increases in glycolysis. Putatively, this may promote a vicious cycle of enhanced glycolysis leading to increased accumulation of pyruvate, which subsequently may stimulate HIF-1␣ and lastly further increase the expression of glycolytic enzymes [57]. Another candidate pathway controlling the glycolysis-dependent phenotype of malignancies, the Akt/PBK (protein kinase B) pathway, has been proposed. Akt/PKB has long been known for its capability of inducing glucose incorporation and glycolytic flux independently of malignancy-promoting capacity [58]. In malignant cells, elevated glycolytic flux was linked to activation of Akt/PKB [59]. Accordingly, blastoma cells lacking constitutive activation of Akt/PKB did not display altered glycolytic flux, whereas expression of Akt/PBK promoted cell death under conditions of glucose deprivation. Moreover, it has recently been shown that the overexpression of Akt/PBK in noninvasive radial-growth melanoma cells triggers an increase in protein markers of glycolysis and in the glycolytic flux of the melanoma concurrently with its transformation to the aggressive vertical-growth invasive behavior [60]. Altogether, these results suggest the possibility that deregulated Akt/PKB signaling might contribute to the Warburg effect in cancer cells. Additionally, interactions of glycolytic enzymes with the well-characterized p53 tumor suppressor protein have been described, further promoting the relevance of cancer-related genes in causing a potential metabolic shift toward increased glycolytic rate. In this regard, a putative role for hexokinase has been proposed [61]. In hepatoma cells, hexokinase was closely associated with the mitochondria, which was not observed in normal (i.e., untransformed) liver cells [62]. A subsequent study revealed that mutations in the p53 tumor suppressor protein might activate

Oxidative Phosphorylation and Cancer: The Ongoing Warburg Hypothesis

9

hexokinase II expression via binding to specific sites of the corresponding hexokinase promoter [63, 64]. In further support of these interesting observations, a protein named TP53-induced glycolysis and apoptosis regulator (TIGAR ), an isoform of phosphofructokinase that can be induced by p53, was identified in recent years [65]. TIGAR acts as an inhibitor of glycolysis by lowering levels of fructose-2,6bisphosphate. Accordingly, in cases of p53 mutations as found in many cancers, activity of TIGAR is downregulated promoting an increase in glycolysis. In summary, a number of cancer-related genes have been shown to promote alterations on the glycolytic phenotype that could explain that particular aspect of the Warburg effect. Aspects other than deregulated glycolysis underscore that oxidative metabolism itself is also impaired in malignant cells, thereby representing another key aspect of Warburg’s hypothesis. The phenomenon of impaired mitochondrial activity [13, 15, 25] appears to be a key feature of numerous cancer specimens [26, 29, 31, 32, 66, 67]. Accordingly, mutations of mitochondrial DNA have repeatedly been associated with induction of tumor growth on a hypothetical basis [67, 68] and later on a factual basis [69–73]. Complexes of the mitochondrial respiratory chain (Fig. 2) including cytochrome c oxidase (COX) exhibit diminished activity in cancers, mainly due to decreased expression of the subunits encoded by the mitochondrial DNA [74]. Likewise, mutations in nuclear genes that affect the bioenergetic function of mitochondria, and thus compromise the provision of metabolic energy, have also been shown to predispose to different types of inherited neoplasia syndromes [75–77]. Moreover, over the past years it has been shown that most prevalent human tumors fit the original Warburg hypothesis because they show a decrease in the expression of ␤-F1-ATPase, which is the catalytic subunit of the mitochondrial H+ ATP synthase and thus a bottleneck of oxidative phosphorylation (CV in Fig. 2), concurrently with an increased expression of several makers of the glycolytic pathway (26, 29, 31 and references therein). This proteomic feature, which was defined as “the bioenergetic signature” of cancer [26], illustrates both alterations in the bioenergetic proteome of the mitochondrion and on the overall mitochondrial activity of the cancer cell [2] and provides markers of the two mechanisms that could interfere with mitochondrial activity in the various types of cancer cells [2]. This is especially relevant because depending upon the tissue type considered, a general reduction in mitochondrial mass or a specific reduction on the expression of the ATP synthase is associated with progression of the neoplasia [2, 26]. Interestingly, these recent findings perfectly complement earlier findings on reduced ATP(synth)ase activity in rodent models of liver carcinogenesis [78]. Additionally, Racker and coworkers also emphasized the putative relevance of ATP(synth)ase in the control of tumor proliferation [79, 80]. As indicated previously, we have recently documented both in vivo using FDGPET imaging in lung cancer patients and in vitro that the rates of glucose capture by lung tumors and of glucose utilization by aerobic glycolysis in cancer cells depend on the expression level of the ␤-F1-ATPase protein and on the activity of oxidative phosphorylation [32]. These results provide strong support to the

10

M. Ristow, J.M. Cuezva

original Warburg hypothesis and to the principle of metabolic regulation known as the Pasteur effect [81]. This principle of metabolism, which basically states that “the metabolic flux of glycolysis in eukaryotic cells depends on the energy provided in the form of ATP by mitochondrial activity,” is what was used by Warburg as the “first stone” for the building of his seminal and outstanding hypothesis in the early dates of biochemistry [2]. Remarkably, the bioenergetic signature has been shown to provide a relevant marker of disease progression in colon [26], lung [30, 32], and breast [31] cancer patients. Furthermore, it has been documented that the bioenergetic signature also provides a marker of the cellular response to chemotherapy in colon [82] and liver [83] cancer cells that might allow its further use as a predictive marker of therapeutic intervention and/or as a target of future cancer treatments. Because it appears that alterations in mitochondrial energetic physiology contribute to the Warburg effect, that is, an impaired oxidative phosphorylation in mitochondria promotes the upregulation of glycolysis (Fig. 1), at least two questions arise: (i) what are the mechanism(s) that impair the OXPHOS activity of mitochondria, and (ii) what is the advantage and mechanistic contribution of the downregulation of OXPHOS for the cancer cell? These will be partially addressed in the following. Supported by previous findings on p53 as mentioned before, it has been shown that this tumor suppressor protein may directly be involved in regulating mitochondrial oxygen consumption through regulation of COX activity [84]. Accordingly, cancer cell lines lacking p53 showed decreased rates of OXPHOS as reflected by impaired ATP synthesis and decreased respiratory rate, and maintenance of cellular ATP levels was dependent on a concurrent increase in glycolysis [85, 86]. This specific regulation was promoted by Synthesis of Cytochrome c Oxidase 2 SCO2, which has previously been shown to be involved in the assembly of complex IV of the respiratory chain [86]. Nevertheless, it remains to be shown whether this particular activation can be directly linked to mitochondrial activity. Therefore, this tumor suppressor, p53, not only regulates glycolysis as shown above but possibly also directly affects mitochondrial oxygen consumption. Not only p53 has been shown to influence both glycolysis and mitochondrial activity. Similar conjunct activities have been put forward for both HIF-1␣ and Akt/PKB signaling pathways. In this regard, HIF-1␣ has previously been demonstrated to activate pyruvate dehydrogenase kinase (PDK), which is known to inactivate pyruvate dehydrogenase, preventing the decarboxylation of pyruvate and hence its further oxidation in the mitochondria having a negative impact on mitochondrial oxygen consumption [87, 88]. Likewise, the above-mentioned tumor suppressor protein VHL not only ubiquitinates HIF-1␣ but also promotes mitochondrial biogenesis and activity. Accordingly, loss of VHL, as repeatedly described in various types of cancer, might possibly impair mitochondrial activity strongly promoting the Warburg effect [66]. Moreover, reduced OXPHOS capacity has previously been suggested to promote phosphorylation and hence activation of Akt [89], a well-defined oncogene, in a Phosphatase and Tensin Homolog PTEN-dependent manner [90]. An additional alternative to explain the “Warburg phenotype” of the cancer cell is the one we have recently put forward [32] after the characterization of the biogenesis and functional development of mitochondria during the cell cycle [91]. We have

Oxidative Phosphorylation and Cancer: The Ongoing Warburg Hypothesis

11

suggested that the downregulation of oxidative phosphorylation and the concurrent upregulation of glycolysis is the phenotype that identifies the energetic metabolism of most proliferating cells. In other words, glycolysis is the metabolic pathway that supports proliferation and tumor growth. In this regard, it is well established that lymphocytes switch to glycolysis as the energy-producing pathway during proliferation [92], and progression through the S/G2/M phases of the cell cycle, the socalled reductive phases of the “metabolic cycle” in which most of the mitochondrial components are being synthesized [91], is supported by nonrespiratory modes of energy generation [93]. In agreement with these findings, it has recently been shown that cyclin D1, which is involved in the phosphorylation and inactivation of the retinoblastoma protein marking in this way the entry of cells into the S phase, represses mitochondrial function in vivo [94, 95]. We presume that the effects that oncogenes and tumor suppressors could have on mitochondrial activity might be superimposed to the natural metabolic phenotype of proliferating cells. Mechanistically, and by analogy with findings on the biogenesis of mitochondria in rat hepatomas [27], in fetal rat liver [96–98] and during the cell cycle [91], the repression of ␤-F1-ATPase expression in human tumors [26, 29–31] could be exerted by translation masking (silencing) of the ␤-F1-ATPase mRNA, a condition that might also affect other OXPHOS mRNAs of the cancer cell [2]. According to Warburg’s hypothesis, impaired mitochondrial function should be considered a key property of cancer cells (i.e., an additional hallmark of cancer to be added to the list) [99]. Strongly supporting this hypothesis, it was shown that induction of mitochondrial energy conversion by forced expression of the mitochondrial protein frataxin [100] leads to reduced growth of cancer cells [101]. Frataxin has been shown to be involved in Fe-S cluster biogenesis [102] and induces OXPHOS [100]. In this regard, and as a novel pathway connecting induction of mitochondrial metabolism and decreased growth rates, mitogen activated protein kinase p38 has been proposed [101, 103]. This protein p38 has repeatedly been observed to act as an efficient tumor suppressor protein [104, 105]. In full accordance with these observations, hepatocyte-specific inactivation of murine frataxin reduces the activity of Fe-S cluster–containing enzymes within the mitochondria, promoting hepatic adenoma formation in such mice [103]. Consistently, defective Krebs cycle activity has been observed in colorectal cancer samples including reduced activity of the Fe-S cluster–containing enzyme aconitase [106]. Taken together, these findings indicate that activation of mitochondrial energy metabolism has a tumor suppressor effect that opposes the Warburg phenotype and could be a therapeutic tool in cancer therapy. Indeed, the execution of cell death requires an efficient oxidative phosphorylation [107–110], the molecular components of the H+ -ATP synthase being specifically required [111–113]. In this regard, we have recently shown that the activity of the H+ -ATP synthase is necessary for the efficient execution of apoptosis in cells that depend on oxidative phosphorylation for the provision of metabolic energy, whereas it is dispensable in those cells that rely heavily on glycolysis [83]. The role of the H+ -ATP synthase in the execution of cell death is mediated by controlling the generation of ROS [83], which in turn promote a severe oxidative damage on mitochondrial proteins, favoring in this way the release of apoptogenic molecules from the organelle [83]. Consistently, highly glycolytic cells

12

M. Ristow, J.M. Cuezva

with an arrested mitochondrial biogenesis and thus with no dependence on oxidative phosphorylation for energy provision do not produce ROS and are resistant to mitochondria-geared cell death stimuli [83]. Altogether, these results provide additional evidence linking metabolism to cell death [114–117] and support strongly that the acquisition of a Warburg phenotype is another strategy of the cancer cell in order to ensure its perpetuation. In recent years, AMP activated protein kinase (AMPK) has been established as a key regulator of cellular energy balance controlling a significant number of pathways that control cellular ATP content [118]. AMPK is activated under conditions of low energy supply, induced by stressors like reduced nutrient availability or hypoxia [118]. Activation of AMPK is initiated by the upstream tumor suppressor kinase, LKB1, which phosphorylates AMPK [119]. Of note, a mutation of LKB1 has been shown to cause Peutz-Jeghers syndrome, a disease that has been linked to increased incidence of cancers. Moreover, two other downstream targets of AMPK, the tumor suppressors TSC1 and 2, have been shown to be mutated in tuberous sclerosis [120]. One of the major outcomes of AMPK and/or activation of its downstream substrates is the induction of mitochondrial biogenesis and/or activity [121]. Not surprisingly, AMPK appears to be relevant in the control of protein synthesis and proliferation [122], and may activate p53 causing cell cycle arrest and/or cellular senescence in states of decreased glucose availability [123]. Given the connection of AMPK on the one hand and several tumor suppressors, including TSCs and LKB1 (see above), on the other hand, as well as the fact that AMPK itself is of eminent relevance to mitochondrial activity and energy balance, disruption of this pathway might be considered of importance in regard to the molecular causes of the Warburg effect in states of cancer and malignant growth. In summary, though the Warburg hypothesis was formulated more than 80 years ago, nowadays it still (or rather again) is a subject of extensive debate. The recent findings on the metabolic signature of tumors and malignant cells on the one hand and several molecular pathways linked to derangements of cellular energy flux in transformed state on the other hand strongly support the overdue importance of one of the most long-standing hypotheses in oncology—Warburg’s hypothesis. Acknowledgments We thank members of our laboratories for their contributions and encouraging discussions on the subject of this review. This review article was written while the research activity in the authors’ laboratory was supported by grants from the German government to M.R. and from the Ministerio de Educaci´on y Ciencia (BFU2007-65253) and Fundaci´on Mutua Madrile˜na to J.M.C.

References 1. Ristow M. Oxidative metabolism in cancer growth. Curr Opin Clin Nutr Metab Care 2006; 9:339–345. 2. Cuezva JM, Sanchez-Arago M, Sala S, Blanco-Rivero A, Ortega AD. A message emerging from development: the repression of mitochondrial beta-F1-ATPase expression in cancer. J Bioenerg Biomembr 2007; 39:259–265.

Oxidative Phosphorylation and Cancer: The Ongoing Warburg Hypothesis

13

3. Lichtenstein AH, Appel LJ, Brands M, et al. Diet and lifestyle recommendations revision 2006: a scientific statement from the American Heart Association Nutrition Committee. Circulation 2006; 114:82–96. 4. Pasteur L. Influence de l oxygene sur le developpement de la levure et la fermentation alcoolique. Bulletin de la Societe Chimique de Paris 1861; 79–80. 5. Pfeiffer T, Schuster S, Bonhoeffer S. Cooperation and competition in the evolution of ATPproducing pathways. Science 2001; 292:504–507. 6. Boyer PD. The ATP synthase. A splendid molecular machine. Annu Rev Biochem 1997; 66:717–749. 7. Tovar J, Leon-Avila G, Sanchez LB, et al. Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation. Nature 2003; 426:172–176. 8. Lill R, M¨uhlenhoff U. Iron-sulfur-protein biogenesis in eukaryotes. Trends Biochem Sci 2005; 30:133–141. 9. Rouault TA, Klausner RD. Iron-sulfur clusters as biosensors of oxidants and iron. Trends Biochem Sci 1996; 21:174–177. 10. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 2001; 15: 2922–2933. 11. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281:1309–1312. 12. Jaattela M. Multiple cell death pathways as regulators of tumour initiation and progression. Oncogene 2004; 23:2746–2756. 13. Warburg O. On the origin of cancer cells. Science 1956; 123:309–314. 14. Warburg O. The metabolism of tumours. London: Constable, 1930. 15. Warburg O. On respiratory impairment in cancer cells. Science 1956; 124:269–270. ¨ 16. Warburg O, Posener K, Negelein E. Uber den Stoffwechsel der Tumoren [On metabolism of tumors]. Biochemische Zeitschrift 1924; 152:319–344. 17. Anonymous. The Nobel prize in physiology or medicine 1931. Available at: http://nobelprizeorg/medicine/laureates/1931/. 18. Burk D. A colloquial consideration on the Pasteur and Neo-Pasteur effects. Cold Spring Harbor Symposia on Quantitative Biology 1939; 7:420–460. 19. Burk D, Schade AL. On respiratory impairment in cancer cells. Science 1956; 124: 270–272. 20. Burk D, Woods M. Newer aspects of glucose fermentation in cancer growth and control. Arch Geschwulstforsch 1967; 28:305–319. 21. Weinhouse S. On respiratory impairment in cancer cells. Science 1956; 124:267–269. 22. Zu XL, Guppy M. Cancer metabolism: facts, fantasy, and fiction. Biochem Biophys Res Commun 2004; 313:459–465. 23. Warburg O. Annual meeting of Nobelists at Lindau, Germany, 1966. English edition by D. Burk. Bethesda, MD: National Cancer Institute. Available at: http://www.hopeforcancer. com/OxyPlus.htm. 24. Krebs H. Otto Warburg: cell physiologist, biochemist and eccentric. Oxford, UK: Clarendon, 1981. 25. Pedersen PL. Tumor mitochondria and the bioenergetics of cancer cells. Prog Exp Tumor Res 1978; 22:190–274. 26. Cuezva JM, Krajewska M, de Heredia ML, et al. The bioenergetic signature of cancer: a marker of tumor progression. Cancer Res 2002; 62:6674–6681. 27. L´opez de Heredia M, Izquierdo JM, Cuezva JM. A conserved mechanism for controlling the translation of beta-F1-ATPase mRNA between the fetal liver and cancer cells. J Biol Chem 2000; 275:7430–7437. 28. Altenberg B, Greulich KO. Genes of glycolysis are ubiquitously overexpressed in 24 cancer classes. Genomics 2004; 84:1014–1020. 29. Isidoro A, Martinez M, Fernandez PL, et al. Alteration of the bioenergetic phenotype of mitochondria is a hallmark of breast, gastric, lung and oesophageal cancer. Biochem J 2004; 378:17–20.

14

M. Ristow, J.M. Cuezva

30. Cuezva JM, Chen G, Alonso AM, et al. The bioenergetic signature of lung adenocarcinomas is a molecular marker of cancer diagnosis and prognosis. Carcinogenesis 2004; 25: 1157–1163. 31. Isidoro A, Casado E, Redondo A, et al. Breast carcinomas fulfill the Warburg hypothesis and provide metabolic markers of cancer prognosis. Carcinogenesis 2005; 26:2095–2104. 32. L´opez-R´ıos F, S´anchez-Arag´o M, Garc´ıa-Garc´ıa E, et al. Loss of the mitochondrial bioenergetic capacity underlies the glucosa avidity of carcinomas. Cancer Res 2007; 67:9013–9017. 33. Birnbaum MJ, Haspel HC, Rosen OM. Transformation of rat fibroblasts by FSV rapidly increases glucose transporter gene transcription. Science 1987; 235:1495–1498. 34. Flier JS, Mueckler MM, Usher P, Lodish HF. Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes. Science 1987; 235:1492–1495. 35. Shim H, Dolde C, Lewis BC, et al. c-Myc transactivation of LDH-A: implications for tumor metabolism and growth. Proc Natl Acad Sci USA 1997; 94:6658–6663. 36. Ziegler A, von Kienlin M, Decorps M, Remy C. High glycolytic activity in rat glioma demonstrated in vivo by correlation peak 1H magnetic resonance imaging. Cancer Res 2001; 61:5595–5600. 37. Rigo P, Paulus P, Kaschten BJ, et al. Oncological applications of positron emission tomography with fluorine-18 fluorodeoxyglucose. Eur J Nucl Med 1996; 23:1641–1674. 38. Gambhir SS. Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer 2002; 2:683–693. 39. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer 2004; 4:891–899. 40. Raghunand N, Gatenby RA, Gillies RJ. Microenvironmental and cellular consequences of altered blood flow in tumours. Br J Radiol 2003; 76(Spec No 1):S11–22. 41. Grover-McKay M, Walsh SA, Seftor EA, Thomas PA, Hendrix MJ. Role for glucose transporter 1 protein in human breast cancer. Pathol Oncol Res 1998; 4:115–120. 42. Sakashita M, Aoyama N, Minami R, et al. Glut1 expression in T1 and T2 stage colorectal carcinomas: its relationship to clinicopathological features. Eur J Cancer 2001; 37:204–209. 43. Younes M, Lechago LV, Lechago J. Overexpression of the human erythrocyte glucose transporter occurs as a late event in human colorectal carcinogenesis and is associated with an increased incidence of lymph node metastases. Clin Cancer Res 1996; 2:1151–1154. 44. Osthus RC, Shim H, Kim S, et al. Deregulation of glucose transporter 1 and glycolytic gene expression by c-Myc. J Biol Chem 2000; 275:21797–21800. 45. Govindarajan B, Shah A, Cohen C, et al. Malignant transformation of human cells by constitutive expression of platelet-derived growth factor-BB. J Biol Chem 2005; 280: 13936–13943. 46. Dang CV, Lewis BC, Dolde C, Dang G, Shim H. Oncogenes in tumor metabolism, tumorigenesis, and apoptosis. J Bioenerg Biomembr 1997; 29:345–354. 47. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol 1992; 12:5447–5454. 48. Robey IF, Lien AD, Welsh SJ, Baggett BK, Gillies RJ. Hypoxia-inducible factor-1alpha and the glycolytic phenotype in tumors. Neoplasia 2005; 7:324–330. 49. Ebert BL, Firth JD, Ratcliffe PJ. Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct cis-acting sequences. J Biol Chem 1995; 270: 29083–29089. 50. Firth JD, Ebert BL, Ratcliffe PJ. Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements. J Biol Chem 1995; 270:21021–21027. 51. Semenza GL, Roth PH, Fang HM, Wang GL. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem 1994; 269:23757–23763. 52. Maxwell PH, Dachs GU, Gleadle JM, et al. Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc Natl Acad Sci USA 1997; 94:8104–8109.

Oxidative Phosphorylation and Cancer: The Ongoing Warburg Hypothesis

15

53. Semenza GL. HIF-1, O(2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus. Cell 2001; 107:1–3. 54. Semenza GL. HIF-1 mediates the Warburg effect in clear cell renal carcinoma. J Bioenerg Biomembr 2007; 39:231–234. 55. McKusick VA, Kniffin CL, Tiller GE, et al. Online Mendelian Inheritance in Man: von Hippel-Lindau syndrome (OMIM 193300). Available at: http://www.ncbi.nlm.nih.gov/ entrez/dispomim.cgi?id=193300.> 56. Koukourakis MI, Giatromanolaki A, Sivridis E, Gatter KC, Harris AL. Pyruvate dehydrogenase and pyruvate dehydrogenase kinase expression in non small cell lung cancer and tumor-associated stroma. Neoplasia 2005; 7:1–6. 57. Lu H, Dalgard CL, Mohyeldin A, McFate T, Tait AS, Verma A. Reversible inactivation of HIF-1 prolyl hydroxylases allows cell metabolism to control basal HIF-1. J Biol Chem 2005; 280:41928–41939. 58. Rathmell JC, Fox CJ, Plas DR, Hammerman PS, Cinalli RM, Thompson CB. Akt-directed glucose metabolism can prevent Bax conformation change and promote growth factorindependent survival. Mol Cell Biol 2003; 23:7315–7328. 59. Elstrom RL, Bauer DE, Buzzai M, et al. Akt stimulates aerobic glycolysis in cancer cells. Cancer Res 2004; 64:3892–3899. 60. Govindarajan B, Sligh JE, Vincent BJ, et al. Overexpression of Akt converts radial growth melanoma to vertical growth melanoma. J Clin Invest 2007; 117:719–729. 61. Pedersen PL. Warburg, me and hexokinase 2: multiple discoveries of key molecular events underlying one of cancers most common phenotypes, the ”Warburg Effect,” i.e., elevated glycolysis in the presence of oxygen. J Bioenerg Biomembr 2007; 39:211–222. 62. Bustamante E, Morris HP, Pedersen PL. Energy metabolism of tumor cells. Requirement for a form of hexokinase with a propensity for mitochondrial binding. J Biol Chem 1981; 256:8699–8704. 63. Mathupala SP, Heese C, Pedersen PL. Glucose catabolism in cancer cells. The type II hexokinase promoter contains functionally active response elements for the tumor suppressor p53. J Biol Chem 1997; 272:22776–22780. 64. Smith TA, Sharma RI, Thompson AM, Paulin FE. Tumor 18F-FDG incorporation is enhanced by attenuation of P53 function in breast cancer cells in vitro. J Nucl Med 2006; 47:1525–1530. 65. Bensaad K, Tsuruta A, Selak MA, et al. TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell 2006; 126:107–120. 66. Hervouet E, Demont J, Pecina P, et al. A new role for the von Hippel-Lindau tumor suppressor protein: stimulation of mitochondrial oxidative phosphorylation complex biogenesis. Carcinogenesis 2005; 26:531–539. 67. Wu M, Neilson A, Swift AL, et al. Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am J Physiol Cell Physiol 2007; 292:C125–136. 68. Hoberman HD. Is there a role for mitochondrial genes in carcinogenesis? Cancer Res 1975; 35:3332–3335. 69. Baggetto LG. Role of mitochondria in carcinogenesis. Eur J Cancer 1992; 29A:156–159. 70. Carew JS, Huang P. Mitochondrial defects in cancer. Mol Cancer 2002; 1:9. 71. Petros JA, Baumann AK, Ruiz-Pesini E, et al. mtDNA mutations increase tumorigenicity in prostate cancer. Proc Natl Acad Sci USA 2005; 102:719–724. 72. Singh KK. Mitochondrial dysfunction is a common phenotype in aging and cancer. Ann N Y Acad Sci 2004; 1019:260–264. 73. Taylor RW, Turnbull DM. Mitochondrial DNA mutations in human disease. Nat Rev Genet 2005; 6:389–402. 74. Herrmann PC, Herrmann EC. Oxygen metabolism and a potential role for cytochrome c oxidase in the Warburg effect. J Bioenerg Biomembr 2007; 39:247–250. 75. Baysal BE, Ferrell RE, Willett-Brozick JE, et al. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 2000; 287:848–851.

16

M. Ristow, J.M. Cuezva

76. Habano W, Sugai T, Nakamura S, Uesugi N, Higuchi T, Terashima M, Horiuchi S. Reduced expression and loss of heterozygosity of the SDHD gene in colorectal and gastric cancer. Oncol Rep 2003; 10:1375–1380. 77. Neumann HP, Pawlu C, Peczkowska M, et al. Distinct clinical features of paraganglioma syndromes associated with SDHB and SDHD gene mutations. JAMA 2004; 292:943–951. 78. Deml E, Oesterle D, Wolff T, Greim H. Age-, sex-, and strain-dependent differences in the induction of enzyme-altered islands in rat liver by diethylnitrosamine. J Cancer Res Clin Oncol 1981; 100:125–134. 79. Racker E. Bioenergetics and the problem of tumor growth. Am Sci 1972; 60:56–63. 80. Racker E, Spector M. Warburg effect revisited: merger of biochemistry and molecular biology. Science 1981; 213:303–307. 81. Lehninger AL. Biochemistry. New York: Worth Publishers, 1970. 82. Shin YK, Yoo BC, Chang HJ, et al. Down-regulation of mitochondrial F1F0-ATP synthase in human colon cancer cells with induced 5-fluorouracil resistance. Cancer Res 2005; 65: 3162–3170. 83. Santamaria G, Martinez-Diez M, Fabregat I, Cuezva JM. Efficient execution of cell death in non-glycolytic cells requires the generation of ROS controlled by the activity of mitochondrial H+-ATP synthase. Carcinogenesis 2006; 27:925–935. 84. Zhou S, Kachhap S, Singh KK. Mitochondrial impairment in p53-deficient human cancer cells. Mutagenesis 2003; 18:287–292. 85. Ma W, Sung HJ, Park JY, Matoba S, Hwang PM. A pivotal role for p53: balancing aerobic respiration and glycolysis. J Bioenerg Biomembr 2007; 39:243–246. 86. Matoba S, Kang JG, Patino WD, et al. p53 regulates mitochondrial respiration. Science 2006; 312:1650–1653. 87. Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 2006; 3:177–185. 88. Papandreou I, Cairns RA, Fontana L, Lim AL, Denko NC. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab 2006; 3:187–197. 89. Pelicano H, Xu RH, Du M, et al. Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism. J Cell Biol 2006; 175:913–923. 90. Stiles B, Groszer M, Wang S, Jiao J, Wu H. PTENless means more. Dev Biol 2004; 273: 175–184. 91. Martinez-Diez M, Santamaria G, Ortega AD, Cuezva JM. Biogenesis and dynamics of mitochondria during the cell cycle: significance of 3 UTRs. PLoS ONE 2006; 1:e107. 92. Wang T, Marquardt C, Foker J. Aerobic glycolysis during lymphocyte proliferation. Nature 1976; 261:702–705. 93. Chen Z, Odstrcil EA, Tu BP, McKnight SL. Restriction of DNA replication to the reductive phase of the cell cycle protects genome integrity. Science 2007; 316:1916–1919. 94. Sakamaki T, Casimiro MC, Ju X, et al. Cyclin d1 determines mitochondrial function in vivo. Mol Cell Biol 2006; 26:5449–5469. 95. Wang C, Li Z, Lu Y, et al. Cyclin D1 repression of nuclear respiratory factor 1 integrates nuclear DNA synthesis and mitochondrial function. Proc Natl Acad Sci USA 2006; 103:11567–11572. 96. Luis AM, Izquierdo JM, Ostronoff LK, Salinas M, Santar´en JF, Cuezva JM. Translational regulation of mitochondrial differentiation in neonatal rat liver. Specific increase in the translational efficiency of the nuclear-encoded mitochondrial beta-F1-ATPase mRNA. J Biol Chem 1993; 268:1868–1875. 97. Izquierdo JM, Ricart J, Ostronoff LK, Egea G, Cuezva JM. Changing patterns of transcriptional and post-transcriptional control of beta-F1-ATPase gene expression during mitochondrial biogenesis in liver. J Biol Chem 1995; 270:10342–10350.

Oxidative Phosphorylation and Cancer: The Ongoing Warburg Hypothesis

17

98. Izquierdo JM, Cuezva JM. Control of the translational efficiency of beta-F1-ATPase mRNA depends on the regulation of a protein that binds the 3 untranslated region of the mRNA. Mol Cell Biol 1997; 17:5255–5268. 99. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57–70. 100. Ristow M, Pfister MF, Yee AJ, et al. Frataxin activates mitochondrial energy conversion and oxidative phosphorylation. Proc Natl Acad Sci USA 2000; 97:12239–12243. 101. Schulz TJ, Thierbach R, Voigt A, et al. Induction of oxidative metabolism by mitochondrial frataxin inhibits cancer growth: Otto Warburg revisited. J Biol Chem 2006; 281:977–981. 102. M¨uhlenhoff U, Richhardt N, Ristow M, Kispal G, Lill R. The yeast frataxin homolog Yfh1p plays a specific role in the maturation of cellular Fe/S proteins. Hum Mol Genet 2002; 11:2025–2036. 103. Thierbach R, Schulz TJ, Isken F, et al. Targeted disruption of hepatic frataxin expression causes impaired mitochondrial function, decreased life span, and tumor growth in mice. Hum Mol Genet 2005; 14:3857–3864. 104. Bulavin DV, Fornace AJ Jr. p38 MAP kinase s emerging role as a tumor suppressor. Adv Cancer Res 2004; 92:95–118. 105. Timofeev O, Lee TY, Bulavin DV. A subtle change in p38 MAPK activity is sufficient to suppress in vivo tumorigenesis. Cell Cycle 2005; 4:118120. 106. Bi X, Lin Q, Foo TW, et al. Proteomic analysis of colorectal cancer reveals alterations in metabolic pathways: mechanism of tumorigenesis. Mol Cell Proteomics 2006; 5:1119–1130. 107. Dey R, Moraes CT. Lack of oxidative phosphorylation and low mitochondrial membrane potential decrease susceptibility to apoptosis and do not modulate the protective effect of Bcl-x(L) in osteosarcoma cells. J Biol Chem 2000; 275:7087–7094. 108. Kim JY, Kim YH, Chang I, et al. Resistance of mitochondrial DNA-deficient cells to TRAIL: role of Bax in TRAIL-induced apoptosis. Oncogene 2002; 21:3139–3148. 109. Park SY, Chang I, Kim JY, et al. Resistance of mitochondrial DNA-depleted cells against cell death: role of mitochondrial superoxide dismutase. J Biol Chem 2004; 279: 7512–7520. 110. Tomiyama A, Serizawa S, Tachibana K, et al. Critical role for mitochondrial oxidative phosphorylation in the activation of tumor suppressors Bax and Bak. J Natl Cancer Inst 2006; 98:1462–1473. 111. Matsuyama S, Xu Q, Velours J, Reed JC. The mitochondrial F0F1-ATPase proton pump is required for function of the proapoptotic protein Bax in yeast and mammalian cells. Mol Cell 1998; 1:327–336. 112. Gross A, Pilcher K, Blachly-Dyson E, et al. Biochemical and genetic analysis of the mitochondrial response of yeast to BAX and BCL-X(L). Mol Cell Biol 2000; 20:3125–3136. 113. Harris MH, Vander Heiden MG, Kron SJ, Thompson CB. Role of oxidative phosphorylation in Bax toxicity. Mol Cell Biol 2000; 20:3590–3596. 114. Plas DR, Thompson CB. Cell metabolism in the regulation of programmed cell death. Trends Endocrinol Metab 2002; 13:75–78. 115. Danial NN, Gramm CF, Scorrano L, et al. BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis. Nature 2003; 424:952–956. 116. Azoulay-Zohar H, Israelson A, Abu-Hamad S, Shoshan-Barmatz V. In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondriamediated apoptotic cell death. Biochem J 2004; 377:347–355. 117. Vahsen N, Cande C, Briere JJ, et al. AIF deficiency compromises oxidative phosphorylation. EMBO J 2004; 23:4679–4689. 118. Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 2005; 1:15–25. 119. Alessi DR, Sakamoto K, Bayascas JR. LKB1-dependent signaling pathways. Annu Rev Biochem 2006; 75:137–163. 120. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell 2003; 115:577–590.

18

M. Ristow, J.M. Cuezva

121. Reznick RM, Shulman GI. The role of AMP-activated protein kinase in mitochondrial biogenesis. J Physiol 2006; 574:33–39. 122. Shaw RJ. Glucose metabolism and cancer. Curr Opin Cell Biol 2006; 18:598–608. 123. Jones RG, Plas DR, Kubek S, et al. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol Cell 2005; 18:283–293.

The Electron Transport Chain and Carcinogenesis Jean-Jacques Bri`ere, Paule B´enit and Pierre Rustin

Abstract Major metabolic changes that affect the balance between respiration and glycolysis occur during carcinogenesis. It is therefore not surprising that it has long been suggested that abnormal activity of the mitochondrial electron transfer chain could play a role in the underlying pathophysiologic process. However, it has only recently been demonstrated that the specific impairment of one component of the electron transfer chain, namely complex II, can indeed be the primary event triggering carcinogenesis. Unexpectedly, rather than superoxide overproduction, the organic acid imbalance resulting from the complex II blockade appears to be instrumental in the early step of tumorigenesis. Nevertheless, because an abnormal handling of oxygen is frequently suspected of being associated with electron transfer dysfunction, a renewed interest is observed for the putative instability of the mitochondrial genome often reported in tumor tissues. In this chapter, we review and discuss the evidence advocated for the implication of the electron transfer chain in carcinogenesis. Keywords Carcinogenesis · Mitochondria · Organic acids · Superoxides · Electron transfer

1 Introduction Mitochondria play an irreplaceable role in all aerobic organisms (i.e., the handling of oxygen by the electron transfer chain for the mutual benefit of the cell host and of these Proteobacteria-derived organelles) [1]. In addition, through a fascinating and complex network of interactions, mitochondria have emerged as central executioners, or possibly even decision makers, for cell death under a large number of conditions [2]. In keeping with this paradigm, a set of mitochondria-releasable

P. Rustin (B) INSERM U676, Hˆopital Robert Debr´e, 75019 Paris, France e-mail: [email protected] S.P. Apte, R. Sarangarajan (eds.), Cellular Respiration and Carcinogenesis, DOI 10.1007/978-1-59745-435-3 2,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

19

20

J.-J. Bri´ere et al.

factors have been shown to be bifunctional proteins [3–5]. Involved in electron transfer chain activity or maintenance, both cytochrome c and apoptosis inducing factor (AIF) readily trigger apoptosis when released in the cytosol upon opening of the elusive mitochondrial permeability transition pore (MPTP) [6]. Furthermore, a severe shortage of mitochondrial ATP production, which presumably occurs when the electron transfer chain is deficient, is supposedly sufficient to cause cell necrosis [7]. However, more recently, it was discovered that respiratory chain complex II deficiency can favor cell proliferation and tumor formation rather than cell death [8]. This has led to a renewed interest in the potential role of respiratory chain dysfunction in tumor promotion.

2 Mitochondria and Cancer: A Long History Otto Warburg performed the seminal observation that, even under aerobic conditions, cancer cells shift their metabolism from an oxidative one (through the mitochondrial respiratory chain) to a highly glycolytic metabolism, thereby producing elevated levels of lactate [9]. This paradoxical effect, wherein oxygen is not utilized even though it is available, was thereafter referred to as the Warburg effect.This phenomenon is at variance with the Pasteur effect according to which glycolysis is naturally favored by conditions that turn off the oxygen-dependent mitochondrial respiratory chain. As a result of the shift of metabolic flux from respiration to glycolysis, the former would become of secondary importance, and this logically suggested the idea that the fate of the mitochondria in tumor tissues would be somewhat irrelevant to tumor development. Accordingly, up to very recently, the word cancer was indeed largely absent from mitochondrial disease–devoted meetings and reviews. Even the reports of decreased respiratory chain activities or of quantitative changes in mitochondrial DNA (mtDNA)-encoded mRNA in tumors were insufficient to really draw the attention of the mitochondria-oriented scientific community. However, several features of the mitochondrial electron transfer chain that could play a role in triggering, or participating in, tumorigenesis deserve to be presented.

3 The Respiratory Chain: Biochemical and Genetic Features The respiratory chain (RC) is constituted by an association of supercomplexes whose spatial organization favors a rapid exchange of electrons. Such a mechanism may serve to avoid—or at least to limit—direct reactions between reduced RC carriers and molecular oxygen that can produce superoxides [10]. Two major entities have been described: the respirasome consisting of the association of RC complexes I/III/IV and presumably a subfraction of cytochrome c; and the ATPasome, consisting of complex V plus the adenylate and the phosphate carriers. The

The Electron Transport Chain and Carcinogenesis

21

respirasome channels the electrons from respiratory substrates to molecular oxygen to form water with a concomitant proton extrusion from the mitochondrial matrix space to the intermembrane space (Fig. 1). This creates an electrochemical gradient across the inner membrane that can be further used by the ATPasome to generate ATP from ADP and inorganic phosphate. Additional processes make use of the electrochemical gradient, ensuring the transport of various components through the membrane, including various cations (e.g., calcium), anions (e.g., organic acids, fatty acids), or proteinaceous material (e.g., mitochondrial protein precursors) [1]. Dissipation of the electrochemical gradient can also be an aim per se, allowing the uncoupling of electron flow from the phosphorylation process [11]. This process liberates heat and mechanically favors the oxidized state of the RC

Fig. 1 The dual genetic origin and the organization of the respiratory chain. (A) Respiratory chain components are encoded by both the nuclear and the mitochondrial (mt) genomes. (B) A schematic view of the organization of the respirasome and the ATPasome. CI–CV, the various complexes of the respiratory chain; cyt c, cytochrome c; IM, inner membrane; Q: ubiquinone pools

22

J.-J. Bri´ere et al.

components, thus reducing the chance to generate superoxides. UnCoupling Proteins (UCP), a large family of proteins, mediate this process, with a high tissue specificity. The physiologic function of a number of these UCPs is still a subject of debate [12]. The respirasome interacts with several other dehydrogenases (e.g., complex II, the electron transfer flavoprotein [ETF], the glycerol-3-phosphate and the dihydroorotate dehydrogenases) through the kinetically compartmentalized quinone pool in the inner mitochondrial membrane (Fig. 1) [12]. Depending on their specific metabolic demand, tissues are variably endowed with these dehydrogenases [13]. Redox status of the quinone pool therefore depends on the respective activities of the reducing enzymes (the various dehydrogenases) and of the oxidizing part of the RC (i.e., the cytochromic segment). This redox status is also important because it helps to reconstitute the reducing power of lipophilic antioxidants, such as tocopherol (vitamin E) [14]. This complex machinery constituted of about 100 proteins is encoded by both the mitochondrial genome (mtDNA) and the nuclear genome (Fig. 1) [15]. The maternally inherited mtDNA ranges from a few hundred (lymphocytes) to more than 100,000 (oocytes) copies per cell [16]. Each circular molecule consists of 16,569 base pairs with 37 genes encoding 13 proteins (polypeptides), 22 transfer RNA (tRNAs), and 2 ribosomal RNAs (rRNAs) [15]. In one cell, the mtDNA copies can be identical (a situation referred as cellular homoplasmy) or not (a situation known as heteroplasmy). In the particular case of deleterious mutations harbored by the mtDNA, a threshold between wild type (wt) and mutant mtDNA species has to be reached before an RC phenotype can be observed [17]. Because of the random distribution of mitochondria during cell division, an individual can have variable levels of heteroplasmy from tissue to tissue. The involvement of a given tissue containing mutant mtDNA will therefore depend on the actual level of heteroplasmy, the threshold value required for RC impairment, and the susceptibility of the tissue to the many consequences of an RC dysfunction (e.g., ATP shortage, superoxide overproduction, metabolic blockade, etc.). The remaining components of the RC are encoded by genes present in the nuclear genome. According to the theory of the symbiotic origin of the mitochondria, these genes have supposedly left the mitochondria to be inserted in the nuclear chromosomes during evolution [18]. Noticeably, nonfunctional copies of genes encoding mitochondrial proteins are frequently encountered in the nuclear genome [19]. This also stands true for genes still harbored by the mtDNA, a feature that should be carefully recorded when studying these genes [20]. Accordingly, the authenticity of the increased levels of mutant mtDNA frequently reported in tumor tissues has been recently questioned [21]. Finally, if as mentioned earlier, the RC does much more than to only transfer electrons and synthesize ATP, its functioning also requires much more than the hundreds of genes that encode its constituents. Indeed about 1500 genes on the 30,000 genes of the human genome are believed to be necessary for the sustained building, function and maintenance of mitochondrial functions.

The Electron Transport Chain and Carcinogenesis

23

4 Mutations in Three Nuclear Genes Encoding Respiratory Chain Complex II Can Promote Tumor Formation Mutations in three of the four genes encoding complex II have been shown to cause tumor formation and cancer. Mutations of succinate dehydrogenase (SDH) are frequently encountered in patients with an apparently sporadic form of paragangliomas (PGLs) or pheochromocytomas (PHEOs) [22]. PGLs are neuroendocrine tumors that may secrete catecholamines [23]. They occur most frequently in the head (glomus tympanicum and jugulare), neck (carotid body and glomus vagale), adrenal medulla, and extra-adrenal sympathetic ganglia. PGLs are generally benign, highly vascularized tumors occurring close to the major blood vessels and cranial nerves [24]. About 10% of these tumors are malignant. PGLs are usually diagnosed on the basis of the presence of a mass in the neck, pulsatile tinnitus, or hearing loss. According to current nomenclature, the term pheochromocytomas should be restricted to secreting PGLs evolving from the adrenal medulla, whereas a functional PGL refers to an extra-adrenal secreting PGL, which was previously known as an ectopic PHEO. The tumors now described as carotid body PGL were formerly known as chemodectoma due to the chemoreceptor function of the carotid body. PGLs seem to be inherited in about 30% of cases. The hereditary form of PGL is usually characterized by an early onset and a more severe presentation than that of the sporadic form. These tumors often display bilateral and multiple locations and may be recurrent or malignant. The presence of one or several secreting tumors is not rare and contributes to the severity of inherited PGL [23]. Such PGLs constitute a genetically heterogeneous group of diseases as four different loci have been implicated: PGL1 on 11q23, PGL2 on 11q13, PGL3 on 1q21, and PGL4 on 1p36. In 2000, linkage analysis and positional cloning allowed Baysal et al. [8] to report the first deleterious mutations in the SDHD gene, corresponding with the PGL1 locus. The use of a candidate gene approach has led to the identification of mutations in SDHC (PGL3) and SDHB (PGL4) [24, 25]. Several mutations in these three genes were subsequently reported in patients with hereditary PGL and in apparently sporadic cases [26]. These observations fueled a vigorous debate on the mechanism linking SDH deficiency to tumor formation. Based on previous findings on the potential consequences of a RC blockade, it has been suggested that superoxide overproduction may be at the origin of increased cell proliferation [27]. Accordingly, the mev1 mutant of the worm Caenorhabditis elegans defective in the cytochrome b subunit of CII was found to have a reduced lifespan that was attributed to overproduction of superoxides [28]. In the minds of many investigators, the superoxide overproduction provided a simple explanation linking RC defects to neoplastic transformation. However, no hyperplasia or indications of abnormal cell proliferation were reported in the mev1 mutant at variance with some other C. elegans mutants for proteins that are prone to trigger tumorigenesis when mutated in the homologous protein in mammals (e.g., cul1 mutant) [29]. Soon after the discovery that SDH mutations can result in tumor formation, it was shown in a family harboring a SDHD mutation that these tumors were highly

24

J.-J. Bri´ere et al.

vascularized and that this corresponded with hypoxia-inducible factor (HIF) stabilization and the activation of the hypoxia pathway [30]. HIFs are composed of two subunits: an ␣ subunit, which is regulated by oxygen, and a ␤-subunit (HIF-1␤, also called the aryl hydrocarbon receptor nuclear translocator, or ARNT), which is constitutively and ubiquitously expressed. Three ␣-subunits have been identified to date (HIF-1␣, HIF-2␣, and HIF-3␣), each encoded by a different gene. Under normoxic conditions, HIF-␣ is continuously ubiquitinated and subsequently degraded by the proteasome [31]. The process of ubiquitination is started by their recognition by the von Hippel–Lindau (VHL) protein, which in turn requires the hydroxylation of two proline residues on HIF-␣ [32]. The very first step of HIF-␣ degradation under normoxic conditions is thus dependent on this hydroxylation, which is catalyzed by HIF prolyl hydroxylases (PHDs). PHDs belong to the superfamily of Fe(II)-dependent oxygenases and require reduced iron as a cofactor, alpha-ketoglutarate (␣-KG) and oxygen as cosubstrates, with carbon dioxide and succinate being the products of the reaction [33]. Under hypoxic conditions, the absence of oxygen prohibits PHD activity, and HIF-␣ is thus stabilized, allowing for its nuclear translocation and the subsequent activation of its target genes. The involvement of HIFs has been observed in numerous types of tumors, playing an active role in the progression of neoplasia [34]. Besides oxygen, others factors, which intervene in the reaction catalyzed by the PHD enzyme, can also affect its activity. Consistent with this hypothesis, it was shown that elevated levels of succinate, the product of the PHDs reaction, can result in the blockade of its activity with the consequent stabilization of the HIF-1␣ protein (Fig. 2) [35, 36]. A significant accumulation of succinate was found in SDH-deficient cells and tumors. Moreover, the addition of ␣-ketoglutarate, the substrate of the PHD,

Fig. 2 The interaction between the mitochondrion and type II hexokinase. The channeling of mitochondrial ATP by type II hexokinase favors the production of glucose 6-phosphate (G 6-P) and ultimately glycolysis. Ant, adenylate carrier; HKII, type II hexokinase; I–V, the various complexes of the respiratory chain; Q, ubiquinone; VDAC, voltage-dependent anion channel

The Electron Transport Chain and Carcinogenesis

25

was shown to abolish the nuclear translocation of HIF-1␣ in the nucleus of SDHdefective cells [35]. This provided an alternative explanation linking SDH deficiency to tumor formation and an explanation of the restriction of the induction of tumors to RC complex II blockade. Finally, the respiration of SDH-defective cells was shown to be not significantly decreased indicating that the electron flow to oxygen and the phosphorylation process are not significantly affected [37]. Additional support in favor of this latter hypothesis came from the observation that fumarase defect can lead to leiomyomatosis and renal cell cancer (HLRCC) syndrome [38]. In this latter case, fumarate, accumulated because of fumarase inactivation, was found to act as a competitive inhibitor of the PHD, thus inducing the abnormal stabilization of HIF-1␣. Noticeably, other structurally related organic acids can also inhibit PHD [39]. Therefore, a tricarboxylic acid cycle (TCA) cycle blockade may result in the induction of angiogenesis and in the upregulation of glycolysis during tumorigenesis, being at the origin of the Warburg effect. These observations support the view that rather than a blockade of the electron flow (potentially associated with all subtypes of RC defects), the impairment of a particular segment of the Krebs cycle can be at the origin of tumor formation [40]. It would therefore be at least rather imprudent to invoke SDH mutations as a general proof that a RC defect results in tumor formation.

5 Does a Defective Respiratory Chain Trigger Cell Proliferation? To date, there is no conclusive evidence that a perturbation of the electron flow through the RC is sufficient to increase cell proliferation and to constitute the primary cause of tumor formation. On one hand, none of the nuclear genes encoding RC components or involved in the building or maintenance of the chain have been demonstrated to be tumor suppressors, with the exception of genes encoding RC complex II (or fumarase; see above) [41]. Noticeably, the accumulation of ␣-ketoglutarate is a feature frequently associated with RC dysfunction, which will not favor HIF-1␣ stabilization through PHD inhibition. On the other hand, RC-specific poisons are not known as carcinogenic (e.g., the complex I–specific inhibitor rotenone shown to possibly induce parkinsonism in mammals). Finally, patients harboring deleterious mutation in genes encoding RC components are not known to be particularly at risk for tumor formation. This is even true for mutations affecting RC proteins such as ATPase (complex V) components that result in high levels of superoxide production [42]. These observations lend less credibility to the idea that an impairment of the RC function could per se be at the origin of cell proliferation and tumor formation. Even more puzzling is the fact that in a subset of tumor cells (liver and pancreatic tumors), the enhanced glycolysis might well require an active production of ATP by the RC. The hexokinase specifically expressed in most tumor cells uses both ATP and glucose to produce ADP and glucose-6-phosphate. This tumor-specific hexokinase is characterized by its poor affinity (high Km , about 10 mM) for glucose, similar to the hexokinase IV (glucokinase) found in normal hepatocytes [43].

26

J.-J. Bri´ere et al.

Fig. 3 Induction of the hypoxia path by succinate through HIF-1␣ stabilization. Upon succinate dehydrogenase (complex I) blockade, succinate accumulates and is exported to the cytosol. There, it inhibits the prolyl hydroxylase (PHD) triggering HIF-1␣ stabilization. The nuclear translocation of this latter factor induces an increased transcription of the hypoxia pathway components. VHL, von Hippel–Lindau; I–V, the various respiratory chain complexes

The Electron Transport Chain and Carcinogenesis

27

It possibly allows for an additional capacity for glucose uptake from plasma and increased glucose phosphorylation by displacement of the cell glucose equilibrium. Interestingly, whereas the isoform with a low affinity is expressed in most tumor cells, in the tumoral liver, similarly to tumoral pancreatic cells, it is largely replaced by a high-affinity form (hexokinase II; HKII). This latter form can readily bind voltage-dependent anion channel (VDAC) at the outer mitochondrial membrane and utilize the mitochondrial ATP to produce glucose-6-phosphate thus favoring an aerobic glycolysis [44] so long as the electron transfer chain is functional (Fig. 3). Any significant impairment of RC activity would then tend to decrease the ATP produced by the RC and oppose the upregulation of glycolysis. In addition, it has been suggested that, upon binding to VDAC, HKII would be protected from product inhibition because of a conformational change and in turn may reduce the chance of VDAC to open and to facilitate release of proapoptotic factors [43, 45]. In this case, not only would carcinogenesis not be triggered by mitochondrial dysfunction, but also it would require the preservation of the respiratory chain function. Finally, there is ample evidence that an impairment of RC function under most conditions leads to cell death, tissue necrosis, and the related wide range of clinical symptoms observed in patients harboring mutations in genes necessary for the synthesis of the electron transfer chain [46]. Potential overproduction of superoxides has been shown to be responsible for triggering apoptotic cell death, even if low amount of superoxides might be required for normal cell division, and can target various cell cycle checkpoints. It therefore appears that a defective RC is probably not per se a primary cause of tumor formation. On the contrary, it could in some cases even constitute an obstacle to this process!

6 Mitochondria, Superoxides, Hypoxia The possibility nevertheless exists that low oxygenation in tumors may secondarily affect mitochondrial functioning favoring the formation of superoxides and peroxides by the RC (Fig. 4). In principle, activated oxygen species may in turn upregulate both oncogene growth factors and their tyrosine kinase receptors thus driving cell transformation [47], simultaneously promoting HIF-1␣ stabilization by inhibiting prolyl hydroxylase. In any event, superoxide production that may promote tumorigenesis would have to be restricted to a very narrow window of concentration, large overproduction readily favoring cell death rather than proliferation. The suggested role of superoxides in triggering tumorigenesis has long been advocated in support for a therapeutic use of antioxidants [48]. Actually, a wide range of antioxidant molecules is available that can potentially interfere with cell free radicals, possibly released by mitochondria. However, contrasting results from in vitro and in vivo (intervention trials) experiments have raised some doubt about the ability of antioxidants to actually fight cancer by such a mechanism. Indeed, most antioxidant molecules also act as prooxidants; this possibly accounts

28

J.-J. Bri´ere et al.

Fig. 4 Linking electron transfer chain to cell death and proliferation. (A) According to a first hypothesis, the level of mitochondrial superoxide production largely determines the fate of the cells. A low superoxide production favors cell division, whereas increasing this production triggers dysplastic lesion and tumor formation. An excessive production rather results in cell death. (B) According to this second scheme, the blockade of the prolyl hydroxylase by organic acids (succinate, fumarase) results in HIF-1␣ stabilization and the activation of the hypoxia path allowing for cell proliferation. Release of mitochondrial proapoptotic factors (AIF, cytochrome c) and overproduction of superoxide (blockade of the ATPase; complex V) result in cell death

for their potential antitumoral activity. For instance, an antioxidant molecule like melatonin would exercise its antiproliferative effect on the growth of rat pituitary prolactin-secreting tumor cells in vitro by damaging mitochondria rather than by quenching superoxides [49]. Thus even if an increased superoxide production is observed in a subset of cancer cells, more evidence is needed to establish that, as a general rule, these superoxides are instrumental in triggering tumorigenesis. Recent studies however have established that overexpression of thioredoxin, manganese-dependent superoxide dismutase (MnSOD), glutathione peroxidase, or catalase all tend to reduce cell proliferation in different animal or cell models

The Electron Transport Chain and Carcinogenesis

29

suggesting that handling of superoxides or their derivatives might be critical at some point in a subset of cases [50, 51] (see, however, Refs. 52, 53].

7 Targeting the Respiratory Chain to Kill Tumors Although it is not clear that a defective respiratory chain actually favors tumor formation, mitochondria might represent the Achilles’ heel of cancer cells (Fig. 5). Indeed, mitochondria house several proapoptotic factors that are simultaneously

Fig. 5 Mitochondria and apoptosis. Release of intermembrane space components (such as AIF or cytochrome c) and/or the loss of membrane potential (⌬⌿) under the effect of numerous stimuli trigger cell commitment to die through an apoptotic process. Loss of membrane potential and of various matrix cofactors can result from the opening of the mitochondrial permeability transition pore (PTP). The balance between the proapoptotic and antiapoptotic members of the Bcl-2 family controls the opening of the pore. Alternatively, the members of this family can form channels that may also allow for the release of proapoptotic components present in the intermembrane space. PTP would be a complex formed between the voltage-dependent anion channel (VDAC; outer membrane) and the adenylate carrier (ANT; inner membrane), associated with several additional proteins. AIF, apoptosis inducing factor; Br, benzodiazepine receptor; CI–CV, the various complexes of the respiratory chain; c, cytochrome c; IM, inner membrane; OM, outer membrane

30

J.-J. Bri´ere et al.

components of (or closely associated with) the electron transfer chain, and targeting tumors with reagents that induce release of these components has became a fashionable idea [54]. Accordingly, cisplatin, one of the most important chemotherapeutic agents ever developed, has been shown to readily interact with mitochondria to trigger apoptosis [55]. Resveratrol, a natural polyphenolic antioxidant, has been reported to possess a cancer chemopreventive potential that has been attributed to its ability to trigger mitochondrial dysfunction and cell apoptosis [56]. Actually, an exploding list of promising therapeutic and preventative agents is being shown (or has been suggested) to actually act through a quite similar mechanism, with or without the involvement of superoxide production.

8 Conclusion Mutations in both nuclear and mitochondrial genes encoding respiratory chain components have been suggested to be causative for triggering cancer. However, despite the numerous mtDNA mutations reported in many tumors, only germ-line mutations in the complex II–encoding genes (SDHB,C,D) have been conclusively shown as the primary cause of tumor formation. Interestingly enough, no inherited mtDNA mutation has been observed to cause tumor to date, contrasting the high frequency of mutant mtDNA in various type of tumors (see, however, Ref. 21). Although these may have a role in the oncogenic process at some point, they may be a secondary event totally irrelevant for this process. As a general rule, it seems that targeting respiratory chain function, except for complex II activity, provides a mechanism to inhibit rather than to promote tumor formation.

References 1. Tzagoloff A. Mitochondria. New York: Plenum Press, 1982. 2. Green DR, Kroemer G. The pathophysiology of mitochondrial cell death. Science 2004; 305(5684):626–629. 3. Krippner A, Matsuno-Yagi A, Gottlieb RA, Babior BM. Loss of function of cytochrome c in Jurkat cells undergoing fas-mediated apoptosis. J Biol Chem 1996; 271(35):21629–21636. 4. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281(5381):1309–1312. 5. Vahsen N, Cande C, Briere JJ, et al. AIF deficiency compromises oxidative phosphorylation. EMBO J 2004; 23(23):4679–4689. 6. Garrido C, Kroemer G. Life s smile, death s grin: vital functions of apoptosis-executing proteins. Curr Opin Cell Biol 2004; 16(6):639–646. 7. Golstein P, Kroemer G. Cell death by necrosis: towards a molecular definition. Trends Biochem Sci 2007; 32(1):37–43. 8. Baysal BE, Ferrell RE, Willett-Brozick JE, et al. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 2000; 287(5454):848–851. 9. Warburg O, Wind F, Negelein E. The metabolism of tumors in the body. J. General Physiol 1926; 8(6):519. 10. Schagger H, Pfeiffer K. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 2000; 19(8):1777–1783. 11. Pecqueur C, Couplan E, Bouillaud F, Ricquier D. Genetic and physiological analysis of the role of uncoupling proteins in human energy homeostasis. J Mol Med 2001; 79(1):48–56.

The Electron Transport Chain and Carcinogenesis

31

12. Gutman M. Electron flux through the mitochondrial ubiquinone. Biochim Biophys Acta 1980; 594(1):53–84. 13. Pette D, Klingenberg M, Buecher T. Comparable and specific proportions in the mitochondrial enzyme activity pattern. Biochem Biophys Res Commun 1962; 7:425–429. 14. Geromel V, Darin N, Chretien D, et al. Coenzyme Q(10) and idebenone in the therapy of respiratory chain diseases: rationale and comparative benefits. Mol Genet Metab 2002; 77(1–2):21–30. 15. Larsson NG, Clayton DA. Molecular genetic aspects of human mitochondrial disorders. Annu Rev Genet 1995; 29:151–178. 16. Chen X, Prosser R, Simonetti S, Sadlock J, Jagiello G, Schon EA. Rearranged mitochondrial genomes are present in human oocytes. Am J Hum Genet 1995; 57(2):239–247. 17. Bourgeron T, Chretien D, Rotig A, Munnich A, Rustin P. Fate and expression of the deleted mitochondrial DNA differ between human heteroplasmic skin fibroblast and Epstein-Barr virus-transformed lymphocyte cultures. J Biol Chem 1993; 268(26):19369–19376. 18. Rustin P, Jacobs HT, Dietrich A, Lightowlers RN, Tarassov I, Corral-Debrinski M. Targeting allotopic material to the mitochondrial compartment: new tools for better understanding mitochondrial physiology and prospect for therapy. Med Sci (Paris) 2007; 23(5):519–525. 19. Ricchetti M, Tekaia F, Dujon B. Continued colonization of the human genome by mitochondrial DNA. PLoS Biol 2004; 2(9):E273. 20. Parfait B, Rustin P, Munnich A, Rotig A. Co-amplification of nuclear pseudogenes and assessment of heteroplasmy of mitochondrial DNA mutations. Biochem Biophys Res Commun 1998; 247(1):57–59. 21. Salas A, Yao YG, Macaulay V, Vega A, Carracedo A, Bandelt HJ. A critical reassessment of the role of mitochondria in tumorigenesis. PLoS Med 2005; 2(11):e296. 22. Briere JJ, Favier J, Gimenez-Roqueplo AP, Rustin P. Tricarboxylic acid cycle dysfunction as a cause of human diseases and tumour formation. Am J Physiol Cell Physiol 2006; 291(6): 1114–1120. 23. Favier J, Briere JJ, Strompf L, et al. Hereditary paraganglioma/pheochromocytoma and inherited succinate dehydrogenase deficiency. Horm Res 2005; 63(4):171–179. 24. Astuti D, Latif F, Dallol A, et al. Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. Am J Hum Genet 2001; 69(1):49–54. 25. Niemann S, Muller U. Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nat Genet 2000; 26(3):268–270. 26. Nakamura E, Kaelin WG Jr. Recent insights into the molecular pathogenesis of pheochromocytoma and paraganglioma. Endocr Pathol 2006; 17(2):97–106. 27. Rustin P. Mitochondria, from cell death to proliferation. Nat Genet 2002; 30(4):352–353. 28. Senoo-Matsuda N, Yasuda K, Tsuda M, et al. A defect in the cytochrome b large subunit in complex II causes both superoxide anion overproduction and abnormal energy metabolism in Caenorhabditis elegans. J Biol Chem 2001; 276(45):41553–41558. 29. Piva R, Liu J, Chiarle R, Podda A, Pagano M, Inghirami G. In vivo interference with Skp1 function leads to genetic instability and neoplastic transformation. Mol Cell Biol 2002; 22(23):8375–8387. 30. Gimenez-Roqueplo AP, Favier J, Rustin P, et al. The R22X mutation of the SDHD gene in hereditary paraganglioma abolishes the enzymatic activity of complex II in the mitochondrial respiratory chain and activates the hypoxia pathway. Am J Hum Genet 2001; 69(6):1186–1197. 31. Hickey MM, Simon MC. Regulation of angiogenesis by hypoxia and hypoxia-inducible factors. Curr Top Dev Biol 2006; 76:217–257. 32. Kaelin WG. Proline hydroxylation and gene expression. Annu Rev Biochem 2005;74: 115–128. 33. Lee JW, Bae SH, Jeong JW, Kim SH, Kim KW. Hypoxia-inducible factor (HIF-1)alpha: its protein stability and biological functions. Exp Mol Med 2004; 36(1):1–12. 34. Gordan JD, Simon MC. Hypoxia-inducible factors: central regulators of the tumor phenotype. Curr Opin Genet Dev 2007; 17(1):71–77.

32

J.-J. Bri´ere et al.

35. Briere JJ, Favier J, Benit P, et al. Mitochondrial succinate is instrumental for HIF1alpha nuclear translocation in SDHA-mutant fibroblasts under normoxic conditions. Hum Mol Genet 2005; 14(21):3263–3269. 36. Selak MA, Armour SM, MacKenzie ED, et al. Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell 2005; 7(1):77–85. 37. Bourgeron T, Rustin P, Chretien D, et al. Mutation of a nuclear succinate dehydrogenase gene results in mitochondrial respiratory chain deficiency. Nat Genet 1995; 11(2):144–1449. 38. Tomlinson IP, Alam NA, Rowan AJ, et al. Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet 2002; 30(4):406–410. 39. MacKenzie ED, Selak MA, Tennant DA, et al. Cell-permeating alpha-ketoglutarate derivatives alleviate pseudohypoxia in succinate dehydrogenase-deficient cells. Mol Cell Biol 2007; 27(9):3282–3289. 40. Briere JJ, Favier J, El Ghouzzi V, et al. Succinate dehydrogenase deficiency in human. Cell Mol Life Sci 2005; 62(19–20):2317–2324. 41. Kroemer G. Mitochondria in cancer. Oncogene 2006; 25(34):4630–4632. 42. Geromel V, Kadhom N, Cebalos-Picot I, et al. Superoxide-induced massive apoptosis in cultured skin fibroblasts harboring the neurogenic ataxia retinitis pigmentosa (NARP) mutation in the ATPase-6 gene of the mitochondrial DNA. Hum Mol Genet 2001; 10(11):1221–1228. 43. Brandon M, Baldi P, Wallace DC. Mitochondrial mutations in cancer. Oncogene 2006; 25(34):4647–4662. 44. Bustamante E, Pedersen PL. High aerobic glycolysis of rat hepatoma cells in culture: role of mitochondrial hexokinase. Proc Natl Acad Sci USA 1977; 74(9):3735–3739. 45. Mathupala SP, Ko YH, Pedersen PL. Hexokinase II: cancer s double-edged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria. Oncogene 2006; 25(34):4777–4786. 46. Munnich A, Rustin P. Clinical spectrum and diagnosis of mitochondrial disorders. Am J Med Genet 2001; 106(1):4–17. 47. Aslan M, Ozben T. Oxidants in receptor tyrosine kinase signal transduction pathways. Antioxid Redox Signal 2003; 5(6):781–788. 48. Nishikawa M, Hashida M. Inhibition of tumour metastasis by targeted delivery of antioxidant enzymes. Expert Opin Drug Deliv 2006; 3(3):355–369. 49. Yang QH, Xu JN, Xu RK, Pang SF. Antiproliferative effects of melatonin on the growth of rat pituitary prolactin-secreting tumor cells in vitro. J Pineal Res 2007; 42(2):172–179. 50. Weydert CJ, Waugh TA, Ritchie JM, et al. Overexpression of manganese or copper-zinc superoxide dismutase inhibits breast cancer growth. Free Radic Biol Med 2006; 41(2):226–237. 51. Li GX, Hirabayashi Y, Yoon BI, et al. Thioredoxin overexpression in mice, model of attenuation of oxidative stress, prevents benzene-induced hemato-lymphoid toxicity and thymic lymphoma. Exp Hematol 2006; 34(12):1687–1697. 52. Lu YP, Lou YR, Yen P, et al. Enhanced skin carcinogenesis in transgenic mice with high expression of glutathione peroxidase or both glutathione peroxidase and superoxide dismutase. Cancer Res 1997; 57(8):1468–1474. 53. Welsh SJ, Bellamy WT, Briehl MM, Powis G. The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis. Cancer Res 2002; 62(17):5089–5095. 54. Galluzzi L, Larochette N, Zamzami N, Kroemer G. Mitochondria as therapeutic targets for cancer chemotherapy. Oncogene 2006; 25(34):4812–4830. 55. Cepeda V, Fuertes MA, Castilla J, Alonso C, Quevedo C, Perez JM. Biochemical mechanisms of cisplatin cytotoxicity. Anticancer Agents Med Chem 2007; 7(1):3–18. 56. Fulda S, Debatin KM. Resveratrol modulation of signal transduction in apoptosis and cell survival: a mini-review. Cancer Detect Prev 2006; 30(3):217–223.

Respiratory Control of Redox Signaling and Cancer Pauline M. Carrico, Nadine Hempel and J. Andr´es Melendez

Abstract In the past several decades, elegant work has been performed in a variety of organisms suggesting that decreases in oxidant burden can prolong life span and decrease the severity of age-associated degenerative disorders (Finkel T, Holbrook NJ. Nature 2000; 408:239–247). Cancer is primarily an age-related disease and is associated with the altered production of oxidants (Cerutti PA. Science 1985; 227:375–381). Compounds that generate reactive oxygen species (ROS) promote skin tumors in mice. Treatment with antioxidants that terminate the chain reactions initiated by ROS antagonizes this process. Many tumor cell types have also been shown to have an increased oxidant radical load relative to normal tissue. Thus, tumor cells have adapted to cope with an increased oxidant burden compared with that of normal nonmalignant tissue and may even harness the increased oxidant load to modulate signaling pathways that are critical for their survival. With the tight connection between oxidant production and tumor promotion and growth, it is quite surprising that antioxidant-based therapeutics have not become pervasive in treatments for many cancers (for a concise review on this topic, see Seifried HE, et al. Cancer Research 2003; 63:4295–4298). Hydrogen peroxide (H2 O2 ) is the electron-neutral, 2e- reduction product of oxygen that is generated both chemically and enzymatically and can modulate the activity of a variety of signaling molecules. This chapter will summarize how the metabolic production of H2 O2 can impact distinct signaling pathways that are essential for successful tumorigenesis and metastatic progression and will identify redoxsensitive pathways that may be targets for antioxidant-based cancer therapy. Keywords Reactive oxygen species · Cancer · Antioxidants · Redox signaling · Hydrogen peroxide

J.A. Melendez (B) Center for Immunology and Microbial Disease MC151, Albany Medical College, Albany, NY 12208 e-mail: [email protected] S.P. Apte, R. Sarangarajan (eds.), Cellular Respiration and Carcinogenesis, DOI 10.1007/978-1-59745-435-3 3,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

33

34

P.M. Carrico et al.

1 ROS and Redox Signaling All aerobic organisms generate ROS as by-products of normal cellular metabolism. Although the concentrations of ROS are normally low but measurable, accumulation of these species can be toxic to the cells. To protect themselves from the deleterious effects of ROS, cells have evolved an impressive repertoire of enzymatic and nonenzymatic antioxidant defenses. In normal cells, a balance between the rates of ROS production and the scavenging capacity by antioxidants is required to maintain the cellular redox homeostasis. An imbalance resulting from either an increase in ROS concentration or a decrease in the antioxidant capacity leads to the modulation of redox-dependent signaling events. ROS production can arise from a variety of sources in nonphagocytic cells. Most ROS are produced as by-products of the electron transport chain of the mitochondria at the level of complex I (NADH/ubiquinone oxidoreductase) and complex III (ubiquinol/cytochrome c oxidoreductase). Outside the mitochondria, ROS can originate from enzymes such as NADPH oxidase, dual oxidases, and lipoxygenases. At the cell surface, ROS production can be initiated after a variety of stimuli, such as integrin signaling, growth factors, and cytokines, such as TNF-␣, which activate NADPH oxidase. ROS can also arise from arachidonic acid metabolism via 5-lipoxygenase. Both cytoplasmic and mitochondrial pathways create the highly unstable superoxide anion (O2 -. ), which is either chemically or enzymatically converted to H2 O2 . The enzyme responsible for this dismutation is superoxide dismutase, isoforms of which can be found in the cytoplasm (copper zinc/sod1) or the mitochondria (manganese /sod2). Conditions where mitochondrial-derived ROS are produced at a rate that exceeds their scavenging capacity are commonly associated with metabolic perturbations that impact signaling. Recent evidence also suggests that ROS produced as a result of normal metabolic flux affect signaling. For example, Nemoto et al. [1] demonstrated that the addition of extracellular pyruvate to HeLa cells resulted in a dramatic increase in the phosphorylation/activation of JNK1, a modest increase in ERK activity, and no effect on JNK2 or p38. This activation was accompanied by an increase in mitochondrial oxidants and was reversible by the addition of N-acetylcysteine (NAC), overexpression of glutathione S-transferase, or extracellular catalase. These data suggest that the addition of pyruvate results in an increase in H2 O2 levels and the subsequent phosphorylation of JNK1. Furthermore, the activation of JNK1 coincided with the inhibition of glycogen synthase kinase 3-␤ and glycogen synthase. This work provided convincing evidence that ROS produced as a result of normal cellular metabolism could act as a regulator of signal transduction. The principle mediator of ROS-dependent signaling is H2 O2 . H2 O2 generated in response to receptor stimulation has been shown to be an efficient signal transducing molecule by its ability to reversibly oxidize active site cysteines [2]. Many protein tyrosine phosphatases (PTPs) are particularly susceptible to H2 O2 -dependent inactivation because of the lowered pKa of the active site cysteine. The essential cysteine residue in the PTP signature active-site motif Cys-(X)5 -Arg exists as a thiolate anion (Cys-S- ), which, at neutral pH, is susceptible to nucleophilic attack by H2 O2 .

Respiratory Control of Redox Signaling and Cancer

35

Oxidation of the active-site cysteine generates a sulfenic derivative (Cys-SOH) leading to enzyme inactivation that can be reversed by cellular thiols. This mode of inactivation is similar to that described for both bacterial and eukaryotic peroxiredoxin family members that are responsible for detoxification of organic hydroperoxides, H2 O2 and ONOO- [3]. A unique feature of the eukaryotic peroxiredoxins (PRXs) is their sensitivity to inactivation by H2 O2 levels that exceed steady state. This sensitivity to inactivation has led to the “floodgate” hypothesis, whereby PRX inactivation can lead to a rapid rising burst of signaling H2 O2 , which may lead to PTP inactivation and subsequent enhancement of kinase signaling [4]. Several PTP family members have been shown to be oxidatively inactivated by physiologic stimuli in vivo including the dual lipid protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN), the low-molecular-weight PTPs (LMW-PTPs), and the mitogen activated protein (MAP) kinase phosphatase family members [5]. Such redox-based signaling, arising from a chronic shift in ROS homeostasis that results from metabolic changes or altered antioxidant scavenging, can impact critical components of the malignant phenotype. Tumor cell proliferation, survival, vascular recruitment, and migration are components that drive the malignant phenotype. Below we discuss how alterations in metabolic ROS production can regulate these mechanisms.

2 Mitochondrial ROS and Cell Cycle ROS have been shown to be potent mitogens. Effective detoxification of H2 O2 by catalase inhibits proliferation in Her-2/neu–transformed Rat-1 fibroblasts. Similarly, addition of exogenous H2 O2 at low concentrations and its intracellular production by the noninflammatory NADPH oxidase family members (Nox1-5) is also mitogenic [6]. H2 O2 may directly induce cell proliferation, however, it also acts as a downstream effector of platelet-derived growth factor (PDGF)-induced mitogenic signaling. In Rat-1 cells treated with PDGF, the SH2 domain–containing PTP, SHP-2, can be oxidatively inactivated, resulting in the phosphorylation and activation of MAPK. Both the inactivation of SHP-2 and resulting activation of MAPK is abrogated by treatment of cells with the H2 O2 scavenger NAC, demonstrating a regulatory role of H2 O2 in cell proliferation through the reversible oxidation of PTPs [7]. In this context, antioxidant strategies can attenuate the proliferative capacity of numerous tumor cell lines [8]. H2 O2 detoxification by the use of adenoviral catalase can effectively inhibit DNA synthesis and decrease the proliferation of human aortic endothelial cells. Goswami and co-workers have demonstrated that intracellular H2 O2 is also required for G1-S transit in mouse embryonic fibroblasts. Thus, in contrast with the growth inhibition commonly associated with the bolus addition of H2 O2 , the intracellular production of submicromolar concentrations of H2 O2 is mitogenic and stimulates proliferation, and its effective removal can prevent cell cycle progression. It is likely that the intracellular redox state fluctuates as a result of alterations in production or removal of mitochondrial-derived ROS. Menon and

36

P.M. Carrico et al.

Goswami have proposed that periodic fluctuations in metabolic ROS production likely control cell proliferation via redox-sensitive cell cycle regulatory proteins [9]. The rapid turnover of antioxidants may give rise to acute shifts in cellular oxidant production that can control normal cell cycle progression. The elucidation of oxidant scavenging or oxidant generating enzymes whose levels fluctuate with respect to cell cycle would give credence to this hypothesis. During the process of carcinogenesis, a similar response may occur as a result of the loss of tumor suppressor function or oncogene activation. Thus, the enhanced proliferative capacity of tumor cells may be attributed to chronic shifts in respiratory ROS production due to perturbations in metabolic or antioxidant function.

3 Mitochondrial ROS and Cell Survival H2 O2 has been implicated as a strong inducer of apoptosis in various cell types. Apoptosis or programmed cell death refers to a series of tightly coordinated events designed to eliminate potentially dangerous cells and cells that have reached the end of their life cycle. Apoptosis allows an organism to tightly control cell number and tissue size, as well as to protect itself from cells that interfere with homeostasis. Apoptosis is a tightly regulated process whereby a set of cysteine proteases (caspases) are made active through a complex signaling cascade resulting in degradation of cellular nuclear DNA [10]. In contrast with necrosis, apoptosis destroys only the cell in question with no effect on surrounding cells. Apoptosis can be triggered by both extracellular stimuli as well as intracellular events that mark cellular dysfunction. The hallmarks of apoptosis include membrane blebbing, cytoskeletal disorganization, and loss of mitochondrial integrity and DNA fragmentation. The ultimate step in apoptosis is the activation of caspases, which act on a number of downstream cellular proteins inactivating them by catalytic cleavage at an Asp-Xxx sequence. The pro/inactive forms of these enzymes are activated via cleavage by other upstream caspases or via autoactivation. Caspases are involved in the degradation of important cellular proteins as well as activation of caspase-activated DNAses that cleave mitochondrial DNA, ultimately leading to cell death. Neoplastic cells are able to evade apoptosis and circumvent cell death, an essential survival step during tumorigenesis. TNF-␣ is produced by cells under stress and induces signaling via binding to its cell surface receptor. TNF-␣ along with cycloheximide (CHX) has been established as a potent inducer of apoptosis in various tumor cell lines. One of the primary mechanisms of TNF-␣/CHX–induced apoptosis involves activation of the receptorassociated death domains that recruit and activate caspase-8, leading to activation of downstream caspases and finally of the executioner caspase, caspase-3. In addition, CHX enhances the induction of apoptosis via TNF-␣ by inhibition of new protein synthesis. One of the proteins that was first discovered to be essential for protection from TNF-mediated cell death is the mitochondrial manganese containing superoxide dismutase (Sod2) [11]. Since this initial observation by Wong and

Respiratory Control of Redox Signaling and Cancer

37

Goeddel, numerous reports have demonstrated a role for Sod2 in protection from many apoptotic stimuli. The antiapoptotic effects of Sod2 suggest that either the removal of O2 -. or the generation of H2 O2 via Sod2 may have an inhibitory effect on programmed cell death. The mitochondrial generation of H2 O2 has been shown to confer resistance to apoptotic stimuli. Procaspases contain an active site cysteine that is susceptible to oxidation thereby preventing their activation via proteolytic cleavage. This may explain the ability of oxidants in some studies to deter apoptotic cell death. Cederbaum and Bai have demonstrated that catalase overexpression in HepG2 hepatocarcinoma cells increases their susceptibility to TNF-␣–induced apoptosis. Fas-induced apoptotic cell death can be enhanced by overexpression of mitochondrial-targeted catalase. The antioxidant flavonoid and terpenoid compounds derived from the Ginkgo biloba plant have been shown to enhance the expression of proapoptotic molecules as a result of its ability to scavenge H2 O2 in distinct cancer cell types. Recent studies from this lab indicate that efficient and mitochondrial-targeted H2 O2 detoxification can reverse the antiapoptotic properties of Sod2 overexpression [12]. Another mechanism whereby H2 O2 may augment cell survival is via oxidative inactivation of the LMW-PTPs. LMW-PTP activity is dependent upon the tyrosine phosphorylation on Tyr131 and Tyr132; phosphorylation on Tyr131 results in a 25fold increase in enzymatic activity, and phosphorylation of Tyr132 serves as a scaffold for the SH2 domain of the Grb2 adaptor protein. In PDGF-treated NIH-3T3 or human prostatic carcinoma PC3 cells, inactivation of LMW-PTP by glucose oxidase (G/O)-induced oxidative stress inhibits LMW-PTP auto-dephosphorylation on Tyr131 and Tyr132, thus enhancing the recruitment of Grb2 to LMW-PTP and, subsequently, ERK activation and cell survival [13]. Thus, even though it has been observed that bolus additions of H2 O2 induce apoptosis, these studies have established that submicromolar concentrations of mitochondrial-derived H2 O2 may provide a selective inhibition of programmed cell death and that effective H2 O2 detoxification may also sensitize cancer cells to apoptotic stimuli.

4 Mitochondrial ROS and Angiogenesis H2 O2 has been shown to regulate proangiogenic responses in a variety of systems including human retinal pigment epithelial cells, cultured keratinocytes, and bovine pulmonary artery endothelial cells. Monte et al. have demonstrated that H2 O2 contributes to promoting the angiogenic activity of tumor-bearing lymphocytes and that this activity can be inhibited by coadministration of H2 O2 -detoxifying enzyme catalase but not superoxide dismutase. In vitro angiogenesis of bovine thoracic aorta can be induced with relatively low concentrations (1 ␮M) of H2 O2 and blocked by catalase. The discovery of a noninflammatory NADPH oxidase (Nox1-5) has provided an additional source for the production of O2 -. and H2 O2 in a variety of tumor cell

38

P.M. Carrico et al.

types [14]. The Nox1-dependent generation of H2 O2 is a potent trigger of angiogenesis, increasing the vascularity of tumors and inducing molecular markers of angiogenesis including vascular endothelial growth factor (VEGF), VEGF receptors, and matrix metalloproteinase (MMP) activity in cultured cells and in tumors. Coexpression of catalase blocks the increased activity of the angiogenic markers, indicating that H2 O2 signals part of the switch to the angiogenic phenotype. Furthermore, MMPs are important contributors to the angiogenic switch, and their expression is redox-responsive [15]. The transcription factor Ets-1 is also an important regulator of angiogenesis and is sensitive to H2 O2 production [15]. The role of Ets-1 family members in tumorigenesis is largely due to their ability to activate the transcription of a number of genes involved in matrix degradation, such as MMP-1, 2, 3, and 9 as well as urokinase plasminogen activator. These proteases contribute to tumor invasion and the early phases of blood vessel formation [16]. In cisplatin-resistant ovarian carcinoma variants, elevations in H2 O2 are attributable to alterations of mitochondrial function relative to parental cell lines leading to an increase in transcription of Ets-1. The increase in transcription enhances binding of Nrf2, a key transcription factor in the cellular response to oxidative stress, to an antioxidant response element (ARE) in the promoter of Ets-1. Ets-1 expression has been correlated with poor prognosis in breast and ovarian cancer. Furthermore, the development of cisplatin resistance is a major limitation of this chemotherapy regime, which is widely used in the treatment of testicular and ovarian cancer. These observations suggest that shifts in the steadystate production of H2 O2 in response to receptor engagement or altered mitochondrial function resulting from the development of cisplatin resistance can enhance the expression of the angiogenic transcription factor Ets-1. VEGF is a key mediator in regulating the vascular recruitment of tumor endothelial cells. The transcriptional activity of VEGF is directly responsive to H2 O2 and involves a GC-rich region of the promoter between −95 and −51. Detailed characterization of the promoter has identified two SP1 and SP3 sites at −73/−66 and −58/−52 that represent the core mechanism of oxidative stress–triggered VEGF transactivation. Connor et al. have also established that mitochondrial-derived H2 O2 resulting from the dismuting activity of Sod2 was responsible for enhanced VEGFdependent angiogenic activity. The mechanism involved the H2 O2 -dependent oxidative inactivation of the dual lipid-protein phosphatase PTEN, leading to increase in activity of the serine/threonine-specific protein kinase AKT and subsequent VEGF expression [17]. Hypoxia inducible factor 1␣ (HIF-1␣), the master regulator of cellular responses to hypoxia, responds to shifts in the metabolic production of ROS [18]. HIF-1␣ regulates the transcriptional activity of genes that participate in angiogenesis, iron metabolism, glucose metabolism, and cell proliferation/survival. The regulation of HIF-1␣ in response to hypoxia is controlled by its hydroxylation state. Hydroxylation is controlled by a family of prolyl hydroxylases (PHDs) that require molecular oxygen for full activity. In the presence of oxygen, PHD activity is maximal, leading to HIF-1␣ hydroxylation. Hydroxylated HIF-1␣ is then targeted for polyubiquitination leading to its degradation by the cellular proteasome complex. In the absence of

Respiratory Control of Redox Signaling and Cancer

39

O2 , hydroxylation is inhibited, leading to HIF-1␣ stabilization and activation of its target genes. Studies have determined that efficient mitochondrial ROS scavenging can prevent the hypoxic induction of HIF-1␣. This ROS-dependent stabilization of HIF-1␣ involves mitochondrial complex III–dependent production of superoxide and subsequent inhibition of PHD activity. The mechanisms for how mitochondrial ROS regulate HIF-1␣ are not clear but may involve a shift in the oxidation state of PHD-bound iron. It also possible that the hypoxic production of ROS may lead to the oxidative inactivation of phosphatases, such as PTEN, that contribute to the maximal activation of VEGF. Thus, it appears that angiogenic signaling is particularly sensitive to alterations in the metabolic production of oxidants and that mitochondrial free radicals may play a role in preserving levels of their initial substrate, oxygen, via HIF-1␣.

5 Mitochondrial ROS and Invasion/Migration A hallmark of malignancy and a primary cause of morbidity and mortality in cancer patients is metastasis. Tumor metastasis is a multistep process initiated by migration and invasion of cells into the surrounding vasculature. This is followed by extravasation from the circulation and proliferation at a secondary site where the formation of a metastatic lesion occurs. A key process in the initial movement and subsequent invasion of neoplastic cells into the circulatory system is the remodeling of the extracellular matrix (ECM). Emerging evidence has implicated ROS and the activation of redox-sensitive signaling pathways in invasion and migration. Intrinsic antioxidant enzymes are vital to the regulation of oxidative stress within cells. In vitro studies have shown that a number of cancer cell lines contain elevated levels of Sod2 and decreased levels of catalase and that this change in steady-state levels of H2 O2 correlates with increased metastasis, proliferation, and resistance to apoptosis. An integral part of migration and adhesion is the dynamic rearrangement of focal adhesion complexes in the cell. The role of Rho GTP-binding proteins (Rho, Rac, and Cdc42) in regulating cytoskeletal dynamics by activation of Src and focal adhesion kinase (FAK) and initiating remodeling of integrins has been firmly established. In adherent cells, mature focal adhesions have been shown to be regulated by RhoA, whose activation results in actin stress fiber formation. Rac and Cdc42 are considered to be promigratory resulting in the formation of focal complexes of lamellipodia and filopodia, respectively. It has been shown that ROS modulate Rac and Rho signaling. Rac can specifically downregulate RhoA in a ROS-dependent manner through oxidative inactivation of LMW-PTP, which normally dephosphorylates and inactivates the Rho GTPase-activating protein, p190Rho-GAP. Activation of p190Rho-GAP results in the conversion of the GTP-bound to the GDP-bound form of Rho, and its inactivation contributes to a loss of adhesion and a transition to a migratory phenotype. While ROS appear to disrupt the formation of mature focal adhesions of adherent cells, there is evidence that suggests a role of ROS in the turnover of leading edge focal complexes observed in lamellipodia of migrating cells. Rac has been shown to activate NADPH oxidase, which is spatially localized

40

P.M. Carrico et al.

to membrane ruffles of the leading edge of migrating cells, and was shown to be associated with cell migration. In this context, oxidants can reversibly inactivate the protein tyrosine phosphatase PTP-PEST that normally dephosphorylates and inactivates FAK, leading to a more rapid turnover of active FAK at focal complexes. It is also possible that mitochondrial-derived ROS are involved in disrupting mature focal adhesions yet contribute to active remodeling of focal complexes of the lamellipodia required for migration. Lipid signaling molecules also play an important role in regulating the migratory phenotype. Studies in phagocytic cells and the amoeba Dictyostelium discoideum demonstrate that phosphoinositide (3,4,5) 3-phosphate (PtdIns[3,4,5]P3 ) accumulates at the front of chemotaxing cells. Concomitant with the distribution of PtdIns(3,4,5)P3 to the leading edge is a redistribution of PTEN to the remaining outside perimeter of the cell where it degrades PtdIns(3,4,5)P3 by removing the 3phosphate. Alterations in the steady-state levels of H2 O2 can inactivate the tumor suppressor PTEN leading to an augmentation of AKT signaling and redistribution of phosphoinositides at the plasma-lamellar surface [17]. It is intriguing to hypothesize that receptor engagement in response to chemotactic triggers may lead to the local oxidant-dependent inactivation of PTEN and rapid reproportioning of lipid signaling molecules that promote the migratory phenotype. An essential and rate-limiting step in metastasis is the remodeling and degradation of the ECM and basement membrane by proteolytic enzymes. In this model, cells must interact with surrounding stromal cells, leading to the loss of matrix function and resulting in a compromised matrix boundary. MMPs are major contributors of stromal degradation and are vital to the process of cellular invasion. MMP-1/interstitial collagenase has a specificity for type I collagen, the primary collagen of the interstitial matrix; though it has been shown to degrade collagen types II, III, VII, and X. Studies using antisense mRNA against MMP-1 in melanoma cells have shown a significant attenuation in the capacity of these cells to invade type I collagen and Matrigel in vitro. Moreover, the expression of MMP-1 has been shown to be a definitive prognostic marker for breast lesions that will develop into cancer suggesting that the expression and regulation of MMP-1 may be important in malignancy. Studies from this and other laboratories have demonstrated that the Sod2dependent production of H2 O2 leads to increased expression of MMP family members and that there is a strong correlation between this increase in MMP levels and enhanced metastasis. Interestingly, the Sod2-dependent increases in MMP expression can be reversed by the H2 O2 detoxifying enzyme, catalase, or glutathione peroxidase [15]. Thus, H2 O2 -dependent upregulation of MMPs may, in part, contribute to increased invasion and metastatic capacity of tumors displaying elevated Sod2 levels. Maximal mitochondrial H2 O2 -dependent transcription of MMP-1 transcription is achieved by a proximal activator protein 1 (AP-1) site at –1602 and a single nucleotide polymorphism (SNP) guanine insertion at –1607, which creates an Ets-1 binding site [15]. This redox-sensitive MMP-1 protein expression requires activation of both ERK1/2 and JNK pathways. JNK signaling is largely responsible for

Respiratory Control of Redox Signaling and Cancer

41

the H2 O2 sensitivity of the MMP-1 promoter, whereas ERK1/2 contributes to both its basal and H2 O2 dependence [19]. The ability of a tumor cell to migrate and invade through the extracellular matrix is attributed to local shifts in signaling that regulate the distribution of phospholipid signaling molecules, rearrangement of focal adhesion complexes, and matrix degradation. All of these processes are redox-sensitive and are likely responsive to metabolic shifts in the steady-state production of H2 O2 . As many of the processes are highly compartmentalized, it is possible that vicinal mitochondria may participate in these migratory signaling events by augmenting their local production of H2 O2 . With the development of fluorescent reporter molecules that detect focal shifts in the production of many of the migratory signaling intermediates and new redox-sensing mitochondrial targeted GFP constructs, we will be able to determine if these events are juxtaposed.

6 Mitochondrial ROS and Cancer Stem Cells Recently, the hypothesis has emerged that conventional therapies may fail to completely eradicate cancer because cancer stem cells, which are thought to act as cancer-initiating cells, are insensitive to these treatments. Work in leukemia, breast, brain, and lung cancers have provided further understanding of the role cancer stem cells play in malignancies. Although cancer stem cells appear to have some of the phenotypic and functional properties of embryonic stem cells, such as the potential to give rise to a larger population of cells, survival of these two different cell types requires different pathways that are frequently at odds with one another. Recent research indicates that signaling by ROS may regulate growth and differentiation in both hematopoietic stem cells and cancer stem cells. PTEN, like p53 is one of the most common targets for mutation in sporadic human cancers. The oxidative inactivation of PTEN and subsequent activation of the AKT signaling pathway may lead to the transformation of adult hematopoietic stem cells (HSCs) into leukemias. Transgenic mice with conditional deletions of PTEN in adult HSCs develop leukemias within 4 to 6 weeks along with a depletion of normal HSCs. These defects are dependent upon the downstream effector mammalian target of rapamycin (mTOR), a serine/threonine kinase that regulates metabolism and that has also been shown to generate ROS [20]. The oxidative inactivation of PTEN and subsequent activation of the AKT signaling pathway leads to the phosphorylation of the class O of forkhead transcription factors (FoxO), resulting in the retention of FoxO in the cytoplasm through interactions with 14-3-3 proteins [21]. FoxOs, which have also been postulated to act as tumor suppressors, regulate the transcription of genes involved in cell cycle arrest, apoptosis, glucose metabolism, and stress response. In C. elegans, the FoxO ortholog DAF-16 has been shown to affect life span through resistance to oxidative stress and adaptation to food shortage. DAF-16 promotes resistance to oxidative stress by activating transcription of Sod2 and catalase. In mammals, the FoxO

42

P.M. Carrico et al.

subgroup consists of four members (FoxO1, FoxO3, FoxO4, and FoxO6), of which three (FoxO1, FoxO3, FoxO4) overlap in function and expression patterns. Individual deletion of FoxO proteins in mice does not result in a tumor-prone phenotype, however, mice containing conditional deletions of FoxO1, FoxO3, and FoxO4 display a cancer-prone condition characterized by thymic lymphomas and hemangiomas [22]. In addition, mice with conditional deletions of FoxO1, FoxO3, and FoxO4 exhibited premature aging of HSCs, which was shown to be at least partly due to an increase in ROS levels in the HSC compartment and attributable to a decrease in expression of antioxidant genes [23]. Treatment of these mice with NAC restored the HSC compartment size, reestablished stem cell cycling, and normalized HSC apoptosis. FOXO3a is also regulated via p66Shc, which belongs to a family of adaptors that function in signal transduction of mitogenic and apoptotic responses, and whose absence has been shown to prolong life span both in cell culture and mouse models. Although p66Shc is activated through tyrosine phosphorylation in response to extracellular signals such as EGF and insulin, phosphorylation of p66Shc at serine 36 in response to oxidative stress leads to the subsequent phosphorylation and sequestration of FOXO3a in the cytosol [24]. This may further increase levels of ROS as FOXO3a has been shown to activate a reporter under the control of the human catalase promoter [24]. In a hypoxic environment, p66shc functions to promote survival of stem cells and mammary cancer stem cells through the upregulation of Notch-3 and carbonic anhydrase IX (CA-IX) [25]. In contrast, as discussed above, inhibition of FOXO proteins in response to H2 O2 augments apoptosis of stem cells. Recently, p66shc was also shown to regulate mitochondrial metabolism [26]. In immortalized mouse embryonic fibroblasts (MEFs) lacking p66shc, oxygen consumption is reduced 30% to 50% with a coincident increase in glycolysis and decrease in NADH metabolism. In addition, Giorgio et al. [27] demonstrated that p66shc utilizes reducing equivalents of the mitochondrial electron transfer chain through the oxidation of cytochrome c to generate mitochondrial H2 O2, which functions as a signaling molecule for apoptosis.

7 Conclusion The role of ROS in the process of carcinogenesis has evolved from damaging lipid, proteins, and nucleic acids that in turn promote tumor development to regulators of signal transduction events that control key facets of the malignant phenotype. Many of the redox-sensitive signaling proteins drive the expression or activation of genes and proteins that regulate tumor cell proliferation, survival, vascular recruitment, and migration. The source of the ROS that regulate these signaling networks has in many cases been assigned to the ubiquitous family of noninflammatory respiratory burst oxidases. However, emerging data indicates that mitochondrial-derived ROS also play a prominent role in regulating the tumorigenic phenotype. A number of

Respiratory Control of Redox Signaling and Cancer

43

signaling proteins like the dual lipid protein phosphatase PTEN and the transcription factor Ets-1 are sensitive to perturbations in mitochondrial oxidant production and are involved in regulating numerous aspects of the tumorigenic phenotype. H2 O2 dependent activation of Ets-1 leads to enhanced tumor angiogenesis, migration, and invasion. Oxidative inactivation of PTEN leads to an increase in active AKT thereby promoting tumor cell survival, proliferation, and angiogenesis. This implies that alterations in mitochondrial function that enhance respiratory ROS production can control signaling molecules that synergize and potentially drive the malignant phenotype. PTEN contains an active site cysteine that renders it redox-sensitive. Ets-1 contains several cysteines that are essential for its DNA binding, and it is likely that oxidation would lead to its inactivation. The fact that oxidants promote Ets-1 activity suggest some as yet undefined oxidant-sensitive signaling molecule may control the redox-responsiveness of this transcription factor independent of its DNA-binding activity. It is clear that many facets of the tumorigenic phenotype are ROS responsive. The angiogenic factor VEGF is controlled by oxidants through promoter activation, stabilization of transcription factors (HIF-1␣), and activation of upstream signals. Ets-1 activation also contributes to the early phases of blood vessel formation. Thus, angiogenic signals appear to be highly sensitive to shifts in oxidant production and may be highly responsive to shifts in respiratory oxidant production. Further characterization and identification of oxidant-responsive molecular targets that control tumor progression and eradication of cancer stem cells will undoubtedly lead to the development of novel antioxidant-based therapies for the treatment of malignancy.

References 1. Nemoto S, Takeda K, Yu ZX, Ferrans VJ, Finkel T. Role for mitochondrial oxidants as regulators of cellular metabolism. Mol Cell Biol 2000; 20(19):7311–7318. 2. Rhee SG, Chang TS, Bae YS, Lee SR, Kang SW. Cellular regulation by hydrogen peroxide. J Am Soc Nephrol 2003; 14(8 Suppl 3):S211–S215. 3. Wood ZA, Poole LB, Karplus PA. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science 2003; 300(5619):650. 4. Poole LB. Bacterial defenses against oxidants: mechanistic features of cysteine-based peroxidases and their flavoprotein reductases. Arch Biochem Biophys 2005; 433(1):240–254. 5. Tonks NK. Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol 2006; 7(11):833–846. 6. Arnold RS, Shi J, Murad E, et al. Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1. Proc Natl Acad Sci USA 2001; 98(10): 5550–5555. 7. Tonks NK. Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol 2006; 7(11):833–846. 8. Floyd RA. Antioxidants, oxidative stress, and degenerative neurological disorders. Proc Soc Exp Biol Med 1999; 222(3):236–245. 9. Menon SG, Goswami PC. A redox cycle within the cell cycle: ring in the old with the new. Oncogene 2007; 26(8):1101–1109. 10. Adams JM. Ways of dying: multiple pathways to apoptosis 2. Genes Dev 2003; 17(20): 2481–2495.

44

P.M. Carrico et al.

11. Wong GHW, Goeddel DV. Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science 1988; 242:941–944. 12. Dasgupta J, Subbaram S, Connor KM, et al. Manganese superoxide dismutase protects from TNF-alpha-induced apoptosis by increasing the steady-state production of H2 O2 . Antioxid Redox Signal 2006; 8(7–8):1295–1305. 13. Chiarugi P, Buricchi F. Protein tyrosine phosphorylation and reversible oxidation: two crosstalking posttranslation modifications. Antioxid Redox Signal 2007; 9(1):1–24. 14. Lambeth JD, Cheng G, Arnold RS, Edens WA. Novel homologs of gp91phox. Trends Biochem Sci 2000; 25(10):459–461. 15. Nelson KK, Melendez JA. Mitochondrial redox control of matrix metalloproteinases. Free Radic Biol Med 2004; 37(6):768–784. 16. Lincoln DW, Bove K. The transcription factor Ets-1 in breast cancer. Front Biosci 2005; 10:506–511. 17. Connor KM, Subbaram S, Regan KJ, et al. Mitochondrial H2 O2 regulates the angiogenic phenotype via PTEN oxidation. J Biol Chem 2005; 280(17):16916–16924. 18. Brunelle JK, Bell EL, Quesada NM, et al. Oxygen sensing requires mitochondrial ROS but not oxidative phosphorylation. Cell Metab 2005; 1(6):409–414. 19. Nelson KK, Ranganathan AC, Mansouri J, et al. Elevated sod2 activity augments matrix metalloproteinase expression: evidence for the involvement of endogenous hydrogen peroxide in regulating metastasis. Clin Cancer Res 2003; 9(1):424–432. 20. Kim JH, Chu SC, Gramlich JL, et al. Activation of the PI3K/mTOR pathway by BCRABL contributes to increased production of reactive oxygen species. Blood 2005; 105(4): 1717–1723. 21. Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999; 96(6):857–868. 22. Paik JH, Kollipara R, Chu G, et al. FoxOs are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell 2007; 128(2):309–323. 23. Tothova Z, Kollipara R, Huntly BJ, et al. FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress. Cell 2007; 128(2):325–339. 24. Nemoto S, Finkel T. Redox regulation of forkhead proteins through a p66shc-dependent signaling pathway. Science 2002; 295(5564):2450–2452. 25. Sansone P, Storci G, Giovannini C, et al. p66Shc/Notch-3 interplay controls self-renewal and hypoxia survival in human stem/progenitor cells of the mammary gland expanded in vitro as mammospheres. Stem Cells 2007; 25(3):807–815. 26. Nemoto S, Combs CA, French S, et al. The mammalian longevity-associated gene product p66shc regulates mitochondrial metabolism. J Biol Chem 2006; 281(15):10555–10560. 27. Giorgio M, Migliaccio E, Orsini F, et al. Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis. Cell 2005; 122(2):221–233.

Cellular Respiration and Dedifferentiation Roberto Scatena, Patrizia Bottoni, and Bruno Giardina

Abstract Mitochondria are far more than the “powerhouse” of the cell as they have classically been described. They are also the location where various catabolic and anabolic processes, calcium fluxes, reactive oxygen and nitrogen species, and numerous signal transduction pathways interact to maintain cell homeostasis and to modulate cellular responses to diverse stimuli. Because of the mitochondrion’s vital roles in cell physiology, this organelle squarely falls within the ambit of cancer pathophysiology. Indeed cancer-associated alterations in cell energy metabolism related to mitochondrial dysfunction have been recognized for many years in the socalled Warburg effect. Furthermore, these organelles appear to play a fundamental role in determining whether a cell will undergo cell death by apoptosis or necrosis; and key metabolic enzymes of the tricarboxylic acid (TCA) cycle, the fundamental oxidative pathway of the cell, may act as oncosuppressors. Recent findings indicate that modulation of mitochondrial activities in general, and of its electron respiratory chain in particular, could induce differentiation and/or death of cancer cells. Thus it may be possible to induce anticancer activities by deliberate modulation of cellular respiration. Elucidation of the role of mitochondria in cancer cell dedifferentiation processes may provide a new definition of the dedifferentiation/differentiation of cancer cells as well as important information about the pathophysiology of so-called stem cells, with important diagnostic, prognostic, and therapeutic consequences. Keywords Cancer cell differentiation · Oxidative phosphorylation · NADH dehydrogenase · Heat shock proteins · Reactive oxygen species · Warburg effect · Nitric oxide · Oncogenes · Oncosuppressors · Biomarkers

R. Scatena (B) Dipartimento di Medicina di Laboratorio, Universit`a Cattolica del Sacro Cuore, 0168 Rome, Italy e-mail: [email protected] S.P. Apte, R. Sarangarajan (eds.), Cellular Respiration and Carcinogenesis, DOI 10.1007/978-1-59745-435-3 4,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

45

46

R. Scatena et al.

1 Introduction Cancer can be defined as a genetic disease characterized by dysregulation of various cellular pathways that orchestrate cell proliferation, differentiation, and death [1]. A cell becomes cancerous when a series of oncogenes and/or oncosuppressors become dysfunctional and hence induce a neoplastic phenotype in that cell. In this dramatic clonal disorder, the cell acquires high proliferation kinetics and loses cell-cell contact inhibition. These properties are progressively achieved by a series of genetic “hits,” including mutations and or other epigenetic modifications [1]. The final result is often defined as dedifferentiation of a neoplastic cell that morphologically becomes anaplastic. The grade of dedifferentiation (anaplasia) differs among cancer cell specimens and is often correlated with the malignancy of the neoplasia. In the dramatic perturbation of cell homeostasis that accompanies carcinogenesis, mitochondria are involved in various relevant, interconnected, but also indirect pathophysiologic roles. In this context, the significance of mitochondrial respiration has received inadequate attention. In spite of an abundance of data that link mitochondria to cancer, there have been few studies that have investigated the interrelationships between cellular respiration and cellular dedifferentiation. Here we briefly summarize some molecular mechanisms, which will be extensively considered in other chapters, that link cancer cell dedifferentiation to mitochondrial oxidative metabolism to provide an introduction to concepts that facilitate the understanding of these aspects of oncology:

1. Peculiar modification of cell metabolism (Warburg effect). Cancer cells generally produce ATP mainly through so-called aerobic glycolysis, a metabolic shift characterized by high glucose uptake and increased production of lactate. Originally, this altered metabolic state was considered to be the result of adaptation (via HIF and/or AKT) to the new potentially anoxic environment of a neoplastic lesion [2]. However it has become evident that the aerobic glycolysis of cancer cells is an epiphenomenon that results from a more complex metabolic rearrangement in which not only glycolysis but also Krebs cycle, beta-oxidation, and anabolic metabolism in general are altered to support the new primary function of the cell (e.g., uncontrolled proliferation) by providing not only energy but also the building blocks for the synthesis of nucleic acids, peptides, and lipids [3–5]. 2. Distinct properties of cancer cell mitochondria. Emerging data suggest that mitochondria have an active pathogenic role in the induction and maintenance of the Warburg effect. This role is emphasized by some typical metabolic peculiarities of mitochondria that enable cancer cells to be functionally distinguished from normal cells. Cancer cells exhibit reduced mitochondrial respiration (both in terms of ATP turnover and proton leakage), increased NADH/NAD+ ratios, and a pronounced increase in membrane potential [6]. Other morphologic (number, size, shape) and structural differences (phospholipids, cholesterol, and

Cellular Respiration and Dedifferentiation

3.

4.

5.

6.

47

membrane proteins) have been described, but their pathogenic roles are not understood [7]. Mitochondrial DNA (mtDNA) mutations. Mutations of mtDNA have been linked to some tumors. In particular, intragenic deletions, missense and chainterminating point mutations, and alterations of homopolymeric sequences have been described in various sporadic tumors. Intriguingly, mutations have been described in the hypervariable regions of the mitochondrial D-loop, the section of DNA that controls mtDNA transcription and replication. Mutations have also been described in all tRNAs, in rRNAs, and in all 13 of the mtDNA-encoded subunits of the electron respiratory chain [8]. However, Salas et al. [9] recently suggested that most of the reported mtDNA mutations in tumors may be due to laboratory-induced artifacts. Others contend that mtDNA mutations play a real pathogenetic role in cancer [10]. Hence there is ongoing debate about the role of mtDNA mutations and the consequent respiratory dysfunction in cancer cells. Nuclear DNA mutations and mitochondrial proteins. Succinate dehydrogenase (SDH) and fumarate hydratase (FH), mitochondrial enzymes of the tricarboxylic acid (TCA) cycle, can act as classic tumor suppressors, and mutations of the genes that encode them have been associated with various benign and malignant tumors. These observations have drawn attention to the potential pathogenetic role of the mitochondrion, especially of mitochondrial oxidative metabolism, in cancer [11, 12]. Given the role of SDH (complex II) in the electron respiratory chain, such findings suggest that there may be critical interrelationships between mitochondrial energy metabolism and timely surveillance of the differentiation/dedifferentiation state of cells. Apoptosis. Mitochondria have a well-defined role in caspase-mediated induction of the intrinsic pathway of apoptosis initiated by the release of cytochrome c and second mitochondrial activator of caspases (SMAC) from the mitochondrial intermembrane space in response to a variety of noxious stimuli, including DNA damage, loss of adherence to the extracellular matrix (ECM), oncogeneinduced proliferation, and growth factor deprivation. Perturbation in the release of apoptosis-inducing proteins regulated by proapoptotic and antiapoptotic members of the Bcl-2 family (e.g., Bcl-2, Bcl-XL, and Mcl-1) seems to influence not only etiopathogenesis but also cancer prognosis and therapeutic efficacy. Accordingly, derangements of the permeability transition pore (PTP) and electrochemical gradient, which strictly depend on mitochondrial respiration, have been implicated in these phenomena [13]. Free radicals (reactive oxygen species [ROS] and reactive nitrogen species [RNS]). Mitochondria are the main source of free radicals in cells. Thus mitochondrial dysfunction, especially at the level of the electron respiratory chain, can result in increased production of ROS and RNS. ROS are considered classic carcinogens because they can directly induce mutagenesis and thereby promote tumorigenesis and cancer progression [14]. However, the role of ROS and RNS in cancer is complex. In fact, the growth-promoting effects of ROS have been also related to activation of redox-responsive signaling cascades (MAPK, PKC,

48

R. Scatena et al.

AP-1, NF-␬B). Paradoxically, ROS can also act as tumor suppressors, activating via a p16ink4a -Rb pathway an irreversible cellular process of senescence that establishes stable G1 phase cell-cycle arrest [15]. 7. Heat shock proteins (HSPs). HSPs represent a ubiquitous and evolutionarily conserved family of proteins (classified according to their molecular weight) that were originally discovered because they can be induced by heat stress. Their production confers a peculiar cellular resistance to further stresses (heat or chemical). Initially, these proteins were considered to be chaperones—molecular guides acting to ensure proper protein folding. Other functions have since been discovered, including roles in gene expression regulation, DNA replication, signal transduction, cell differentiation, metastasis, apoptosis, senescence, immortalization, and, intriguingly, multidrug resistance development. However, the precise pathophysiologic roles of HSPs in general, and of mitochondrial Heat Shock Proteins (mitHSPs) in particular, are not well understood. Studies employing several methodological approaches (immunochemical, immunohistochemical, proteomic) have shown that HSP expression gradually increases from normal to dysplastic and then to neoplastic tissues, such that HSP overexpression correlates with cancer evolution. Hence some of these proteins (i.e., HSP60, HSP10, HSP70, PHB1, and PHB2) have been implicated in cancer diagnosis, prognosis, and therapy [16]. Overexpression of HSP70 (also known as 75-kDa glucose regulated protein, HSP70-9, HSP70-9B, mortalin 2) may be particularly important in cancer cell proliferation and dedifferentiation; its role may go beyond generic cytoprotection with respect to apoptosis and involve a more specific role in the pathogenesis of the peculiar cellular metabolic shift that characterizes the cancer cell phenotype [17]. 8. Hexokinase II. The cancer cell glycolytic phenotype is supported by overexpression of the hexokinase II isoenzyme, which is linked to the outer mitochondrial membrane via the porin-like protein voltage-dependent anion channel (PPVAC). Hexokinase II-PPVAC binding may have significant functional implications in cancer because it facilitates glucose phosphorylation and thus glucose utilization for energetic and biosynthetic purposes [18]. 9. Calcium. Because of the pivotal roles mediated by calcium ions in signal transduction pathways, calcium has been considered to be a potential physiopathogenetic marker in several chronic and acute cellular diseases. However, research findings have not confirmed the originally grand expectations for calcium in disease processes, at least in oncology. When Scorrano et al. [19] examined the interrelationships between calcium, mitochondrial respiration, and dedifferentiation, they found only an indirect role of Bax/Bak via regulation of calcium release from the endoplasmic reticulum. Interestingly, however, chemotherapeutic-resistant tumor cells sequester cytosolic calcium (Cacyt ) more efficiently than do normal cells; they show reduced release of calcium ions from intracellular storage sites upon apoptosis induction [20]. It has been proposed that transition to more invasive cellular behavior may be related to impaired release of calcium from the endoplasmic reticulum of various kinds of cancer cells. On the contrary, Amuthan et al. [21] showed in tumorigenic

Cellular Respiration and Dedifferentiation

49

human pulmonary carcinoma A549 cells that mitochondrial stress induced by damaging mtDNA (with ethidium bromide treatment) activates a mitochondria to nucleus signaling pathway mediated by increased Cacyt concentration and that activation of this pathway induces cell dedifferentiation associated with phenotypic changes and cell invasion.

2 Cellular Respiration and Dedifferentiation Although the connection between mitochondrial respiration and cancer cell dedifferentiation (i.e., resistance to apoptosis, cell proliferation, metabolic shift, cell invasion, genetic instability) is fundamental to cancer pathogenesis, the precise molecular interrelationships and regulatory processes are far from clarified. This lack of understanding is related, at least in part, to the traditional view that the socalled Warburg effect was essentially a reflection of mitochondria being damaged and dysfunctional organelles in cancer cells. Recent studies are reexamining mitochondrial function, especially cellular respiration, in cancer. However, a consensus among the findings has yet to be achieved [6, 21–25]. Herein some important findings linking cellular respiration to cell dedifferentiation in cancer are provided. It is worth noting that this putative link may have crucial pharmacologic and clinical implications. The work of Amuthan et al. [21] is particularly relevant in this regard because the findings provide mechanistic insight into how cancer progression and tumor invasion may be directly related to chemical damage of mtDNA that results in mitochondrial respiratory chain dysfunction, loss of mitochondrial membrane potential, and reduction of ATP synthesis. This mitochondrial derangement also causes calcium release into the cytoplasm, which activates a cell dedifferentiation program. Amuthan et al. [21] showed in C2C12 myoblasts and in human pulmonary carcinoma A549 cells that thus-induced dedifferentiation programs can transform cells into a more aggressive phenotype with the following properties: • induction of hexokinase, the enzyme that primes glucose by consuming ATP; • induction of phosphoenolpyruvate carboxykinase (PEPCK), which regulates gluconeogenesis, glyceroneogenesis, and many other anaplerotic reactions; • induction of tumor invasion markers (i.e., cathepsin L, TGF-␤1, ERK1, ERK2, calcineurin) and antiapoptotic proteins (Bcl2 and Bcl-XL ); • reduction of levels of the proapoptotic proteins Bid and Bax; • reduction of cell growth and occurrence of morphologic changes. In summary, the findings of Amuthan et al. [21] demonstrate that, at least in this set of experimental conditions, a generic mitochondrial stress caused by mtDNA depletion deranges cellular respiration and alters patterns of nuclear gene expression and thereby induces tumorigenic properties.

50

R. Scatena et al.

In general, it is well-known that mtDNA depletion can also render cells more sensitive to apoptotic stimuli. Amuthan et al. [21] explain that the outcome of the stress (cell dedifferentiation vs. apoptosis) could be related to the relative resistance to apoptosis and/or to the capacity of the adopted cellular models to buffer efficiently the secondary metabolic stresses. Furthermore, evidence obtained from this combination of methodological approaches demonstrated the existence of mitochondriato-nucleus stress signaling, through which alterations in cellular respiration have the ability to change nuclear gene expression and selectively promote the growth of more aggressive clones. These results compel us to consider other research demonstrating that mitochondria can mediate the proliferative or inhibitory actions of different physiologic or pharmacologic molecules that influence cellular respiration. Interestingly, it has been reported recently that some “nongenomic” effects of estrogens, that is; effects mediated by mitochondria rather than estrogen receptors, seem to modulate the expression of nuclear cell cycle genes and human breast tumor growth. Estrogens cause increases in the transcription of various complex subunits of the electron respiratory chain in mitochondria. For example, estradiol treatment (0.5 nM for 6 days) is associated with a 16-fold increase in cytochrome oxidase II (COII) mRNA in rat pituitary tumor cells. Similarly, ethinyl estradiol treatment (0.5 to 10 ␮M for 40 hours) caused a two- to threefold increase in cytochrome oxidase I (COI), COII, and NADH dehydrogenase subunit I. Estrogens seem to negatively affect mitochondria at the protein level as well. Several studies have shown that estrogens can inhibit mitochondrial respiratory complexes I, II, III, IV, and V. This perturbation of the electron respiratory chain can induce mitochondrial ROS production, which could promote the generation and progression of some cancers via oxidative stress and/or ROS-related signaling pathways [23]. Given the suggested role of estrogens in some cancers (i.e., breast cancer) and the discussed pathogenetic mechanisms at the basis of oncopromotion and oncoprogression activity, further evidence should be gathered to determine the relevance of these findings. The ability of lipophilic molecules to derange cellular respiration and alter cell differentiation/dedifferentiation processes is a neglected pathophysiologic facet of the mitochondrial respiratory chain. On the basis of the cytopathology of liver disease associated with use of fibrates (lipid-reducing drugs) and some experimental data indicating that fibrate binding to human hemoglobin can functionally modify the hemoprotein, we postulated that fibrates, and their thiazolidinedione derivatives, could interact with many hydrophobic protein domains and especially with complex I subunits of the mitochondrial electron respiratory chain. We found that this molecular interaction is rapid (<1 hour from administration) and occurs in numerous cell types in varying degrees. The following human cell lines showed derangement of varying degrees of complex I and cell differentiation: K-562 human erythroleukemia, HL-60 human myeloid leukemia, U-937 human monocytic leukemia, TE-671 human rhabdomyosarcoma, HepG2 human hepatocarcinoma, and SK-N-BE[2] human neuroblastoma [22, 24, 25]. In fact, these molecules, by their lipophilic properties, diffuse freely into lipid bilayers in general and in mitochondria in particular also because of interplay

Cellular Respiration and Dedifferentiation

51

between their logD and mitochondrial electrochemical gradient, altering in such a way the function of proteins embedded in membranes. One of the larger protein components of mitochondria membranes is complex I (NADH-ubiquinone oxidoreductase, subunits >30, MW 850 kDa), which because of these features may act as the main lipophilic molecular sink in mitochondria [26]. The rapid iatrogenic respiratory derangement caused by introduction of these lipophilic molecules can induce metabolic and ROS-mediated stress that provokes a differentiation process, also mitochondrial HSP-mediated, in human tumor cell lines. In particular, the derangement of mitochondrial complex I may induce ROS production and may impair NADH oxidation resulting in an energetic shortage in cancer cells. Importantly, considering that complex I is only partially deranged, the oxidative and energetic stress are in general not lethal. In this condition, cell growth is hampered and phenotypic differentiated features may reappear. This implies that there is modification of the actual differentiation/dedifferentiation definition and thus suggests that a redefinition of the role of some oncogenes and oncosuppressors is appropriate. Data confirming that fibrates and thiazolidinediones inhibit, via nongenomic activity, complex I–impairing NADH oxidation at the mitochondrial level can explain some of the pharmacologic activities of this class of drugs (i.e., antihyperlipidemia, insulin sensitization). The findings also may account for some adverse side effects of fibrates and thiazolidinediones, including myocardiotoxicity, hepatotoxicity, and, most relevant to this discussion, peroxisome proliferation, carcinogenicity, and cancer differentiating properties, considered typical peroxisome proliferator activated receptor (PPAR)-␣–mediated effects in rodents [27]. The differentiating activity of fibrates and thiazolidinediones on human tumor cell lines has been shown to be tightly correlated with the level of mitochondrial complex I derangement. This differentiating activity is independent of the expression level of PPARs, as well as the embryonic origin of the tissue. Stress-related differentiation in human tumor cell lines, including some human leukemia cell lines, is a well-known occurrence [28]. Fibrate/thiazolidinedione-induced differentiation is unusual; it is linked to the metabolic status of differentiated cells that shift toward a more glycolytic metabolism (paradoxical Warburg effect) and thus showing that original neoplastic phenotype yet used mitochondria. Moreover, metabolic parameters recorded during drug-induced cell differentiation (residual activity of the mitochondrial electron respiratory chain components glycerol 3-phosphate dehydrogenase and succinate dehydrogenase; levels of glycolytic and nonglycolytic acetate, pyruvate, and alanine) suggest that the so-called Warburg effect is really an epiphenomenon of a more complex metabolic rearrangement of the neoplastic cell. This cancer-associated metabolic rearrangement is induced by the cancer’s rigid genetic selection program, characterized by a high level of cell proliferation and diffusion, which is normally present only during embryonic and fetal development. Our understanding of the functional connections between alterations in cell metabolism and mitochondrial physiology in cancer is under profound revision. And all mitochondrial physiology is strongly affected by electron respiratory chain activities. The recent results of Fantin et al. [6] are consistent with the view that tumor

52

R. Scatena et al.

progression is strongly associated with the so-called Warburg effect and mitochondrial metabolism. These authors showed that attenuation of lactate dehydrogenase A (LDH-A) expression by LDH-A short hairpin RNAs in tumor cell lines reactivates mitochondrial respiration, in terms of proton leakage and ATP turnover, and restores the normally depolarized state of mitochondrial membranes. In this situation, cancer cells can no longer oxidize NADH via LDH but must rely on the mitochondrial respiratory chain. Interestingly, this abruptly induced change in metabolism inhibited cell growth. The authors concluded that these results demonstrate a fundamental role of aerobic glycolysis in general and LDH in particular in tumor maintenance and progression and further show that cancer-associated slowdown of mitochondrial respiration is not due to structural damage of the organelles but rather results from altered metabolic signals. NADH/NAD+ ratio and modulation of related mitochondrial shuttle systems are likely involved in these signals. The fundamental role of cellular respiration in cancer cell dedifferentiation is further emphasized by a recent study that examined energy metabolism in breast cancer brain metastasis, which undoubtedly represents a clear in vivo model of bioenergetic adaptation of dedifferentiated cells [29]. Interestingly, these metastatic cells showed increased expression of enzymes involved in glycolysis, in accordance with the Warburg effect, but also showed increased expression of enzymes involved in the TCA cycle, pentose phosphate pathways, beta-oxidation, and oxidative phosphorylation. Intriguingly, the authors also reported induction of enzymatic pathways related to glutathione synthesis, which together with activation of the pentose phosphate pathway could indicate the presence of antioxidant defense mechanisms to minimize production of ROS derived from enhanced oxidative metabolism [29].

3 Conclusion Emerging evidence, despite conflicts likely due to experimental differences, is shedding light on the interrelationships between cellular respiration and cancer cell dedifferentiation, in particular: • Confirms a more active role of mitochondrial respiration to maintain the cellular differentiated phenotype and at the same time underline the pathogenetic significance of perturbations of mitochondrial electron respiratory chain in promotion and progression of cancer. • Stresses the importance of the shift of all cellular metabolism to ensure the new energetic and structural needs of neoplastic cell. • Justifies some debated metabolic and functional evidence peculiar to cancer cell (i.e., induction of hexokinase II and its binding to ANT). • Enhances the role of NADH and NADH/NAD+ ratio in modulation of a signal transduction pathway that controls differentiation/dedifferentiation status. • Emphasizes the interplay of complex I and complex II to adapt cell metabolism to different cellular physiopathologic conditions.

Cellular Respiration and Dedifferentiation

53

• Last but not least, addresses the new research, which explores the interrelationships between cellular respiration and cell dedifferentiation, toward the regulatory function of complex I, complex II, and above all pushes to investigate deeper, in terms of cancer cell metabolism adaptions, the other pathways of electron transport (i.e., alpha glycerophosphate and fatty acyl CoA).

In conclusion, it is clear that there exists a significant link between cellular respiration and cell differentiation/dedifferentiation. Elucidating the molecular mechanisms at the basis of this link may have critical clinical implications in terms of cancer diagnosis, prognosis, and, most importantly, therapy. Disentangling the relationship between cancer cell oxidative metabolism and cellular differentiation state is fundamental not only for oncology but also for properly understanding and using all potential clinical applications related to the physiopathology of stem cells.

References 1. Bale AE, Brown SJ. Etiology of cancer: cancer genetics. In: De Vita VT, Hellman S, Rosenbreg SA, eds. Cancer—Principles & practice of oncology, 6th ed. Philadelphia: Lippincot Williams & Wilkins, 2001:170–183. 2. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer 2004; 4:891–899. 3. Moreno-Sanchez R, Rodriguez-Enriquez S, Marin-Hernandez A, Saavedra E. Energy metabolism in tumor cells. FEBS J 2007; 274:1393–1418. 4. Kim JW, Dang CV. Cancer’s molecular sweet tooth and the Warburg effect. Cancer Res 2007; 66:8927–8930. 5. Garber K. Energy deregulation: licensing tumor to grow. Science 2006; 312:1158–1159. 6. Fantin VR, St-Pierre J, Leder P. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell. 2006; 9:425–434. 7. Modica-Napolitano JS, Singh KK. Mitochondrial dysfunction in cancer. Mitochondrion 2004; 4:755–762. 8. Brandon M, Baldi P, Wallace DC. Mitochondrial mutations in cancer. Oncogene 2006 7; 25:4647–4662. 9. Salas A, Yao YG, Macaulay V, Vega A, Carracedo A, Bandelt HJ. A critical reassessment of the role of mitochondria in tumorigenesis. PLoS Med 2005; 2(11):e296. 10. Herrnstadt C, Preston G, Howell N. Errors, phantoms and otherwise, in human mtDNA sequences. Am J Hum Genet 2003; 72:1585–1586. 11. King A, Selak MA, Gottlieb E. Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer. Oncogene 2006; 25:4675–4682. 12. Gottlieb E, Tomlinson IP. Mitochondrial tumour suppressors: a genetic and biochemical update. Nat Rev Cancer 2005; 5:857–866. 13. Cheng WC, Berman SB, Ivanovska I, et al. Mitochondrial factors with dual roles in death and survival. Oncogene 2006; 25:4697–4705. 14. Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2002; 82: 47–95. 15. Takahashi A, Ohtani N, Yamakoshi K, et al. Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence. Nat Cell Biol 2006; 8:1291–1297. 16. Calderwood SK, Khaleque MA, Sawyer DB, Ciocca DR. Heat shock proteins in cancer: chaperones of tumorigenesis. Trends Biochem Sci 2006; 31:164–172.

54

R. Scatena et al.

17. Czarnecka AM, Campanella C, Zummo G, Cappello F. Mitochondrial chaperones in cancer: from molecular biology to clinical diagnostics. Cancer Biol Ther 2006; 5:714–720 18. Mathupala SP, Ko YH, Pedersen PL. Hexokinase II: cancer s double-edged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria. Oncogene 2006; 25(34):4777–4786. 19. Scorrano L, Oakes SA, Opferman JT, et al BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science 2003; 300:135–139. 20. Chandra D, Liu JW, Tang DG. Early mitochondrial activation and cytochrome c up-regulation during apoptosis. J Biol Chem 2002; 277:50842–50854. 21. Amuthan G, Biswas G, Ananadatheerthavarada HK, Vijayasarathy C, Shephard HM, Avadhani NG. Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells. Oncogene 2002; 21:7839–7849. 22. Scatena R, Bottoni P, Vincenzoni F, et al. Bezafibrate induces a mitochondrial derangement in human cell lines: a PPAR-independent mechanism for a peroxisome proliferator. Chem Res Toxicol 2003; 16:1440–1447. 23. Felty Q, Roy D. Estrogen, mitochondria, and growth of cancer and non-cancer cells. J Carcinog 2005; 4:1–18 24. Bottoni P, Giardina B, Martorana GE, et al. A two-dimensional electrophoresis preliminary approach to human hepatocarcinoma differentiation induced by PPAR-agonists. J Cell Mol Med 2005; 9:462–467. 25. Scatena R, Nocca G, Sole PD, et al. Bezafibrate as differentiating factor of human myeloid leukemia cells. Cell Death Differ 1999; 6:781–787. 26. Scatena R, Bottoni P, Martorana GE, et al. Mitochondria, ciglitazone and liver: a neglected interaction in biochemical pharmacology. Eur J Pharmacol 2007; 567:50–58. 27. Michalik L, Desvergne B, Wahli W. Peroxisome proliferator-activated receptor and cancers: complex stories. Nat Rev Cancer 2004; 4:61–70. 28. Richards FM, Watson A, Hickman JA. Investigation of the effects of heat shock and agents which induce a heat shock response on the induction of differentiation of HL-60 cells. Cancer Res 1988; 48:6715–20. 29. Chen EI, Hewel J, Krueger JS, et al. Adaptation of energy metabolism in breast cancer brain metastases. Cancer Res 2007; 67:1472–1486.

Cellular Adaptations to Oxidative Phosphorylation Defects in Cancer Sarika Srivastava and Carlos T. Moraes

Abstract Mitochondrial DNA (mtDNA) somatic mutations or mutations in nuclear genes encoding mitochondrial proteins important for the assembly, activity, or maintenance of the individual oxidative phosphorylation (OXPHOS) complexes have been observed in tumors. Although the functional consequence of such mutations is unclear at the moment, retrograde signaling in response to OXPHOS defects can activate various nuclear genes and signaling pathways that alter mitochondrial function, tumor invasion, metastasis, redox-sensitive pathways, programmed cell death pathways, calcium signaling pathways, and cellular pathways leading to global changes in cellular morphology and architecture. In addition, we have found that some cancer cell lines harboring deleterious mtDNA mutations upregulate the expression of members of the peroxisome-proliferator activated ␥ coactivator 1 family of coactivators, probably to sustain the necessary ATP production for cell proliferation. In this chapter, we describe such cellular adaptations and changes in response to OXPHOS defects that are associated with a variety of cancer cell types. Keywords mtDNA · Retrograde signaling · OXPHOS · Cancer · Mitochondria

1 Mammalian Oxidative Phosphorylation Mammalian mitochondrial oxidative phosphorylation (OXPHOS) occurs in five multimeric enzyme complexes (complexes I, II, III, IV, and V) using two electron transport carriers (ubiquinone or coenzyme Q10 and cytochrome c). Electrons from reduced equivalents (NADH and FADH2 ) are transported along these complexes via ubiquinone and cytochrome c to molecular oxygen, producing water. At the same time, protons are pumped across the mitochondrial inner membrane (from the matrix to intermembrane space) by enzyme complexes I, III, and IV thereby generating a C.T. Moraes (B) University of Miami Miller School of Medicine, Miami, FL 33136 e-mail: [email protected] S.P. Apte, R. Sarangarajan (eds.), Cellular Respiration and Carcinogenesis, DOI 10.1007/978-1-59745-435-3 5,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

55

56

S. Srivastava, C.T. Moraes Intermembrane space H+

H+

H+

eO2 e-

e-

e-

e-

H2 O

e-

Cyt c

CoQ10

ATP

ADP

H+ Complex I

Complex II

Complex III

Complex IV

Complex V

Matrix

Fig. 1 Schematic representation of mammalian mitochondrial OXPHOS. Mammalian electron transport chain (ETC) consists of four multimeric complexes (complexes I, II, III, and IV). Electrons from the reduced equivalents, NADH and FADH2 , enter the ETC and reduce complex I and complex II, respectively. Coenzyme Q10 or ubiquinone is an electron carrier in the inner mitochondrial membrane that accepts an electron from either complex I or complex II and donates it to complex III. Cytochrome c, another electron carrier in the mitochondrial intermembrane space, accepts an electron from complex III and donates it to complex IV. Complex IV donates electrons to molecular O2 , which results in the formation of H2 O. During the electron flow, complexes I, III, and IV pump the protons from the matrix toward the intermembrane space thereby generating an electrochemical gradient across the mitochondrial inner membrane. The energy in the electrochemical gradient is harnessed by complex V to generate ATP from ADP (oxidative phosphorylation)

proton gradient or membrane potential (⌬⌿m). The energy in this gradient is harnessed by complex V (ATP synthase) to synthesize ATP from ADP, a phenomenon termed OXPHOS, during which protons flow back from the mitochondrial intermembrane space to the mitochondrial matrix (Fig. 1). Mitochondria are also important for cytosolic Ca+2 buffering and are the predominant source of ROS production in most cell types. Superoxide anions are generated at the level of complex I and complex III in the inner mitochondrial membrane.

2 OXPHOS Defects in Cancer OXPHOS defects in cancer cells were first reported more than 50 years ago by Warburg who stated that “cancer cells are impaired in respiratory chain function and are very glycolytic” [1]. Studies on various types of cancer cells have found

Cellular Adaptations to OXPHOS Defects in Cancer

57

this hypothesis to be correct in several but not all tumor cells. Several independent groups have also reported that many cancer cells possess a full complement of respiratory chain enzymes and can couple electron transport chain to ATP production [2]. Isolated tumor mitochondria have also been found to respire on a variety of substrates. Specific changes that occur in the energy metabolism of tumor cells have not yet been established. In the past 10 years, studies on mitochondrial DNA (mtDNA) mutations that cause OXPHOS defects and their potential role in cancer have regained momentum. Mitochondrial DNA mutations have been increasingly identified in various types of cancer cells [3, 4]. Polyak et al. were first to report a detailed analysis of somatic mtDNA mutations in human colorectal cancer cells by sequencing their complete mitochondrial genome [3] (somatic mtDNA mutations are those present in the tumor tissue but absent from the adjacent normal tissue). After this initial finding, mtDNA from various tumor cell types was sequenced by different groups, and mtDNA mutations have now been reported in esophageal, ovarian, thyroid, head, neck, lung, bladder, and renal cancer cells [4–6]. The majority of the mtDNA mutations reported in different tumor cell types are transitions (G-to-A or T-to-C), a feature that is characteristic of reactive oxygen species (ROS)-derived mutations. Further, most of the somatic mtDNA mutations have been reported to be homoplasmic suggesting their dominance at intra- and intercellular levels.

2.1 Functional Significance of OXPHOS Defects in Cancer Although in the past decade, the list of mtDNA mutations affecting OXPHOS in cancer cells has increased steadily, the functional relevance of these mutations in tumor promotion or formation process are yet obscure. Because the majority of the somatic mtDNA mutations found in tumor cells have been reported to be homoplasmic, one of the existing hypotheses is that the homoplasmic levels of these mutations were probably achieved through some kind of replication or growth advantage that the mutant mtDNA harbored over the wild type in the tumor progenitor cells. It has also been suggested that mtDNA mutations may cause a moderate increase in ROS production that in turn stimulates cell growth [3, 4]. Low levels of ROS have been shown to stimulate mitosis in various cell types [7, 8]. In contrast with this hypothesis, Coller and co-workershave demonstrated that there is a sufficient opportunity for a tumor progenitor cell to achieve homoplasmy through unbiased mtDNA replication and segregation during cell division [9]. Coller et al.constructed a theoretical model based on computer simulation studies and showed that there is a good correlation between the reported frequency of homoplasmic mutations in tumor cells and the predicted frequency of homoplasmy in tumor progenitor cells that can be attained in the absence of any selection [9]. The authors suggested that mtDNA mutations such as silent substitutions of amino acid coding regions or alterations in the length of polynucleotide tracts that are polymorphic in healthy individuals are the ones that are unlikely to confer any selective advantage to the mtDNA or the

58

S. Srivastava, C.T. Moraes

host cell. However, the authors did not exclude the possibility that certain mtDNA point mutations that cause functional alterations (e.g., mutations affecting the conserved amino acid regions in the coding region of a protein) might be segregated in a nonrandom manner and may provide a selective growth advantage to the host cell [9]. mtDNA mutations leading to OXPHOS dysfunction have also been shown to increase tumorigenicity in certain tumor cell types. Mutations in the mtDNA COXI gene were shown to increase tumorigenicity in prostate cancer patients [10]. Petros et al. reported that ∼11% to 12% of prostate cancer patients harbor pathogenic COXI mtDNA mutations that alter the conserved amino acid regions in the protein and suggested that these mutations may play a role in the etiology of prostate cancer [10]. mtDNA ATP6 gene mutation (T8993G) has also been shown to increase tumorigenicity by two independent groups. The first study was from Petros and coworkers who showed that PC3 prostate cancer cybrid cells harboring T8993G ATP6 mutant mtDNA when introduced in nude mice formed tumors that were ∼7 times larger than cells harboring the wild-type mtDNA suggesting a role of mtDNA mutation in tumor promotion process [10]. They also showed that tumor cells harboring ATP6 mutant mtDNA generated significantly higher levels of ROS than did those harboring wild-type mtDNA, further suggesting that high levels of ROS promoted tumor cell growth. The second study was from Shidara and co-workers. They constructed HeLa transmitochondrial cybrids harboring homoplasmic T8993G ATP6 mtDNA mutation and showed that these cybrids grew much faster in culture as well as in nude mice forming tumors that were larger in size compared with the cybrids harboring the wild-type mtDNA suggesting that the mtDNA ATP6 mutation in these cells conferred a selective growth advantage [11]. Further, transfection of a wild-type nuclear version of the mitochondrial ATP6 gene in these HeLa cybrids harboring homoplasmic ATP6 mutant mtDNA followed by their transplantation in nude mice was shown to slow down the tumor cell growth. Conversely, the expression of a nuclear version of the mutant ATP6 gene in the wild-type cybrids was found to accelerate tumor cell growth thereby suggesting that the growth advantage in these tumor cells depended on the mitochondrial ATP6 function [11]. OXPHOS dysfunction caused by partial mtDNA depletion (genetic stress) or treatment with mitochondrial-specific inhibitors (metabolic stress) has been shown to induce cell invasion and tumor progression in otherwise noninvasive cells [12, 13]. A correlation between OXPHOS impairment and tumor aggression has also been reported in renal carcinomas [14]. Simonnet et al. studied the mitochondrial respiratory chain enzyme content in three different renal tumors and found that the OXPHOS impairment increased from the less aggressive to the most aggressive types of renal carcinomas [14]. They also found that renal oncocytomas (benign tumors) are exclusively deficient in mitochondrial complex I activity, whereas the activity of all other OXPHOS complexes are increased in these tumors [15]. These tumors further showed dense mitochondrial proliferation suggesting an increase in mitochondrial biogenesis as an attempt to compensate for the potential loss of OXPHOS function. Complex I deficiency was also found in normal cells adjacent to the tumor tissue leading the authors to suggest that the complex I deficiency could be

Cellular Adaptations to OXPHOS Defects in Cancer

59

an early event in the formation of renal oncocytomas [15]. Similarly, OXPHOS dysfunction and mitochondrial proliferation have also been reported in thyroid oncocytomas [16]. The current studies on potential effects of mtDNA mutations and OXPHOS dysfunction in cancer cells therefore suggest their role in tumor promotion and/or progression. It is believed that nuclear DNA mutations and not mtDNA mutations control the tumor initiation process. Akimoto and co-workers have tested this hypothesis [17]. They showed that cells carrying nuclear DNA from tumor cells and mtDNA from normal cells form tumors in nude mice whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells do not form tumors [17]. However, mutations in the mitochondrial enzymes that are encoded by the nuclear genome have been associated with certain types of cancer. Germ-line mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase have been shown to be associated with uterine fibroids, skin leiomyomata, and papillary renal cell cancer [18], and mutations in the succinate dehydrogenase (SDH) subunits B, C, and D have been shown to be associated with hereditary paragangliomas and pheochromocytomas [19–21]. The potential role of these mutations in the tumor formation process is yet unclear.

3 Cellular Adaptations to OXPHOS Defects in Cancer The cross-talk between mitochondria and nucleus plays an important role in maintaining mitochondrial function and integrity. Mitochondrial dysfunction affects the mitochondrial-nuclear cross-talk leading to altered signaling cascades (Fig. 2). The nuclear-mitochondrial stress signaling, also called retrograde signaling, is a phenomenon that involves changes in the nuclear gene expression in response to

OXPHOS regulatory or structural genes

Altered Gene Expression

Fig. 2 Mitochondrial-nuclear intergenomic signaling in response to OXPHOS defects. Mitochondrial OXPHOS dysfunction in cancer cells can activate a retrograde signaling cascade. Altered retrograde signaling can affect the expression of various nuclear genes and cellular pathways that allow tumors cells to adapt to the environment and/or promote tumor cell growth or progression

Calcium signaling pathway Redox signaling / antioxidant defense pathway Cellular morphology and architecture Tumor invasion or metastatic genes

Nucleus

Retrograde Signaling

OXPHOS Dysfunction

60

S. Srivastava, C.T. Moraes

OXPHOS dysfunction. These nuclear gene expression changes allow the tumor cells to adapt to the environment that may promote tumor cell growth and/or progression. Studies have shown that a variety of nuclear genes are altered in response to OXPHOS defects in different tumor cell types and have implicated their role in tumorigenesis.

3.1 Retrograde Signaling in Cancer Cells Retrograde signaling has been implicated as an important mechanism in carcinogenesis although the precise mechanism(s) of this pathway is yet elusive in mammalian systems. Studies have shown that retrograde signaling in response to mitochondrial dysfunction can alter the expression of nuclear genes involved in energy metabolism, tumor invasion and metastasis, calcium signaling, redox signaling, antiapoptotic genes, as well as genes involved in regulating cellular morphology and architecture. 3.1.1 Altered Expression of OXPHOS Regulatory Genes and Subunits The mitochondrial OXPHOS complexes (I, III, IV, and V) comprise subunits encoded by both mtDNA and nuclear DNA. The expression of nuclear encoded OXPHOS subunits is under a tight regulation by regulatory genes, namely transcription factors and transcriptional coactivators. Nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) are the nuclear transcription factors, and the members of the peroxisome-proliferator activated ␥ coactivator 1 (PGC-1) gene family are the nuclear transcriptional coactivators that directly or indirectly modulate the expression of nuclear encoded OXPHOS genes, respectively. Besides regulating the expression of nuclear encoded OXPHOS genes, the NRFs also exert a regulatory control over the expression of mitochondrial transcription and replication machinery components, the protein import machinery components, and heme biosynthesis. A change in the expression levels or activity of NRFs and/or PGC-1 family of transcriptional coactivators would therefore influence the expression of OXPHOS subunits and mitochondrial biogenesis. Altered expression of OXPHOS subunits and transcriptional regulatory genes has been reported in different tumor cell types [22, 23]. We studied a human colorectal tumor cell line (termed V425) harboring a homoplasmic nonsense mutation in the mitochondrial COXI gene. Despite the mtDNA mutation load, these cells were found to maintain a very high rate of mitochondrial respiration (∼75% of control cells) and a low level of COX activity (∼10% of control cells). Interestingly, osteosarcoma cybrids harboring the V425 mutant mtDNA showed a significant decline in respiration and COX activity [22]. Previous studies have shown that ∼10% COX activity cannot sustain a functional respiratory chain [24]. We found that the steady-state transcript levels of a transcriptional coactivator PGC-1␣ and its homolog PGC-1␤ were highly upregulated in V425 cells. In parallel, we also observed an increase in the steady-state levels of several mitochondrial proteins (e.g., SDH, COXIV, and cytochrome c) in these

Cellular Adaptations to OXPHOS Defects in Cancer

61

cells. Further, the overexpression of PGC-1␣ in osteosarcoma cybrids harboring the V425 mutant mtDNA showed a significant improvement in mitochondrial respiration and an increase in the steady-state levels of several mitochondrial proteins suggesting that PGC-1␣ upregulation can improve mitochondrial respiration in a colorectal cancer cell line harboring COX deficiency [22]. It has also been shown that PGC-1␣ stimulates mitochondrial biogenesis and respiration by activating the expression of NRFs, augmenting their transcriptional activity or activating both the expression and transcriptional activity [25]. Altered expression of PGC-1 related coactivator (PRC), NRF-1, and mitochondrial transcription factor A (TFAM) have also been observed in thyroid oncocytomas harboring an OXPHOS defect [23]. Savagner et al. found that dense mitochondrial proliferation in 90% of the tested oncocytic tumors were associated with overexpression of the PRC compared with the controls suggesting that increased mitochondrial biogenesis might be a feedback mechanism to compensate for the presence of OXPHOS deficit in these tumors. They also found that the increase in PRC levels was associated with a 5-fold increase in NRF-1 transcripts, 10-fold increase in TFAM transcripts, and a 3-fold increase in COX activity further suggesting that the overexpression of PRC pathway is likely responsible for the increased mitochondrial proliferation in thyroid oncocytomas [23]. In contrast with the above studies, low levels of expression of peroxisomeproliferator activated receptor gamma (PPAR-␥) and PGC-1 have also been reported in breast cancer tissues [26]. Jiang et al. showed that the human metastatic breast cancer tissues have low levels of PPAR-␥ and PGC-1 transcripts compared with those of the normal tissues. Further, lower levels of these molecules were found to be associated with poor clinical outcomes in breast cancer patients [26]. Interestingly, agonists of PPAR-␥ have also been shown to inhibit growth and proliferation of cancer cells by inducing apoptosis [27]. 3.1.2 Activation of Genes Involved in Tumor Invasion and Progression OXPHOS dysfunction has been shown to modulate tumor invasive properties by transcriptional upregulation of genes coding for specific members of matrix remodeling pathway and activation of tumor-specific marker genes. van Waveren et al. showed that OXPHOS dysfunction can alter the expression of nuclear genes coding for factors involved in extracellular matrix remodeling in human osteosarcoma cells [28]. These genes included members of the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP) family, urokinase plasminogen activator and its inhibitor, plasminogen-activator inhibitor 1 (PAI-1), and CTGF and CYR61 (members of the cysteine-rich 61, connective tissue growth factor and nephroblastoma-overexpressed [CCN] gene family of growth regulators). Changes at the protein level for some of these factors have also been observed, though at a lesser magnitude. The study also showed that osteosarcoma cells harboring mtDNA mutations were associated with an increased matrigel invasion [28]. Activation of tumor-specific marker genes in response to an OXPHOS dysfunction was observed by Amuthan et al. [12, 13]. They found that partial mtDNA

62

S. Srivastava, C.T. Moraes

depletion (genetic stress) or treatment of cells with mitochondrial-specific inhibitors (metabolic stress) activated Ca2+ -dependent kinases (PKC, ERK1, ERK2, and calcineurin) and signaling pathways that in turn activated the expression of nuclear genes involved in tumor invasion and progression. Among these genes, the extracellular matrix protease cathepsin L, transforming growth factor ␤ (TGF-␤), and mouse melanoma antigen (MMA) were found to be upregulated in both genetically and metabolically stressed rhabdomyosarcoma and pulmonary carcinoma cells. Using in vitro Matrigel invasion assays and in vivo rat tracheal xenotransplants in Scid mice, they further showed that these genetically and metabolically stressed cells were ∼4to 6-fold more invasive than were their respective controls thereby implicating a role of OXPHOS dysfunction in inducing genes involved in tumor invasion and progression. The invasive behavior of mtDNA-depleted rhabdomyosarcoma and pulmonary carcinoma cells was reverted by the restoration of the mtDNA content [12, 13]. These findings clearly established a role of mitochondrial retrograde signaling in inducing phenotypic changes and tumor progression in osteosarcoma, rhabdomyosarcoma, and human pulmonary carcinoma cells. OXPHOS dysfunction was also shown to be associated with tumor aggressiveness in renal carcinoma cells [14]. Retrograde signaling in breast cancer has been reported by Delsite et al. They performed a comparative microarray analysis of the nuclear gene expression changes in a human breast cancer cell line (MDA-435) and its ␳o derivative devoid of mtDNA. They found that the expression of several nuclear genes involved in cell signaling, cell growth, energy metabolism, cell architecture, cell differentiation, and apoptosis were altered in the ␳o derivative of the breast cancer cell line [29]. Functional characterization of these genes is further required to gain a clear insight to the potential role of retrograde signaling in tumorigenesis. 3.1.3 Activation of Calcium Signaling Pathway Changes in intracellular calcium (from the approximate 100 nM at rest) mediate a number of processes that affect tumorigenesis, including angiogenesis, motility, cell cycle, differentiation, transcription, telomerase activity, and apoptosis [30]. Mitochondria take up Ca2+ in a process dependent on a ⌬⌿m [31]. Ca2+ uptake by mitochondria controls organelle metabolic activity, as pyruvate, ␣-ketoglutarate, and isocitrate dehydrogenases are activated by Ca2+ . In addition, some metabolite transporters have been shown to be regulated by Ca2+ as well and to enhance aerobic metabolism. Mitochondria, by buffering local [Ca2+ ] (generated by Ca2+ channels on the plasma membrane or the ER/SR), can also modulate intracellular Ca2+ [31]. Therefore, Ca2+ can regulate cell growth and survival by modulating OXPHOS function. Likewise, a defective OXPHOS impairs Ca2+ buffering. As described above, impaired Ca2+ buffering capacity by defective mitochondria can activate proteins involved in tumor invasion and progression, including PKC, ERK1, ERK2, and calcineurin. OXPHOS dysfunction has been shown to activate calcium-dependent signaling events in several tumor cell types. Avadhani and co-workers investigated the nuclear gene expression changes in C2C12 rhabdomyosarcoma and human A549 pulmonary carcinoma cells in response to OXPHOS dysfunction and found that

Cellular Adaptations to OXPHOS Defects in Cancer

63

calcium-dependent signaling events were activated in these cells [12, 13, 32]. OXPHOS defects associated with decreased mitochondrial membrane potential (⌬⌿m) and ATP synthesis caused a sustained increase in intracellular calcium [Ca2+ ]i levels in these cells. They found that the expression of ryanodine receptor-1 (RyR-1) and ryanodine recptor-2 (RyR-2) genes was upregulated in C2C12 rhabdomyosarcoma and A549 pulmonary carcinoma cells, respectively. Increased RyR gene expression correlated with high levels of Ca2+ release from ER to the cytosol in response to caffeine stimulation (RyR agonist). Avadhani and co-workers suggested that the defect in ⌬⌿m was associated with the observed increase in [Ca2+ ]i levels as mitochondria are key players in sequestering cytosolic Ca2+ and a defect in ⌬⌿m would affect mitochondrial Ca2+ uptake ability. Restoration of the ⌬⌿m in both C2C12 rhabdomyosarcoma and A549 pulmonary carcinoma cells was found to revert the ryanodine receptor gene expression and [Ca2+ ]i levels to near normal levels suggesting a direct link between OXPHOS dysfunction and elevated [Ca2+ ]i in these cancer cells. Ca2+ responsive factors such as calcineurin, calcineurindependent NFATc (cytosolic counterpart of activated T-cell–specific nuclear factor), and JNK (c-Jun N-terminal kinase)-dependent ATF2 (activated transcription factor 2) were also found to be elevated in both C2C12 rhabdomyosarcoma and pulmonary carcinoma cells. Further, Ca2+ -dependent PKC (protein kinase C) and MAPK (mitogen activated protein kinase) genes were activated in C2C12 rhabdomyosarcoma and lung carcinoma cells, respectively. Another study by Avadhani and co-workers has shown that calcineurin that is activated in response to elevated cytosolic Ca2+ inactivates I-␬B␤ thereby leading to an activation of the NF-␬B/Rel family of transcription factors in the OXPHOS-deficient C2C12 rhabdomyosarcoma cells [33]. They suggested that the NF-␬B/Rel family of transcription factors might be involved in the activation of nuclear genes in response to OXPHOS dysfunction in these cells [33]. Rise in cytosolic Ca2+ in response to OXPHOS dysfunction has also been observed in rat pheochromocytoma PC12 cells. Luo et al. showed that the treatment of PC12 cells with mitochondrial uncoupler FCCP releases Ca2+ from internal stores leading to a rise in cytosolic Ca2+ , which in turn activates MAPK also suggesting a potential link between OXPHOS dysfunction and Ca2+ signaling in these cells [34]. 3.1.4 Activation of Redox Signaling Pathway Changes in nuclear gene expression in response to oxidative stress have been observed in a variety of tumor cells. mtDNA mutations or OXPHOS dysfunction predispose mitochondria to the generation of ROS leading to oxidative stress. Cellular oxidative stress has been implicated in initiation, promotion, and progression of carcinogenesis [35]. Low levels of ROS generation have been shown to be mitogenic in a variety of human and mouse cell types [36, 37]. For example, under cell culture conditions, 10 nM to 1 ␮M concentrations of both O2 •- and H2 O2 have been shown to stimulate the growth of hamster and rat fibroblasts [37]. Low levels of ROS were also shown to stimulate the growth of mouse epidermal cells, human fibroblasts, and human astrocytoma cells [38, 39]. The increased tumorigenesis of

64

S. Srivastava, C.T. Moraes

NARP cells with a pathogenic mtDNA mutation described above [10] could also be related to increased ROS, as the mutation in ATPase 6 was reported to significantly increase ROS production [40]. Further, many chemical carcinogens initiate carcinogenesis by stimulating ROS production, which in turn activates the expression of early growth related genes such as c-fos, c-myc, and c-jun [41]. Although the molecular mechanism(s) of ROS-mediated cell growth are yet unclear, cellular ROS have been shown to activate the expression of redox-sensitive nuclear genes and signaling pathways. It has been shown that ROS activate the expression of nuclear factor kappa B (NF-␬B) and activator protein (AP-1) transcription factors [42, 43]. Both NF-␬B and AP-1 are redox-sensitive transcription factors. NF-␬B in its active form consists of two subunits, p50 and p65. In the absence of a stimulus, it is present in the cytoplasm in association with its inhibitory subunit I-␬B. In response to a stimulus that leads to the I-␬B subunit phosphorylation, NF-␬B dissociates from the I-␬B inhibitory complex and translocates to the nucleus where it activates the expression of specific target genes [44]. AP-1 is a heterodimer of transcription factors composed of Jun, Fos, or activating transcription factor (ATF) subunits and binds the AP-1 sites in the promoter region to activate the target gene expression [45]. Constitutive activation of NF-␬B and Ap-1 genes in response to oxidative stress has been observed in a variety of cancer cell types. Shi et al. demonstrated that OH radicals activated the expression of NF-␬B in Jurkat cells, macrophages, and mouse epidermal cells, and antioxidants that scavenge OH radicals inhibited its activation [46]. High levels of ROS were also shown to cause a constitutive elevation of NF-␬B and AP-1 transcription factors in transformed mouse keratinocyte cells [7]. Constitutive elevation of AP-1 activity has also been shown to be associated with the conversion of benign papillomas to malignant carcinomas in mouse epidermal cells suggesting that the target genes induced by activated AP-1 were involved in cell growth and metastasis [47]. Inhibition of Ap-1 activity by a dominant negative c-Jun mutant was shown to suppress the tumorigenic phenotype of the malignant mouse epidermal cells thereby blocking the tumor formation in nude mice [48]. Constitutive activation of NF-␬B has also been shown to induce genes involved in tumor progression and metastasis [49]. Further, treatment with an antioxidant, N-acetylcysteine (NAC), has been shown to inhibit the elevated expression of NF␬B and AP-1 levels in tumor cells implicating the role of ROS in the activation of redox-sensitive signaling pathways [7]. The activities of NF-␬B and AP-1 transcription factors are modulated by phosphorylation. Although the precise signaling events that activate the phosphorylation of these transcription factors in response to increase in cellular ROS levels are yet not fully understood, protein kinases are central in their activation. It has been observed that MAPK pathways are involved in signaling the activation of both NF␬B and Ap-1 transcription factors. There are three subtypes of MAPKs; the extracellular signal regulated kinases (ERKs), the c-jun N-terminal kinases (JNKs), and the p38 MAPKS. Gupta et al. showed that the NF-␬B activation in malignantly progressed mouse keratinocytes was associated with an increase in ERK-1/2 and p38 MAPK activities and that NAC treatment rapidly abolished the increase in MAPK activities [7]. Direct activation of I-␬B by MEKK1 (MAPK/ERK kinase kinase-1)

Cellular Adaptations to OXPHOS Defects in Cancer

65

has also been demonstrated in vitro [50]. Activation of AP-1 by MAPK in response to various stimuli has been demonstrated [51]. There is also evidence that antioxidants can attenuate MAPK activation suggesting that MAPK signaling cascades are activated in response to cellular oxidative stress conditions [7]. 3.1.5 Altered Antioxidant Defense Pathway The antioxidant defense enzymes have been found to be altered in several human tumors. Superoxide dismutases (SODs) are the first line of cellular defense against increase in intracellular ROS. There are two forms of intracellular SODs: a manganese-containing SOD (MnSOD) that localizes to the mitochondria and a copper-zinc–containing SOD that localizes to the cytosol. Most human tumor cell types have been found to be deficient in their antioxidant defense function. Antioxidant enzymes including catalase and glutathione peroxidase have also been shown to be lowered in cancer cells [52, 53] suggesting that tumor cells have a deficient antioxidant function. Studies have shown that antioxidant treatment prevents the malignant transformation of cancer cells. Increased expression of MnSOD has been shown to suppress cancer phenotypes in several murine and human cancer cells. St Clair et al. showed that overexpression of human MnSOD in mouse cells significantly reduced the frequency of radiation-induced neoplastic transformation implicating a direct link between mitochondrial antioxidants and neoplastic transformation [54]. Zhao et al. demonstrated that in response to tumor promoters (phorbol esters), transgenic mice expressing human MnSOD in the skin showed a significant reduction in papilloma formation compared with that of the non-transgenic controls [55]. They further found that the decreased papilloma formation was associated with a delay and reduction in AP-1 transcription factor binding activity suggesting that the MnSOD overexpression suppressed the tumor formation and Ap-1 activation in these transgenic animals [55]. Overexpression of human MnSOD has also been shown to suppress the malignant phenotype of human pancreatic and breast cancer cells. Li et al. demonstrated that MnSOD-overexpressing MCF-7 breast cancer cells showed a marked inhibition in growth rate in vitro under culture conditions and in vivo upon inoculation in nude mice when compared with the wild-type MCF-7 cells [56]. In another study, they demonstrated that the transcriptional and DNA binding ability of NF-␬B and AP-1 were reduced by ∼50% in MnSOD-overexpressing MCF-7 cells [57]. They further showed that the expression of NF-␬B and AP-1 responsive genes was downregulated in these cells when compared with that of the wild-type MCF-7 cells suggesting that the tumor-suppressive effects of MnSOD were associated with the inhibition of NF-␬B and Ap-1 activities that in turn downregulated the genes involved in malignant progression [57]. 3.1.6 Altered Mitochondrial Morphology, Cell Surface, and Architecture Mitochondrial fission and fusion are the central events that regulate mitochondrial morphology. Changes in mitochondrial morphology appear to be a key event during retrograde signaling. Studies have implicated a role of mitochondrial morphology in

66

S. Srivastava, C.T. Moraes

modulating respiratory activity. For example, downregulation of mitofusin 2 (Mfn 2) decreases mitochondrial respiration and increases the cellular glucose uptake levels for glycolysis. Further, ectopic upregulation of Mfn 2 increases the expression of OXPHOS subunits, glucose oxidation, and ⌬⌿m [58]. Optic Atrophy 1 (Opa 1) is an important player in the formation of mitochondrial cristae. Downregulation of Opa 1 has been shown to induce the loss of mitochondrial respiration and increased mitochondrial fragmentation [59]. Changes in mitochondrial morphology and ultrastructure were reported in different tumor cell types over several decades. Tumor cell mitochondria were found to contain abnormal size, shape (dumbbell or cup), cristae organization, or inclusions. For example, rapidly growing hepatomas were found to contain small-size mitochondria with fewer cristae, whereas slowly growing hepatomas were found to contain large-size mitochondria with densely packed cristae [60]. Changes in the mitochondrial morphology in tumor cells could therefore affect their respiratory activity. Conversely, it is also possible that in response to altered respiratory activity or OXPHOS dysfunction, the retrograde signaling events in tumor cells affect the expression of nuclear genes involved in the regulation of mitochondrial morphology and ultrastructure. Changes in cell morphology and architecture are a characteristic feature of many cancer cells. These changes occur in cell surface charge, glycoprotein composition, membrane organization, and/or the ability of cells to agglutinate on lectin. The initial evidence of a potential role of OXPHOS dysfunction in inducing cell surface changes was obtained in yeast. In 1980, Evans and colleagues showed that mitochondrial petite mutations in yeast that cause respiratory deficiency lead to drastic changes in cell surface charge, cell wall organization, and lectin agglutinability in contrast with the wild-type yeast cells suggesting that the OXPHOS deficiency in yeast altered the retrograde signaling pathway leading to gene expression changes and hence altered cell surface properties [61]. Preliminary evidence in literature has also suggested a link between OXPHOS dysfunction and cell surface properties in mammalian cells. For example, Soslau et al. showed that baby hamster kidney cells when treated with ethidium bromide (Etbr) show an altered glycoprotein composition in the plasma membrane [62]. Further, they also showed that the glycopeptide elution profile of these Etbr-treated cells was similar to that of the cells transformed with Rous sarcoma virus. Low concentrations of Etbr are known to intercalate to mtDNA and abrogate mtDNA replication. Exponentially growing cells when treated with Etbr (25 ng/mL to 2 ␮g/mL) undergo mtDNA loss leading to either partial or complete loss of mtDNA molecules, generating an OXPHOS defect. These findings therefore suggest that altered retrograde signaling events during OXPHOS dysfunction could activate nuclear genes or pathways that cause global changes in cell surface properties. 3.1.7 Activation of Antiapoptotic Genes Activation of antiapoptotic genes is a common occurrence in most human cancers. However, retrograde signaling pathway has also been shown to activate

Cellular Adaptations to OXPHOS Defects in Cancer

67

antiapoptotic genes (e.g., Bcl-xL and Bcl-2) in certain cancer cell types [12, 63]. Increased expression of the antiapoptotic genes may also contribute to metabolic homeostasis. It has been shown that members of the Bcl-2 antiapoptotic family of proteins regulate ATP/ADP exchange across the mitochondrial membranes and can prevent the loss of mitochondrial respiration during apoptosis [64]. Manfredi and colleagues showed that Bcl-2/Bcl-xL can improve OXPHOS function in cells harboring pathogenic mtDNA mutations. The effect of Bcl-2 overexpression in mutated cells was found to be independent from apoptosis and was suggested to modulate the adenine nucleotide exchange between mitochondria and cytosol. This study therefore provided evidence that when OXPHOS function is reduced, Bcl-2 expression can improve it by regulating the levels of adenine nucleotides inside the mitochondria [64].

3.2 Mechanism of Retrograde Signaling The mechanism of retrograde signaling was first identified in yeast and it is still poorly understood in other systems. Three retrograde regulatory genes (RTG1, RTG2, and RTG3) play a central role in retrograde signaling in yeast [65] (Fig. 3). Rtg1p and Rtg3p are transcription factors, whereas Rtg2p is mitochondrial function sensor. During normal mitochondrial function, Rtg1p and Rtg3p form heterodimers

P Nucleus

Rtg3p

Rtg1p

Target gene

GTCAC

R box

P Rtg3p

P P P Rtg3p P

Rtg1p

Rtg2p Rtg1p

Sensor

Mitochondrial Dysfunction

Cytoplasm

Fig. 3 Schematic model of retrograde signaling in yeast. RTG genes play a central role in retrograde signaling. Rtg1p and Rtg3p are sequestered in cytoplasm during normal mitochondrial function. Rtg3p is highly phosphorylated during this state. In response to mitochondrial dysfunction (sensed by Rtg2p), the Rtg3p undergoes partial dephosphorylation. Both Rtg3p and Rtg1p translocate to the nucleus and activate target gene expression leading to changes in nuclear genes and/or signaling pathways that in turn can alter cellular function and homeostasis

68

S. Srivastava, C.T. Moraes

and are sequestered in the cytoplasm. Rtg3p is phosphorylated at multiple sites during this state. In response to mitochondrial dysfunction (sensed by Rtg2p), a retrograde signaling pathway gets activated. The Rtg3p undergoes partial dephosphorylation and translocates to the nucleus. Rtg1p also follows and translocates to the nucleus. Both Rtg1p and Rtg3p activate transcription at the target genes. In the absence of Rtg2p, the Rtg1p/Rtg3p complex remains in the cytoplasm during active retrograde signaling pathway implicating its role as mitochondrial function sensor and in signal relay to the Rtg1p/Rtg3p complex [65]. The precise mechanism of retrograde signaling in mammalian systems is yet not known. The mammalian homologs of RTG genes have yet not been identified. However, human MYC protein has been found to share a significant homology with yeast Rtg3p, and it is involved in the regulation of many cellular functions such as regulation of glycolysis, cellular stress response cell cycle progression, and apoptosis [66, 67].

4 Conclusion Mitochondrial dysfunction has been reported in a variety of human cancers. Mitochondrial-nuclear intergenomic signaling is an important phenomenon in regulating mitochondrial and cellular function and homeostasis. Although the role of mtDNA mutations or OXPHOS defects in cancer progression is poorly understood, a considerable body of evidence has now shown that mitochondrial function has an important role in different aspects of tumorigenesis. Processes such as cell survival and cell invasion can be directly influenced by OXPHOS function. Less clear is the association between cell cycle progression and an OXPHOS defect. However, specific examples, such as SDH mutations in paragangliomas and pheochromocytomas, provide proof of principle that OXPHOS defects can somehow stimulate cell division. It appears likely that some properties related to OXPHOS dysfunction can have an advantageous consequence for tumor development. However, this feature could impair cell performance at a different level. Therefore, tumor cells with an OXPHOS defect, in some cases, have compensatory mechanisms, such as an increase in mitochondrial biogenesis via overexpression of PGC-1 family members. Studies on the interplay between these metabolic and signaling pathways will help us better understand the basic mechanisms of tumor progression.

Note A recent study by Ishikawa et al. (Ishikawa K, Takenaga K, Akimoto M, et al. ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis.

Cellular Adaptations to OXPHOS Defects in Cancer

69

Science. 2008;320(5876):661–664) showed that specific mtDNA changes in complex I subunit can increase the metastatic properties of tumor cells.

References 1. Warburg O. On the origin of cancer cells. Science 1956; 123(3191):309–314. 2. Pedersen PL, Greenawalt JW, Chan TL, Morris HP. A comparison of some ultrastructural and biochemical properties of mitochondria from Morris hepatomas 9618A, 7800, and 3924A. Cancer Res 1970; 30(11):2620–2626. 3. Polyak K, Li Y, Zhu H, et al. Somatic mutations of the mitochondrial genome in human colorectal tumours. Nat Genet 1998; 20(3):291–293. 4. Fliss MS, Usadel H, Caballero OL, et al. Facile detection of mitochondrial DNA mutations in tumors and bodily fluids. Science 2000; 287(5460):2017–2019. 5. Liu VW, Shi HH, Cheung AN, et al. High incidence of somatic mitochondrial DNA mutations in human ovarian carcinomas. Cancer Res 2001; 61(16):5998–6001. 6. Nagy A, Wilhelm M, Sukosd F, Ljungberg B, Kovacs G. Somatic mitochondrial DNA mutations in human chromophobe renal cell carcinomas. Genes Chromosomes Cancer 2002; 35(3):256–260. 7. Gupta A, Rosenberger SF, Bowden GT. Increased ROS levels contribute to elevated transcription factor and MAP kinase activities in malignantly progressed mouse keratinocyte cell lines. Carcinogenesis 1999; 20(11):2063–2073. 8. Irani K, Xia Y, Zweier JL, et al. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science 1997; 275(5306):1649–1652. 9. Coller HA, Khrapko K, Bodyak ND, Nekhaeva E, Herrero-Jimenez P, Thilly WG. High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection. Nat Genet 2001; 28(2):147–150. 10. Petros JA, Baumann AK, Ruiz-Pesini E, et al. mtDNA mutations increase tumorigenicity in prostate cancer. Proc Natl Acad Sci USA 2005; 102(3):719–724. 11. Shidara Y, Yamagata K, Kanamori T, et al. Positive contribution of pathogenic mutations in the mitochondrial genome to the promotion of cancer by prevention from apoptosis. Cancer Res 2005; 65(5):1655–1663. 12. Amuthan G, Biswas G, Ananadatheerthavarada HK, Vijayasarathy C, Shephard HM, Avadhani NG. Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells. Oncogene 2002; 21(51):7839–7849. 13. Amuthan G, Biswas G, Zhang SY, Klein-Szanto A, Vijayasarathy C, Avadhani NG. Mitochondria-to-nucleus stress signaling induces phenotypic changes, tumor progression and cell invasion. EMBO J 2001; 20(8):1910–1920. 14. Simonnet H, Alazard N, Pfeiffer K, et al. Low mitochondrial respiratory chain content correlates with tumor aggressiveness in renal cell carcinoma. Carcinogenesis 2002; 23(5): 759–768. 15. Simonnet H, Demont J, Pfeiffer K, et al. Mitochondrial complex I is deficient in renal oncocytomas. Carcinogenesis 2003; 24(9):1461–1466. 16. Savagner F, Franc B, Guyetant S, Rodien P, Reynier P, Malthiery Y. Defective mitochondrial ATP synthesis in oxyphilic thyroid tumors. J Clin Endocrinol Metab 2001; 86(10):4920–4925. 17. Akimoto M, Niikura M, Ichikawa M, et al. Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells. Biochem Biophys Res Commun 2005; 327(4):1028–1035. 18. Tomlinson IP, Alam NA, Rowan AJ, et al. Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet 2002; 30(4):406–410. 19. Astuti D, Latif F, Dallol A, et al. Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. Am J Hum Genet 2001; 69(1):49–54.

70

S. Srivastava, C.T. Moraes

20. Niemann S, Muller U. Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nat Genet 2000; 26(3):268–270. 21. Baysal BE, Ferrell RE, Willett-Brozick JE, et al. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 2000; 287(5454):848–851. 22. Srivastava S, Barrett JN, Moraes CT. PGC-1alpha/beta upregulation is associated with improved oxidative phosphorylation in cells harboring nonsense mtDNA mutations. Hum Mol Genet 2007; 16(8):993–1005. 23. Savagner F, Mirebeau D, Jacques C, et al. PGC-1-related coactivator and targets are upregulated in thyroid oncocytoma. Biochem Biophys Res Commun 2003; 310(3):779–784. 24. Villani G, Greco M, Papa S, Attardi G. Low reserve of cytochrome c oxidase capacity in vivo in the respiratory chain of a variety of human cell types. J Biol Chem 1998; 273(48): 31829–31836. 25. Wu Z, Puigserver P, Andersson U, et al. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 1999; 98(1):115–124. 26. Jiang WG, Douglas-Jones A, Mansel RE. Expression of peroxisome-proliferator activated receptor-gamma (PPARgamma) and the PPARgamma co-activator, PGC-1, in human breast cancer correlates with clinical outcomes. Int J Cancer 2003; 106(5):752–757. 27. Ohta K, Endo T, Haraguchi K, Hershman JM, Onaya T. Ligands for peroxisome proliferatoractivated receptor gamma inhibit growth and induce apoptosis of human papillary thyroid carcinoma cells. J Clin Endocrinol Metab 2001; 86(5):2170–2177. 28. van Waveren C, Sun Y, Cheung HS, Moraes CT. Oxidative phosphorylation dysfunction modulates expression of extracellular matrix–remodeling genes and invasion. Carcinogenesis 2006; 27(3):409–418. 29. Delsite R, Kachhap S, Anbazhagan R, Gabrielson E, Singh KK. Nuclear genes involved in mitochondria-to-nucleus communication in breast cancer cells. Mol Cancer 2002; 1(1):6. 30. Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. Calcium and cancer: targeting Ca2+ transport. Nat Rev Cancer 2007; 7(7):519–530. 31. Giacomello M, Drago I, Pizzo P, Pozzan T. Mitochondrial Ca2+ as a key regulator of cell life and death. Cell Death Differ 2007; 14(7):1267–1274. 32. Biswas G, Adebanjo OA, Freedman BD, et al. Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of interorganelle crosstalk. EMBO J 1999; 18(3):522–533. 33. Biswas G, Anandatheerthavarada HK, Zaidi M, Avadhani NG. Mitochondria to nucleus stress signaling: a distinctive mechanism of NFkappaB/Rel activation through calcineurin-mediated inactivation of IkappaBbeta. J Cell Biol 2003; 161(3):507–519. 34. Luo Y, Bond JD, Ingram VM. Compromised mitochondrial function leads to increased cytosolic calcium and to activation of MAP kinases. Proc Natl Acad Sci USA 1997; 94(18): 9705–9710. 35. Cerutti PA. Prooxidant states and tumor promotion. Science 1985; 227(4685):375–381. 36. Burdon RH, Rice-Evans C. Free radicals and the regulation of mammalian cell proliferation. Free Radic Res Commun 1989; 6(6):345–358. 37. Burdon RH, Gill V, Rice-Evans C. Oxidative stress and tumour cell proliferation. Free Radic Res Commun 1990; 11(1–3):65–76. 38. Arora-Kuruganti P, Lucchesi PA, Wurster RD. Proliferation of cultured human astrocytoma cells in response to an oxidant and antioxidant. J Neurooncol 1999; 44(3):213–221. 39. Murrell GA, Francis MJ, Bromley L. Modulation of fibroblast proliferation by oxygen free radicals. Biochem J 1990; 265(3):659–665. 40. Mattiazzi M, Vijayvergiya C, Gajewski CD, et al. The mtDNA T8993G (NARP) mutation results in an impairment of oxidative phosphorylation that can be improved by antioxidants. Hum Mol Genet 2004; 13(8):869–879. 41. Amstad P, Crawford D, Muehlematter D, Zbinden I, Larsson R, Cerutti P. Oxidants stress induces the proto-oncogenes, C-fos and C-myc in mouse epidermal cells. Bull Cancer 1990; 77(5):501–502.

Cellular Adaptations to OXPHOS Defects in Cancer

71

42. Abate C, Patel L, Rauscher FJ 3rd, Curran T. Redox regulation of fos and jun DNA-binding activity in vitro. Science 1990; 249(4973):1157–1161. 43. Flohe L, Brigelius-Flohe R, Saliou C, Traber MG, Packer L. Redox regulation of NF-kappa B activation. Free Radic Biol Med 1997; 22(6):1115–1126. 44. Baeuerle PA. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim Biophys Acta 1991; 1072(1):63–80. 45. Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 1997; 9(2): 240–246. 46. Shi X, Dong Z, Huang C, et al. The role of hydroxyl radical as a messenger in the activation of nuclear transcription factor NF-kappaB. Mol Cell Biochem 1999; 194(1–2):63–70. 47. Domann FE Jr, Levy JP, Finch JS, Bowden GT. Constitutive AP-1 DNA binding and transactivating ability of malignant but not benign mouse epidermal cells. Mol Carcinog 1994; 9(2):61–66. 48. Domann FE, Levy JP, Birrer MJ, Bowden GT. Stable expression of a c-JUN deletion mutant in two malignant mouse epidermal cell lines blocks tumor formation in nude mice. Cell Growth Differ 1994a; 5(1):9–16. 49. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ Jr, Sledge GW Jr. Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth. Mol Cell Biol 1997; 17(7):3629–3639. 50. Lee FS, Hagler J, Chen ZJ, Maniatis T. Activation of the IkappaB alpha kinase complex by MEKK1, a kinase of the JNK pathway. Cell 1997; 88(2):213–222. 51. Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem 1995; 270(28):16483–16486. 52. Gupta A, Butts B, Kwei KA, et al. Attenuation of catalase activity in the malignant phenotype plays a functional role in an in vitro model for tumor progression. Cancer Lett 2001; 173(2):115–125. 53. Oberley LW, Oberley TD. Role of antioxidant enzymes in cell immortalization and transformation. Mol Cell Biochem 1988; 84(2):147–153. 54. St Clair DK, Wan XS, Oberley TD, Muse KE, St. Clair WH. Suppression of radiation-induced neoplastic transformation by overexpression of mitochondrial superoxide dismutase. Mol Carcinog 1992; 6(4):238–242. 55. Zhao Y, Xue Y, Oberley TD, et al. Overexpression of manganese superoxide dismutase suppresses tumor formation by modulation of activator protein-1 signaling in a multistage skin carcinogenesis model. Cancer Res 2001; 61(16):6082–6088. 56. Li JJ, Oberley LW, St. Clair DK, Ridnour LA, Oberley TD. Phenotypic changes induced in human breast cancer cells by overexpression of manganese-containing superoxide dismutase. Oncogene 1995; 10(10):1989–2000. 57. Li JJ, Oberley LW, Fan M, Colburn NH. Inhibition of AP-1 and NF-kappaB by manganesecontaining superoxide dismutase in human breast cancer cells. FASEB J 1998; 12(15): 1713–1723. 58. Pich S, Bach D, Briones P, et al. The Charcot-Marie-Tooth type 2A gene product, Mfn2, up-regulates fuel oxidation through expression of OXPHOS system. Hum Mol Genet 2005; 14(11):1405–1415. 59. Chen H, Chomyn A, Chan DC. Disruption of fusion results in mitochondrial heterogeneity and dysfunction. J Biol Chem 2005; 280(28):26185–26192. 60. Hruban Z, Swift H, Rechcigl M Jr. Fine structure of transplantable hepatomas of the rat. J Natl Cancer Inst 1965; 35(3):459–495. 61. Evans IH, Diala ES, Earl A, Wilkie D. Mitochondrial control of cell surface characteristics in Saccharomyces cerevisiae. Biochim Biophys Acta 1980; 602(1):201–206. 62. Soslau G, Fuhrer JP, Nass MM, Warren L. The effect of ethidium bromide on the membrane glycopeptides in control and virus-transformed cells. J Biol Chem 1974; 249(10):3014–3020. 63. Dey R, Moraes CT. Lack of oxidative phosphorylation and low mitochondrial membrane potential decrease susceptibility to apoptosis and do not modulate the protective effect of Bcl-x(L) in osteosarcoma cells. J Biol Chem 2000; 275(10):7087–7094.

72

S. Srivastava, C.T. Moraes

64. Manfredi G, Kwong JQ, Oca-Cossio JA, et al. BCL-2 improves oxidative phosphorylation and modulates adenine nucleotide translocation in mitochondria of cells harboring mutant mtDNA. J Biol Chem 2003; 278(8):5639–5645. 65. Sekito T, Thornton J, Butow RA. Mitochondria-to-nuclear signaling is regulated by the subcellular localization of the transcription factors Rtg1p and Rtg3p. Mol Biol Cell 2000; 11(6):2103–2115. 66. Miceli MV, Jazwinski SM. Common and cell type-specific responses of human cells to mitochondrial dysfunction. Exp Cell Res 2005; 302(2):270–280. 67. Epstein CB, Waddle JA, Hale WT, et al. Genome-wide responses to mitochondrial dysfunction. Mol Biol Cell 2001; 12(2):297–308.

Regulation of Glucose and Energy Metabolism in Cancer Cells by Hypoxia Inducible Factor 1 ´ ´ C´esar Ferreira and Elida Tulio Geralda Campos

Abstract A crucial aspect of carcinogenesis is the adaptation of cells to the changing environmental conditions of the tumor mass. To grow and proliferate, the cells need nutrients that have to be delivered by surrounding vessels. In addition, they have to cope with a gradient of oxygen tension as the tumor mass grows. Hypoxia inducible factor 1 (HIF-1) mediates important responses to physiologic and pathologic processes that allow angiogenesis, erythropoiesis, cell proliferation and survival, and metabolic changes to take place. It is a heterodimeric transcription factor regulated by a complex network. Among the major metabolic changes evident in cancer cells is the activation of the glycolytic pathway, even in the presence of oxygen. HIF-1 regulates glucose and energy metabolism by directly activating the gene expression of glycolytic enzymes, regulatory enzymes, and glucose transporters. HIF-1␣ (the regulated subunit of the HIF-1 complex) is regulated mainly at the posttranscriptional level through mechanisms that block its proteasomal degradation depending on the oxygen concentration. HIF-1 is overexpressed in many tumors, and evidence is mounting that HIF-1 regulation of glucose and energy metabolism is important in carcinogenesis and tumor progression. Keywords HIF-1 · HIF-1␣ · Glycolysis · von Hippel–Lindau tumor suppressor protein (pVHL) · Lactate · Glucose transporter · Regulation of gene expression · Hypoxia response element (HRE) · Prolyl hydroxylases

1 Oxygen Homeostasis Cells respond to extracellular and intracellular stimuli to maintain homeostasis, and hypoxia is one of the most fundamental environmental stimuli. Oxygen homeostasis is crucial to maintain the approximately 1014 cells in the adult human body with

´ E.G. Campos (B) Departamento de Biologia Celular, Universidade de Bras´ılia, CEP 70910-900, Bras´ılia, DF, Brazil e-mail: [email protected] S.P. Apte, R. Sarangarajan (eds.), Cellular Respiration and Carcinogenesis, DOI 10.1007/978-1-59745-435-3 6,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

73

74

´ T.C. Ferreira, E.G. Campos

an adequate supply of O2 . The maintenance of the O2 levels within strict limits is essential throughout life but is particularly important during periods of rapid cellular proliferation, such as in normal growth or during the development of neoplasias. In normal tissues, the average oxygen tension is 40 to 50 mm Hg, whereas in tumors these levels are 5 to 10 mm Hg. Control of O2 levels in the tissues of animals occurs through the combination of gene regulation and biochemical and physiologic mechanisms. Low levels of oxygen in the environment lead cells and tissues to adapt to this adverse condition. Significant changes in gene expression and activation of signaling pathways occurs in response to hypoxia, a characteristic condition in solid tumors, resulting in an elevated transcription of angiogenic, hematopoietic, and some metabolic genes. Hypoxia inducible factor 1 (HIF-1) was discovered in 1992 by Gregg L. Semenza and Guang L. Wang at the Johns Hopkins University School of Medicine. It is currently known as the central regulator of the response to low levels of oxygen. HIF-1 is a heterodimeric transcription factor and regulates genes whose products participate in glycolysis, glucose transport, angiogenesis, cell proliferation and survival, pH regulation, and cell migration.

2 HIF-1 Structure HIF-1␣ protein is one of the subunits that form HIF-1 and belongs to the basic helix-loop-helix (bHLH)-PAS family of transcription factors. PAS is an acronym that refers to the Drosophila proteins Period (or PER), ARNT, and Single-minded or (SIM), the first proteins in which this motif was identified. HIF-1␣ has the bHLHPAS domain, which is in the N-terminal region of the protein and has two repeats (A and B) in its structure. This subunit binds to the second subunit that forms HIF-1, the aryl hydrocarbon receptor nuclear translocator (ARNT), also referred as HIF-1␤. ARNT is a nuclear protein constitutively expressed that also participates in the transcriptional response to xenobiotics when interacting with the aryl hydrocarbon receptor (AHR). As with HIF-1␣, the ␤ subunit also belongs to the bHLH-PAS family of transcription factors. HIF-1␣ and HIF-1␤ share some amino acid sequence similarities. The bHLH-PAS domains in both subunits are crucial for the heterodimerization and binding of these proteins to DNA. HIF-1␣ is an 826-amino-acid protein that has two transactivation domains (TADs), one in the N-terminal region (TAD-N) that overlaps with the oxygen degradation dependent (ODD) domain and another in the C-terminal region (TAD-C), the latter also found in the HIF-1␤ (Fig. 1). The TAD domains are localized at amino acids 531 to 575 (TAD-N) and 786 to 826 (TAD-C) of the HIF-1␣. The TAD-C domain of HIF-1␣ interacts with the transcriptional activators CREB binding protein (CBP) and p300. Maintenance of cysteine 800 in the TAD-C in a reduced state is essential for p300/CBP binding. Therefore, HIF-1␣ redox state is involved in its activation. HIF-1␣ and p300 interaction requires a leucine-rich hydrophobic interface that is regulated by the reversible change between hydrophobic and hydrophilic Cys800 of HIF-1␣. The reduced state of this cysteine is maintained by redox factor-1

Regulation of Glucose and Energy Metabolism in Cancer Cells by HIF-1

75

Fig. 1 HIF-1 proteins ␣ and ␤ have similar structural domains. HIF-1␣ (upper drawing) is a 826-amino-acid protein with a basic helix-loop-helix-PAS (bHLH-PAS) domain in the N-terminal and repeats A and B. There are two transactivation domains (TADs), one located in the N-terminal region (TAD-C), which overlaps with the oxygen degradation dependent (ODD) domain, and other in the C-terminal region (TAD-C). Both TADs are connected by an inhibitory domain (ID). HIF1␤ (lower drawing) is a 789-amino-acid protein, which also belongs to the bHLH-PAS family of transcription factors. As with HIF-1␣, HIF-1␤ also has the bHLH-PAS domain in the N-terminal and repeats A and B. However, it has only one TAD, which is located in the C-terminal of the protein (see Color Insert)

[1] and mediated by thioredoxin (TRX). Therefore, compounds that inhibit thioredoxin cause inhibition of HIF-1␣–mediated transactivation of HIF-1 target genes. Both TAD-C and TAD-N domains are connected by a inhibitory domain (ID). The ODD domain is a proline-serine-threonine–rich domain involved in the regulation of protein stability as a function of O2 concentration. In HIF-1␣, the main target amino acids for hydroxylation are localized in this domain (described below). HIF1␣ protein contains two nuclear localization signals (NLS), one in the N-terminal (NLS-N) and the other in the C-terminal, which encompass the amino acids 17 to 23 and 718 to 721, respectively [1].

3 HIF-1␣ Regulation HIF-1␣ regulation is complex and is governed by posttranslational mechanisms (Fig. 2). Protein stability and activity are regulated by covalent modifications such as hydroxylation, acetylation, S-nitrosation, and phosphorylation. Among them, hydroxylation and phosphorylation are the most studied. In normoxia conditions, hydroxylated HIF-1␣ is recognized by the von Hippel–Lindau tumor suppressor protein (pVHL), which is part of a polyubiquitination complex. HIF-1␣ is hydroxylated by a HIF prolyl hydroxylase (PHD). pVHL recognizes the hydroxylated proline residues (Pro402 and Pro564) located in the human HIF-1␣ ODD domain. PHD is a dioxygenase that uses O2 and 2-oxoglutarate (2-OG) as substrates, the latter generated by the tricarboxylic acid (TCA) cycle or by the transamination of amino acids. This enzyme transfers the first oxygen atom to the proline residue, and the second oxygen atom reacts with the 2-OD producing succinate. In addition to oxygen and 2-OG, the PHD proteins require Fe2+ and ascorbate as cofactors [2]. The first 2-OG dioxygenase to be identified was procollagen prolyl hydroxylase, which participates in collagen synthesis and catalyzes trans-4-hydroxylation reactions. There are three PHD isoenzymes: 1, 2, and 3. PHDs hydroxylate specific proline residues inside a highly conserved domain whose sequence is LXXLAP

76

´ T.C. Ferreira, E.G. Campos

Fig. 2 Mechanisms of HIF-1␣ regulation. Under normoxia conditions, HIF-1␣ is hydroxylated by prolyl hydroxylase (PHD), an enzyme that belongs to the family of dioxygenases and requires O2 and 2-oxoglutarate (2-OG) as substrates. This enzyme also uses iron as cosubstrate, which is maintained in the reduced form (Fe+2 ) by ascorbic acid. Iron can be scavenged by treatment of cells in normoxia with desferrioxamine (DFO), mimicking a hypoxia condition. Nickel and cobalt also promote HIF-1␣ stabilization in normoxia condition by competing with iron. The pVHL recognizes the hydroxylated (OH) proline residues 402 and 564. pVHL is a member of E3-ubiquitinligase complex formed by elongin C (EloC), elongin B (EloB), and cullin 2 (CUL2) and RBX1.

Regulation of Glucose and Energy Metabolism in Cancer Cells by HIF-1

77

(where X indicates any amino acid; P indicates the hydroxylated proline; L, leucine and A, alanine). Concerning the cellular localization, PHD1 is found exclusively in the nucleus, PHD2 in the cytoplasm, and PHD3 is found in both cytoplasm and nucleus, with predominance in the nucleus. Selective blockage of HIF-1␣ expression using RNA interference technology leads to a complete loss of PHD2 and PHD3 gene expression under hypoxia, whereas PHD1 mRNA levels remain unchanged [2]. It seems that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-1␣. This negative feedback mechanism probably limits HIF-1␣ accumulation in hypoxia and leads to accelerated degradation on reoxygenation after long period of hypoxia. RNA interference showed the participation of PHD2 on HIF-1␣ hydroxylation. Indeed, the specific silencing of PHD2 stabilized the HIF1␣ in normoxia conditions in several tested human cells. However, the silencing of PHD1 and PHD3 has no effect in HIF-1␣ stabilization in both normoxia and reoxgenation of cells briefly exposed to hypoxia. Therefore, it seems that PHD2 is the critical oxygen sensor that maintains HIF-1␣ in a low level in normoxic conditions [3]. Under normoxia, HIF-1␣ activity is also regulated by hydroxylation of asparagine 803 in the TAD-C domain, catalyzed by an enzyme known as factor inhibiting HIF-1 (FIH), asparaginyl hydroxylase. FIH also belongs to the superfamily of 2-OD–dependent dioxygenases whose activity requires O2 as substrate. The hydroxylation of Asn803 does not lead to HIF-1␣ degradation. Therefore, this residue is not directly involved in the O2 -sensing mechanism. Hydroxylation of Asn803 abolishes the interaction between HIF-1␣ and its transcriptional coactivators CBP and p300. CBP and p300 are coactivators that participate in the activities of many different transcription factors. The CBP/p300 proteins have a key role in coordinating and integrating multiple signal-dependent events. They make possible the adequate expression levels of genes in response to diverse physiologic stimuli and influences; for example, proliferation, differentiation, and apoptosis. 

Fig. 2 (Continued) This complex is responsible for HIF-1␣ polyubiquitination prior to degradation by the proteasome. Factor inhibiting HIF-1 (FIH-1) hydroxylates the asparagine 803 (N803) residue of HIF-1␣ preventing interaction with the CBP/p300. ARD1 acetylates the lysine 532 residue of HIF-1␣ leading to its degradation. Hypoxia, oncogenes, and growth factors can activate the phosphatidylinositol 3-kinase (PI3K) pathway, which has AKT as a downstream target. AKT can also be activated by phosphatidylinositol-dependent protein kinase 1 (PDK-1). PI3K/AKT activation causes HIF-1␣ overexpression mediated by mTOR (mammalian target of rapamycin). MEK (MAPK kinase) participates in the phosphorylation pathway activating the extracellular signal–regulated kinases (ERK1/2), which phosphorylate HIF-1␣. Phosphorylated HIF-1␣ is stabilized and activated, enters into the nucleus, forms the heterodimer with HIF-1␤, is coactivated by CBP/p300 complex, and induces the transcription of more than 100 target genes. A cysteine residue is kept in the reduced state by redox factor-1 (REF-1) mediated by thioredoxin (TRX), which participate in HIF-1␣ stabilization. Under hypoxia, nitric oxide (NO) disfavors HIF-1␣ accumulation (activates PHDs), and under normoxia condition, NO “mimics” hypoxia conditions (inhibits PHDs) and leads to HIF-1␣ accumulation . Some evidence points to the role of reactive oxygen species (ROS) as messengers that regulate HIF-1 activity (see Color Insert)

78

´ T.C. Ferreira, E.G. Campos

pVHL is a component of the E3 ubiquitin-protein ligase complex that drives the proteolysis in the proteasome, preventing the transcriptional activation of its target genes. E3 ubiquitin ligases are a family of proteins that participate in the degradation and activity of many cellular proteins and function together with ubiquitinactivating enzyme E1 and ubiquitin-conjugating enzyme E2. In the hydroxylated HIF-1␣ degradation pathway, E3 ubiquitin-protein ligase complex is formed by pVHL, elongin C, elongin B, cullin 2, and RBX1 (Fig. 2). The E3 complex is capable of functioning with E1 ubiquitin-activation and E2 ubiquitin-conjugating enzymes to mediate the ubiquitination of HIF-1␣ leading to its rapid proteosomal degradation. HIF-1␣ has a half-life of approximately 5 minutes. However, when HIF-1␣ escapes the degradation pathway, its detection by Western blot experiments occurs within minutes. Indeed, HIF-1␣ is detectable in the nucleus of cells after less than 2 minutes of exposure to anoxia or hypoxia. In this case, HIF-1␣ escapes ubiquitination and proteosomal degradation, is stabilized by posttranslational modifications, and enters into the nucleus. A comprehensive study about HIF1␣ turnover (analyzed by electrophoretic mobility shift assay; EMSA) showed that its maximal protein expression occurs in cells exposed for 1 hour in anoxia/hypoxia. These expression levels are maintained after 4 hours at low levels of oxygen exposure. However, after reoxygenation of cells, a decrease in the HIF-1 DNA binding activity is observed after 2 minutes. In addition, the nuclear HIF-1␣ protein was reduced within 4 to 8 minutes, down to a level below the detection limit within 32 minutes [4]. Under hypoxia conditions, HIF-1␣ is no longer degraded; it is phosphorylated in the cytoplasm and activates its target genes inside the nucleus. Once the target genes are expressed, a physiologic response to low oxygen concentration is achieved. Little is known about the fine-tuning of the posttranslational modifications and their role in HIF-1␣ activity. The MAPK (mitogen-activated protein kinase) and phosphatidylinositol-3-kinase (PI3K) pathways are certainly involved in the regulation of HIF-1␣. ERK1/2 (also called p44/42) activation, which is a result of activation of the upstream proteins Ras/Raf-1/MEK-1/ERK1/2, leads to HIF1␣ phosphorylation. Although MAPK phosphorylates HIF-1␣ in vitro, studies on mutations in the phosphorylation sites in HIF-1␣ protein show that its transactivation capacity is not affected or directly mediated by phosphorylation. Regulation of HIF-1␣ by MAPK is still an open issue [5]. Loss of tumor suppressor activity may also contribute to HIF-1␣ overexpression. For example, interaction of the p53 tumor suppressor gene with HIF-1␣ negatively regulates HIF-1␣ protein levels. Therefore, HIF-1␣ overexpression correlates with expression of mutant p53 in human cancer. AKT (protein kinase B), a serine/threonine kinase, is commonly activated in cancer cells. This protein functions downstream to PIP3 (phosphatidylinositol 3,4,5-triphosphate) in the PI3K phosphorylation pathway (Fig. 2). PI3K/AKT signaling pathway regulates vascular endothelial growth factor (VEGF) expression through HIF-1␣ in several types of transformed cells, including ovarian cancer and human gastric adenocarcinoma. Cancer cells have constitutive AKT

Regulation of Glucose and Energy Metabolism in Cancer Cells by HIF-1

79

activity through amplification of PI3K signaling pathway or deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a PI3K antagonist. AKT induction is an important event in tumorigenesis as this pathway leads to a higher level of glycolysis in a dose-dependent manner that correlates with a more aggressive malignancy in vivo. Apparently, AKT has an effect more pronounced on glucose metabolism than it effects on proliferation and survival of these transformed cells. This phenomenon was observed in established human glioblastoma cells, which showed a switch to aerobic glycolysis and glucose dependence [6]. HIF-1␣ is also modified in the lysine residue 532 localized in the ODD domain of HIF-1␣ by an acetyl transferase called arrest defective-1 (ARD1). ARD1 was originally identified in the yeast Saccharomyces cerevisiae and received this name due to the phenotype of mutant yeast defective in the mitotic cell cycle. A human variant of ARD1 is able to partially decrease HIF-1␣ accumulation in hypoxia [7]. Another HIF-1␣ posttranslational modification is S-nitrosation. Nitric oxide (NO) promotes HIF-1␣ stabilization, DNA binding, and transactivation of target genes under normoxic conditions. This NO effect on HIF-1␣ activity was verified in several cell lines, including swine tubular cells (LLC-PK1), human glioblastoma and hepatoma, as well as endothelial cells from bovine pulmonary artery. These cells, when treated with different NO donors, such as S-nitroglutathione, (Z)-1[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (NOC-18), 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5), and S-nitroso -N-acetyl-D,L-penicillamine (SNAP), accumulate HIF-1␣ protein [8]. Therefore, under hypoxia, NO disfavors HIF-1␣ accumulation, whereas under normoxic conditions, NO “mimics” hypoxia conditions and leads to HIF-1␣ accumulation.

4 HIF-1 Isoforms There are two other members of the bHLH-PAS superfamily that share structural similarity to HIF-1␣: HIF-2␣, also named endothelial PAS-domain protein 1 (EPAS1), and HIF-3␣. The roles of the 2␣ and 3␣ subunits are poorly understood. Similarly to HIF-1␣, HIF-2␣ is also regulated by hydroxylation of the conserved proline residue, which causes its degradation under normoxic conditions via ubiquitin-E3-ligase. HIF-2␣ has 48% amino acid sequence identity with HIF-1␣. HIF-2␣ and HIF-3␣ also have the common ability to heterodimerize to HIF-1␤ and bind to the hypoxia response element (HRE) motif in the regulatory regions of target genes. Additionally, an alternative splice variant of HIF-3␣ exists, a protein that lacks the transactivation domain. This splice variant was termed inhibitory PAS (IPAS) protein, and it was identified as the dominant-negative regulator of HIF-1. IPAS interacts with the amino-terminal region of HIF-1␣ and prevents its DNA binding. The effect of the different members of the HIF-1 family on gene expression can vary depending on the cell type. For example, HIF-1␣ and HIF-2␣ are strongly expressed in the kidney, vascular cells, bone marrow, macrophages, lungs,

80

´ T.C. Ferreira, E.G. Campos

endothelium, and carotid body, whereas HIF-3␣ is mainly expressed in Purkinje cells of the cerebellum and mouse cornea epithelial cells. HIF-2␣ has more restrictive distribution inside tissues than does HIF-1 [9].

5 HIF-1␣ Induction by Nonhypoxic Stimuli In addition to hypoxia, HIF-1 is also responsive to several nonhypoxic stimuli, including insulin, platelet-derived growth factor (PDGF), transforming growth factor ␤ (TGF-␤), insulin-like growth factor (IGF-1), thrombin angiotensin II, cytokines, and carbachol (activates the muscarinic acetylcholine receptors). Whereas growth factor stimulation of HIF-1␣ is cell type specific, hypoxia increases HIF-1␣ expression in all cell types. In addition, HIF-1 is activated by some metals such as cobalt, chromium, nickel, arsenic, and iron. Desferrioxamine also activates HIF-1 by mimicking hypoxic conditions. Some evidences point to the role of reactive oxygen species (ROS) as messengers that regulate HIF-1 activity [10]. The oxidative status of the iron ion bound to the PHDs might also regulate HIF-1␣ activity, as these enzymes are Fe2+ -dependent. ROS generated by mitochondria and released into the cytoplasm of cells exposed to hypoxia promote oxidation of Fe2+ to Fe3+ . A decrease in the Fe2+ availability would cause reduction in the PHD activity. This mechanism allows HIF-1 accumulation and stabilization [11].

6 HIF-1 in Cancer Once stabilized and activated, inside the nucleus, HIF-1 recognizes and binds to hypoxia response elements (HREs) present in the regulatory regions of its target genes. The consensus HRE has the sequence 5 -BRCGTGVBBB-3 , where the RCGTG core is the HIF-1 binding site (HBS). Microarray experiments suggest that more than 200 HIF-1 target genes might exist. However, not all of them are likely to be directly regulated by an HRE in their regulatory regions [10]. More than 100 HIF1 target genes are known, including the genes that encode erythropoietin, VEGF, glucose transporters, and glycolytic enzymes. Erythropoietin (EPO) and VEGF play crucial roles in adaptive responses to systemic and local hypoxia. Both are implicated in tumor progression. EPO stimulates erythropoiesis, which increases O2 delivery, whereas VEGF stimulates angiogenesis, which increases delivery of both O2 and energy substrates such as glucose. Rigorous analysis has confirmed the role of HIF-1 in EPO and VEGF transcriptional activation, and consequently in carcinogenesis. HIF-1␣ is overexpressed in many human cancers [12]. Immunohistochemical staining analysis of surgical biopsy specimens show overexpression of HIF-1␣ protein in colon, ovary, lung, prostate, skin, stomach, brain, and breast cancers. Significant associations between HIF-1␣ overexpression and patient mortality have been shown in cancers of the breast, brain, cervix, oropharynx, esophagus, ovary, and

Regulation of Glucose and Energy Metabolism in Cancer Cells by HIF-1

81

uterus. Premalignancy and neomalignant lesions as well as cancer metastases of some cancer types also show increased HIF-1␣ protein. For example, HIF-1␣ is overexpressed in the majority of breast and colon cancer metastases. By contrast, association between HIF-1␣ overexpression and decreased mortality were reported for patients with head and neck cancer and non–small cell lung cancer. Thus, the effect of HIF-1␣ overexpression seems to be dependent on the cancer type. HIF-1␣ can have antiapoptotic or proapoptotic effects on tumor cells [9]. Mutation in the VHL gene results in the so called von Hippel–Lindau syndrome, characterized by the formation of tumors and fluid-filled sacs (cysts) in many different parts of the body. Clear cell renal carcinoma (CCRC) cells lacking pVHL tumor suppressor constitutively express HIF-1␣ and HIF-1 target genes under nonhypoxic conditions [13]. CCRCs present all the main characteristics supposed to be maintained by proteins encoded by genes regulated by HIF-1: increased glycolysis, decreased respiration, increased glucose uptake and lactate production. pVHL lossof-function also occurs in hemangioblastoma. Therefore, at least in these two types of tumors, deregulation of HIF-1␣ regulatory pathway plays a causal role.

7 HIF-1, Cancer, and the Glycolytic Pathway The way cells change their metabolism under hypoxia shares many similarities to the way tumors cells change their metabolism to survive and proliferate. Indeed, many of the known oncogenic signaling pathways overlap with hypoxia-induced pathways. Cancer cells have special metabolic needs for their proliferation and survival. They need to cope with different levels of oxygen and adjust their metabolism to guarantee cell proliferation and minimize apoptosis. The vast majority of human and animal tumors display a high rate of glycolysis, increased glucose uptake, increased lactate production, and decreased respiration under aerobic conditions (a phenomenon known as the Warburg effect) [14]. Aerobic glycolysis provides cancer cells (both nonmetastatic and metastastic) a high competitive capacity over normal cells. This adaptive response can be driven by hypoxia, as well as by mutations that inactivate tumor suppressor genes or activate oncogenes. Hypoxia is a potent inducer of gene expression, especially of genes involved in glycolysis for maintaining cellular energy. The higher glycolytic rate in tumor cells compared with that of nontumorigenic cells can be attained by the increased transcription, followed by translation, of glycolytic genes and glucose transporters associated or not with inhibition of oxidative phosphorylation. Hypoxia has HIF-1 as the key regulator. Evidence is surmounting that HIF-1 regulation of glycolysis is important in carcinogenesis. However, there is much controversy as to whether HIF-1– induced glycolysis is a reactive process or a “cause” of cancer. A definitive solution to this key question is complicated by the fact that hypoxia and HIF-1 are not exclusive regulators of the glycolytic enzymes, and HIF-1 activation is not associated only with hypoxia (oncogenes and growth factors can also activate this pathway). In addition, high pyruvate concentrations result in HIF-1␣ stabilization indepen-

82

´ T.C. Ferreira, E.G. Campos

dently of hypoxia [15].Therefore, although hypoxic HIF-1 activation alone does not explain aerobic glycolysis, other factors may provide feedback to enhance normoxic HIF-1 induction. The structure, regulation, and action of HIF-1 was discussed in detail above and shows a very complex network (Fig. 2). Regardless of which pathway leads to HIF-1 activation, its effect on glucose metabolism is due to the upregulation of specific target genes. Regulation of metabolism by HIF-1␣ was evidenced through investigation of its function in mouse embryonic stem (ES) cells in which the Hif1␣ gene was inactivated by homologous recombination. Analysis of Hif1a+/+ , Hif1a+/- , and Hif1a-/ES cells revealed that HIF-1␣ is required for the induction of several genes whose products are involved in glucose metabolism; more specifically, aldolase A and C, enolase 1, hexokinase I and II, phosphofructokinase L, phosphoglycerate kinase I, pyruvate kinase M, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase A, GLUT1 and GLUT3 [16]. HRE has been characterized in the aldolase A, enolase 1, and lactate dehydrogenase A human promoters and also in the aldolase C mouse promoter. Complete identification of the key gene products specifying HIFdependent tumor metabolism is crucial for therapeutic targeting of HIF-1 signaling in cancer. In the presence of oxygen, glucose is converted to pyruvate by the glycolytic enzymes. Pyruvate is transported to the mitochondrial matrix, where it is converted to acetyl-CoA and metabolized in the TCA cycle. Under hypoxia, pyruvate is converted to lactate through lactate dehydrogenase (LDH) A in the cytoplasm, which is a target for HIF-1 regulation. In cancer cells, aerobic glycolysis and lactate production is favored to the detriment of oxidative phosphorylation. To understand the role of HIF-1 in the regulation of glucose and energy metabolism in cancer cells, we reviewed the literature in search of experimental data demonstrating the direct involvement of HIF-1 in the induction of such genes in these cells. In some cases, the link between HIF-1 and upregulation of a gene in cancer cells was based on the presence of HRE motifs in their regulatory regions. In the next sections, we describe the link found between HIF-1 and the proteins involved in glucose metabolism in cancer cells.

8 Hexokinase Hexokinases (HKs) catalyze the first step in the glycolytic pathway, conversion of glucose to glucose-6-phosphate (G6P). Human cells express four isozymes of hexokinases: types I, II, III, and IV (glucokinase). The key hexokinase isoenzyme involved in cancer promotion is the type II (HKII), the major isozyme overexpressed in tumors exhibiting the high glycolytic phenotype. HKII has been extensively studied by Dr. Peter Pedersen from Johns Hopkins University School of Medicine and his co-workers [17]. They found that HKII binds to transmembrane channels formed by the porin-like protein voltage-dependent anion channel (VDAC) located within the outer mitochondrial membrane. This interaction reduces the enzyme’s sensitivity

Regulation of Glucose and Energy Metabolism in Cancer Cells by HIF-1

83

to product inhibition by G6P and allows the enzyme to have direct and rapid access to mitochondrially generated ATP. It also protects HKII against proteolytic degradation. These combined properties, together with the high content of the enzyme in highly malignant tumors (>100-fold increase), result in the rapid production of G6P. This key metabolic intermediate-precursor serves as a major carbon source for most biosynthetic pathways that are essential for the growth and rapid proliferation of tumors. It also serves as the initial substrate for glycolysis and ATP generation. Enhanced transcription of HKII occurs in malignant tumors, implicating the HKII promoter activation and upregulation during tumorigenesis. Functional HREs (HIF-1 responsive) are located in the distal region of the HKII promoter. HKII can also be transcriptionally activated by mutant p53 or through demethylation of its promoter. Regulation of the gene encoding hepatoma HKII occurs through activation of its promoter that contains in its distal region two overlapping E-box/HIF-1 sequences separated by approximately 50 base pairs. These sequences are known to be associated in other gene promoters with responsiveness to glucose (E-box) or hypoxic conditions (HIF-1). The response of the HKII gene promoter to both glucose and hypoxic conditions is not confined to its distal region but involves significantly the proximal region as well. Elements in this region remain to be identified and studied in detail.

9 Glucose Phosphate Isomerase Glucose phosphate isomerase (GPI) is a housekeeping cytosolic enzyme of glucose metabolism that plays a key role in both glycolysis and gluconeogenesis pathways, catalyzing the reversible isomerization of glucose-6-phosphate and fructose6-phosphate. Upon secretion, GPI also acts as a cytokine also referred to as autocrine mobility factor (AMF) or NLK [18]. AMF was originally isolated as a cytokine autocrine factor that stimulates cell mobility and NLK as a lymphokine and neurotrophic factor for spinal and sensory neurons. GPI/AMF/NLK is associated with the development of tumors, and an increased amount of this protein is present in the urine and serum of patients with various malignancies. Therefore, in addition to its role in the glycolytic pathway, the induction of this gene under hypoxia may play a role in stimulating cell mobility, invasion, and metastasis in tumor cells. In human pancreatic cancer PC-3 cells, GPI mRNA is weakly expressed in cells cultured in normoxic conditions, but its synthesis is markedly increased in hypoxic conditions (1% oxygen plus CoCl2 ). YC-1, 3-[5 -hydroxymethyl-2 furyl]-1-benzyl indazole, a guanylate cyclase activator, reduces the mRNA levels of GPI in PC-3 cells in a dose-dependent manner under hypoxic conditions. Guanylate cyclase activation can increase production of NO, which inhibits the accumulation and activity of HIF-1 in hypoxia. Therefore, GPI transcriptional activation is intimately linked to HIF-1 activity.

84

´ T.C. Ferreira, E.G. Campos

10 Phosphofructokinase Phosphofructokinase 1 (PFK-1) catalyses the rate-limiting phosphorylation of fructose-6-phosphate to fructose-1,6-biphosphate, which is an energy-consuming step in glycolysis. The fructose-2,6-biphosphate is the most potent allosteric activator of glycolysis and exerts control over the rate of glucose utilization. It activates PFK-1 and inhibits the gluconeogenic enzyme fructose-1,6-biphosphatase. Because of the antagonistic effects in these enzymes, the product of the reaction catalyzed by 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKBF) plays an essential role in the opposing glycolytic and gluconeogenic pathways. In human cells, there are at least four different genes encoding PFKFBs (PFKBP-1, PFKBP-2, PFKBP-3, and PFKBP-4). They all respond to hypoxia in vivo, and this response is diverse in different organs. Tissue-specific isoforms of PFKBP are not completely exclusive, and several tissues express more than one isoform. Among the PFKFB isoforms of mammalian origin, PFKFB3 has the highest kinase:phosphatase activity (K/B) and is the most highly expressed isozyme in transformed cells. The PFKFB-4 isozyme lacks a serine phosphorylation residue that is critical for the downregulation of its kinase activity. Therefore, one can conclude that it may have a high K/B ratio, and it should greatly promote glycolysis under conditions of limited oxygen supply. The use of dimethyloxalylglycine (a specific competitive inhibitor of PHDs) demonstrated the role of HIF-1␣ in hypoxic induction of the genes coding for PFKFB3 and PFKFB-4 isoforms (in human hepatoma Hep-3B and HepG2 cell lines, human prostate cancer cell, PC-3 and HeLa cells). A splice variant of PFKFB-4 (PFKFB-4 s) is also induced by hypoxia in DB-1 melanoma, besides the full-length PFKFB-4. Hypoxic induction of the transcription of these genes is mediated by HREs located in their promoter regions. In addition, PFKFB3 and PFKFB4 isoforms are overexpressed in solid malignant tumors from the breast and colon, overexpression of the PFKFB-4 protein being higher compared with that of PFKBF-3 [19].

11 Aldolase Aldolase (ALD) catalyzes the reversible aldol cleavage of fructose-6-phosphate into two trioses, glyceraldehyde-3-phosphate and dihydroxyacetone. It has a role in the glycolytic, gluconeogenic, and fructose metabolic pathways. Three tissue-specific forms exist in higher vertebrates: aldolase A (predominately in muscle and red blood cells), aldolase B (predominately in liver, kidney, and intestine), and aldolase C (predominately in brain). The direct association of aldolase gene expression and HIF-1 in tumors was observed in a study with a mouse hepatoma cell line, Hepa1c1c7, and its mutant derivative, ARNT-deficient c4T. The absence of the ARNT protein completely abolished the oxygen regulation of the aldolase A and phosphoglycerate kinase 1 (PGK1) gene expression, indicating that these genes share a common pathway of response to hypoxia involving activation of HIF-1. Among the three

Regulation of Glucose and Energy Metabolism in Cancer Cells by HIF-1

85

isoenzymes, aldolases A and C seem to have evolved to perform the glycolytic reaction, fructose-1,6-phosphate cleavage, more efficiently than does aldolase B. Aldolase B seems to be more involved with the gluconeogenic pathway [20].

12 Glyceraldehyde Phosphate Dehydrogenase Glyceraldehyde phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis and also a multifunctional protein. It converts glyceraldehyde-3-phosphate into 1,3bisphosphoglycerate. Its gene is constitutively expressed in many tissues. Variations in GAPDH expression levels occurs in tissues at different developmental stages and have been observed in cells treated with a variety of agents such as mitogens, dexamethasone, calcium, insulin, and in cells under hypoxia conditions. Overexpression of GAPDH in tumor cells as a consequence of the development of a hypoxic cellular microenvironment is cell type specific. It has been observed in human prostate adenocarcinoma cells (cell line LNCap), human spontaneous cervical cancer cells (SiHA), but not in human malignant glioma cells (cell lines U373-MG, GaMG, and U251) ([21], [22]). It is not clear if the reason for the upregulation of GAPDH in tumor cells is only for increasing glycolysis. The role of GAPDH as a NADH generator and as a mediator of cell death has been highlighted in several studies, which adds to the complexity of its upregulation in tumor cells.

13 Phosphoglycerate Kinase Phosphoglycerate kinase (PGK) catalyzes the transfer of the phosphoryl group from the acyl phosphate of 1,3-bisphosphoglycerate to ADP. ATP and 3phosphoglycerate are the products. Human PGK1 gene harbors HREs in the 5 flanking and 5 UT regions, and mouse PGK1 harbors three HREs in the 5 flanking sequence [10]. The HREs of the PGK promoter mediate the transcriptional response to hypoxia. Indeed, the induction of PGK in different solid tumor cell lines in hypoxic conditions is under the control of HIF-1␣. Expression of PGK1 increases in mouse hepatoma Hepa1c1c7 cell lines exposed to hypoxia or to treatment with desferrioxamine [20].

14 Enolase Enolase (ENO) is also upregulated in many cancers, including breast, liver, colon, and lung. Direct evidence of HIF-1 upregulation of the enolase gene was obtained using YC-1, a drug that targets HIF-1. Levels of aldolase and enolase mRNAs decrease in YC-1–treated Hep3B hepatoma cells. The inhibitory action of YC-1 on these glycolytic genes probably inhibits cell survival under hypoxia and may promote cell death in hypoxic areas [23].

86

´ T.C. Ferreira, E.G. Campos

15 Pyruvate Kinase Pyruvate kinase (PK) is responsible for net ATP production within the glycolytic pathway. It is consistently altered during tumorigenesis. Four isoenzymes of pyruvate kinase have been identified: type L (present in tissues with gluconeogenesis such as liver and kidney); type R (expressed in erythrocytes); type M1 (muscle and brain); and type M2 (expressed in lung tissues and all cells with high rates of nucleic acid synthesis, such as embryonic cells, adult stem cells, and especially tumor cells). Investigation of the mRNA levels of HIF-1␣, PGK1 and muscle-type pyruvate kinase 2 (PKM2) were performed in various hepatoma cell lines as well as in mouse and human tumor specimens and the corresponding normal tissues. In five of eight human colorectal cancers investigated, PGK1 and PKM2 mRNA levels were increased in comparison with the corresponding normal tissues, whereas HIF1-␣ was not significantly changed [24]. Though probable, these studies do not conclusively demonstrate that the increase in PKM2 gene expression is, in fact, mediated by HIF-1–induced transcriptional activation. However, HIF-1␣ plays a critical role in PK expression during hypoxic challenge in glial cells.

16 Pyruvate Dehydrogenase The pyruvate dehydrogenase (PDH) complex catalyzes the conversion of pyruvate to acetyl-coenzyme A, which enters the TCA cycle, producing NADH and carbon dioxide. The PDH complex is composed of three subunits, E1 (pyruvate decarboxylase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehydrogenase), and is under the control of pyruvate dehydrogenase kinases (PDKs) 1 to 4. PDKs phosphorylate the E1 subunit of PDH causing its inhibition. The activity of PDK is regulated by the concentration of the metabolic products of pyruvate (NADH and acetyl-coA). HIF-1 upregulates the expression of PDK1 [25]. Upregulation of the expression of PDK1 by HIF-1 illustrates how it favors a metabolic switch toward glycolysis to the detriment of oxidative phosphorylation. Hypoxic cells compensate the inefficient glycolytic energy output by increasing glucose uptake, overexpressing and increasing the activities of the glycolytic enzymes.

17 Lactate Dehydrogenase and Lactate Carrier MCT4 Lactate dehydrogenase (LDH) converts pyruvate into lactate. LDH-A converts pyruvate to lactate during glycolysis under anaerobic conditions in normal cells. The other isoenzyme, LDH-B, kinetically favors the conversion of lactate to pyruvate and is found at high levels in aerobic tissues such as heart. Elevated LDH-A expression contributes to the ability of tumor cells to undergo aerobic glycolysis. Once produced, lactic acid exits cancer cells through the MCT4 lactate carrier that is also

Regulation of Glucose and Energy Metabolism in Cancer Cells by HIF-1

87

induced in these cells. Under hypoxia, both pyruvate transformation into lactate with concomitant regeneration of NAD+ and lactate exit from the cell are stimulated because the genes encoding the LDH-A isoform and the lactate carrier MCT4 are HIF-1 targets. The c-myc gene is frequently activated in human cancers. This transcription factor forms a heterodimer with another protein (MAX) and binds to a 5 -CACGTG-3 consensus sequence in its target genes inducing gene expression. In addition to HIF-1, the LDH-A gene is also a c-MYC target. Therefore, the molecular basis for altered tumor glycolysis and lactate production cannot be solely explained by HIF-1 activation of specific genes [26]. In addition to its role in lactate production, LDH-A may also be involved in the regulation of gene expression and/or DNA replication. Therefore, its modulation by HIF-1 and c-MYC could be linked to effects other than to increased lactate production.

18 Glucose Transporters and HIF-1 Glucose enters animal cells through facilitated diffusion via glucose transporters (GLUT1 to GLUT5) and is subsequently phosphorylated by hexokinase. GLUT1 and GLUT3, present in nearly all mammalian cells, are responsible for basal glucose uptake and continually transport glucose into cells at an essentially constant rate. They have a high affinity for glucose, allowing transport of glucose at a high rate under normal physiologic conditions. Cancer cells overexpress both GLUT1 and GLUT3 to increase glucose uptake. Upregulation of glucose transporters has been observed during carcinogenesis in esophageal, gastric, breast, and colon cancers. A major technique used to measure glucose intake in tumor cells is positron emission tomography (PET) with [18 F]fluoro-2-deoxy-D-glucose (FDG). PET is a noninvasive imaging technique used in the clinic to detect malignant tumors, which measures the glucose metabolism in vivo. FDG is a fluorinated glucose analogue and accumulates in tumor cells in proportion to the rate of glucose metabolism. A high uptake of FDG is associated with a higher GLUT1 expression. The enhanced uptake of glucose often reflects tumor aggressiveness in patients with different types of tumors. The glucose transfer from the bloodstream to the inside of cells mediated by GLUT1 plays a central role in the development and malignant behavior of cancer cells. HIF-1 binding to the promoter of glucose transporters was seen in human HepG2 cells (GLUT1 and GLUT3) and mouse hepatoma cells exposed to hypoxia (GLUT1). Studies of GLUT mRNA expression in hypoxic cells have shown that the level of GLUT1 mRNA in human placenta choriocarcinoma cell line BeWo (JCRB911) increases 1.4-fold in the presence of 5% O2 and by 2.2-fold in the presence of 250 ␮M CoCl2 compared with that under 20% O2 . In the rat choriocarcinoma Rcho-1 cell line, the level of GLUT1 mRNA increases 1.8-fold in the presence of 5% O2 and by 3.2-fold in the presence of 250 μ M CoCl2 compared with that under 20% O2 . The GLUT1 protein levels also increase under

88

´ T.C. Ferreira, E.G. Campos

hypoxia and CoCl2 treatment, and these results suggest that GLUT mRNA and protein levels were directly mediated through HIF-1 induction [27].

19 HIF-1␣ Upregulation by TCA Intermediates Is Linked to Carcinogenesis Succinate dehydrogenase (SDH) is a TCA enzyme, which is also part of the mitochondrial respiratory chain. Succinate dehydrogenase deficiency causes either severe encephalomyopathy or tumor formation [28]. Germ-line heterozygote mutations in the SDH subunits cause inherited pheochromocytomas (adrenal gland tumors) and paragangliomas. The effects of SDH deficiency are postulated to be associated with the disturbance of two signaling pathways: one involving the superoxide radical and another involving HIF-1. SDH defect can increase superoxide radical production by the mitochondrial respiratory chain, which is able to trigger both cell death and proliferation. On the other hand, when succinate accumulates inside the mitochondria, it is transported into the cytosol where it can inhibit prolyl hydroxylases (PHD1 to PHD3), the enzymes responsible for HIF-1 destabilization. Upon PHD inhibition, HIF-1␣ is stabilized under normoxic conditions. Heterodimerization of stabilized HIF-1␣ and HIF-1␤ forms active HIF-1, which induces transcription of nuclear genes involved in tumor progression. Mutations in another TCA enzyme, fumarate hydratase (FH), cause cutaneous and uterine leiomyomas, as well as renal cell carcinomas. Mitochondrial proteins may also play a role in the development of sporadic kidney tumor oncocytoma. High concentrations of cytosolic fumarate may inhibit prolyl hydroxylases and have the same consequences as excess succinate on HIF-1 destabilization. Defective SDH and FH activity lead, respectively, to the accumulation of succinate and fumarate. These TCA intermediates, in addition to oxaloacetate, are capable of inhibiting the PHD activity and subsequently inhibiting HIF-1␣ degradation [29].

20 HIF-1 Activity as a Therapeutic Target The prognostic significance of HIF-1␣ expression in cancer cells, its central role in the adaptive response to hypoxia, and its participation in the Warburg effect make it a potential target for anticancer drug development. It is a reasonable speculation that inhibition of HIF-1 activity may be a useful cancer therapy. A number of anticancer agents that may target HIF-1 activity have been described (some cited in this text) and are treated more fully by Patiar and Harris [30]. The challenge remains to prove that the antitumorigenic effect of these drugs is due to HIF-1␣ inhibition. Selectivity may be resolved by the fact that tumor cells are more hypoxic than are normal cells and have specific intrinsic markers of HIF-1␣ activation. Further increase in this difference is possible through the use of angiogenesis inhibitors (e.g., thrombospondin-1) in association with HIF-1␣ inhibition.

Regulation of Glucose and Energy Metabolism in Cancer Cells by HIF-1

89

21 Conclusion HIF-1␣ can be activated by hypoxic and nonhypoxic stimuli, including growth factors, oncogenes, metals, and ROS. Metabolism of glucose and oxidative phosphorylation are directly affected by the HIF-1 signaling pathway. A change in ATP production pathway from oxidative phosphorylation to aerobic glycolysis is a hallmark of cancer cells. There is an evident overlap between HIF-1 function and cancer cell metabolism. Cancer cells have special needs for rapid proliferation and survival. Some of these needs are fulfilled by the HIF-1 signaling cascade. In addition, HIF-1 is located at the center of the major pathways that define cell destiny toward adaptation to fast growth or apoptosis. The available data show that cancer cell survival is a much more pronounced HIF-1 effect than is cell death. Therefore, HIF-1 will continue to be explored as a potential target for cancer therapy.

References 1. Semenza GL. Expression of hypoxia-inducible factor 1: mechanisms and consequences. Biochem Pharmacol 2000;59: 47–53. 2. Metzen E, Berchner-Pfannschmidt U, Stengel P, et al. Intracellular localisation of human HIF-1␣ hydroxylases: implications for oxygen sensing. J Cell Sci 2003; 116:1319–1326. 3. Berra E, Benizri E, Ginouv`es A, Volmat V, Roux D, Pouyss´egur J. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1␣ in normoxia. EMBO J 2003; 22:4082–4090. 4. Jewell UR, Kvietikova I, Scheid A, Bauer C, Wenger RH, Gassmann M. Induction of HIF–1␣ in response to hypoxia is instantaneous. FASEB J 2001; 15:1312–1314. 5. H¨opfl G, Ogunshola O, Gassmann M. HIFs and tumors – causes and consequences. Am J Physiol Regul Integr Comp Physiol 2004; 286:R608–R623. 6. Elstrom RL, Bauer DE, Buzzai M, et al. AKT stimulates aerobic glycolysis in cancer cells. Cancer Res 2004; 64:3892–3899. 7. Kim SH, Park JA, Kim JH, et al. Characterization of ARD1 variants in mammalian cells. Biochem Biophys Res Commun 2006; 340:422–427. 8. Br¨une B, Zhou J. The role of nitric oxide (NO) is stability regulation of hypoxia inducible factor-1␣ (HIF-1␣). Current Med Chem 2003; 10:845–855. 9. Lee J-W, Bae S-H, Jeong J-W, Kim S-H, Kim K-W. Hypoxia-inducible factor (HIF-1) ␣: its protein stabilization and biological functions. Exp Mol Med 2004; 36:1–12. 10. Wenger RH, Stiehl DP, Camenisch G. Integration of oxygen signalling at the consensus HRE. Sci Stke 2005;re12. 11. Pouyssegur J, Mechta-Grigoriou F. Redox regulation of the hypoxia-inducible factor. Biol Chem 2006; 387:1337–1346. 12. Zhong H, De Marzo AM, Laughner E, et al. Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res 1999; 59:5830–5835. 13. Semenza GL. HIF-1 mediates the Warburg effect in clear cell renal carcinoma. J Bioenerg Biomembr 2007; 39:231–234. 14. Warburg O. On the origin of cancer cells. Science 1956; 123:309–314. 15. Lu H, Forbes RA, Verma A. Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates the Warburg effect in carcinogenesis. J Biol Chem 2002; 277:23111–23115. 16. Iyer NV, Kotch LE, Agani F, et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1␣. Genes Dev 1998; 12:149–162.

90

´ T.C. Ferreira, E.G. Campos

17. Mathupala SP, Ko YH, Pedersen PL. Hexokinase II: cancer’s double-edged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria. Oncogene 2006; 25:4777–4786. 18. Funasaka T, Yanagawa T, Hogan V, Raz A. Regulation of phosphoglucose isomerase/autocrine motility factor expression by hypoxia. FASEB J 2005; 19:1422–1430. 19. Minchenko OH, Ochiai A, Opentanova IL, et al. Overexpression of 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase-4 in the human breast and colon malignant tumors. Biochimie 2005; 87:1005–1010. 20. Salceda S, Beck I, Caro J. Absolute requirement of aryl hydrocarbon receptor nuclear translocator protein for gene activation by hypoxia. Arch Biochem Biophys 1996; 334:389–394. 21. Lu S, Gu X, Hoestje S, Epner DE. Identification of an additional hypoxia responsive element in the glyceraldehyde-3-phosphate dehydrogenase gene promoter. Biochim Biophys Acta 2002; 1574:152–156. 22. Said HM, Hagemann C, Stojic J, et al. GAPDH is not regulated in human glioblastoma under hypoxic conditions. BMC Mol Biol 2007; 8:55. 23. Yeo EJ, Chun YS, Cho YS, et al. YC-1: a potential anticancer drug targeting hypoxiainducible factor 1. J Natl Cancer Inst 2003; 95:516–525. 24. Kress S, Stein A, Maurer P, et al. Expression of hypoxia-inducible genes in tumor cells. J Cancer Res Clin Oncol 1998; 124:315–320. 25. Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 2006; 3:177–185. 26. Shim H, Dolde C, Lewis BC, et al. c-Myc transactivation of LDH-A: implications for tumor metabolism and growth. Proc Natl Acad Sci USA 1997; 94:6658–6663. 27. Hayashi M, Sakata M, Takeda T, et al. Induction of glucose transporter 1 expression through hypoxia-inducible factor 1 under hypoxic conditions in trophoblast-derived cells. J Endocrinol 2004; 183:145–154. 28. Selak MA, Armour SM, MacKenzie ED, et al. Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell 2005; 7:77–85. 29. Pollard PJ, Briere JJ, Alam NA, et al. Accumulation of Krebs cycle intermediates and overexpression of HIF1alpha in tumours which result from germline FH and SDH mutations. Hum Mol Genet 2005; 14:2231–2239. 30. Patiar S, Harris AL. Role of hypoxia-inducible factor-1alpha as a cancer therapy target. Endocr Relat Cancer 2006; 13(Suppl 1):S61–S75.

The Role of Glycolysis in Cellular Immortalization Hiroshi Kondoh

Abstract The clinical significance of the Warburg effect is well established, although it is not clear why and when cancer cells start to display a high glycolytic rate in vivo. The discovery of hypoxia-inducible transcriptional factor as a critical regulatory factor for glycolytic genes along with the recent advances in senescence biology imply that the metabolic shift to enhanced glycolysis would be observed in multiple stages during tumorigenesis in vivo. Increased glycolysis would be required in the early step of immortalization to avoid oxidative stress–mediated senescence, followed by the adaptation to hypoxic conditions through increased enhancement of glycolysis