Accepted Manuscript Title: Mitochondrial dysfunction in cancer chemoresistance Author: Nicoletta Guaragnella Sergio Giannattasio Loredana Moro PII: DOI: Reference:
S0006-2952(14)00448-1 http://dx.doi.org/doi:10.1016/j.bcp.2014.07.027 BCP 12043
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Received date: Revised date: Accepted date:
23-6-2014 25-7-2014 28-7-2014
Please cite this article as: Guaragnella N, Giannattasio S, Moro L, Mitochondrial dysfunction in cancer chemoresistance, Biochemical Pharmacology (2014), http://dx.doi.org/10.1016/j.bcp.2014.07.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mitochondrial dysfunction in cancer chemoresistance Nicoletta Guaragnella, Sergio Giannattasio*, Loredana Moro
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Institute of Biomembranes and Bioenergetics, Via Amendola 165/A, 70126 Bari, Italy
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*Corresponding author: Sergio Giannattasio
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Consiglio Nazionale delle Ricerche Istituto di Biomembrane e Bioenergetica
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Via Amendola 165/A
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70126 Bari (ITALY) Email: [email protected]
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Fax +390805443317
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Tel +390805443316
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Abstract
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Mitochondrial dysfunction has been associated with cancer development and progression. Recent evidences suggest that pathogenic mutations or depletion of the mitochondrial genome can
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contribute to development of chemoresistance in malignant tumors. In this review we will
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describe the current knowledge on the role of mitochondrial dysfunction in the development of chemoresistance in cancer. We will also discuss the significance of this research topic in the
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context of development of more effective, targeted therapeutic modalities and diagnostic strategies for cancer patients, with a particular focus on the potential use of PARP inhibitors in
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cancer patients displaying mitochondrial DNA mutations. We will discuss recent studies highlighting the importance of the cross-talk between the tumor microenvironment and
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mitochondrial functionality in determining selective response to certain chemotherapeutic drugs. Finally, owing to the similarities between cancer and yeast cell metabolism, we will point out the
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use of yeast as a model system to study cancer-related genes and for anti-cancer drugs screening.
Keywords: cancer chemoresistance, mitochondrial dysfunction, tumor microenvironment, mitochondrial retrograde signaling, yeast
Chemical compounds cited in this article: 3-bromopyruvate (PubChem CID: 70684 ); 2deoxyglucose (PubChem CID: 439268); dichloroacetate (PubChem CID: 25975); lonidamine (PubChem CID: 39562); rucaparib (PubChem CID: 9931954). 2
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1. Introduction
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“We understand the origin of cancer cells if we know how their large fermentation originates, or, to express it more fully, if we know how the damaged respiration and the
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excessive fermentation of the cancer cells originate” [1]. In this way Warburg described the metabolic feature of tumor cells, which is a metabolic shift from oxidative phosphorylation to
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glycolysis for ATP generation in tumor cells, even in the presence of high oxygen tension,
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termed the "Warburg effect". Originally Warburg proposed that this glycolytic shift was due to defects in mitochondrial function leading to impaired oxidative phosphorylation. It has later been
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appreciated that even tumors with "normal" oxidative phosphorylation and oxygen consumption exhibit increased glycolytic metabolism [2] converting most incoming glucose to lactate rather
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than metabolizing it in the mitochondria through oxidative phosphorylation. New hypothesis
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have been proposed but still there is no definitive conclusion. Zu and Guppy [3] have indicated
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that not all cancer cells exhibit defects in oxidative metabolism. More recently, Moreno-Sanches and co-workers [4] have suggested that cancer cells have a heterogeneous metabolic profile and some cancer cell populations would use glycolysis as main energetic pathway, while others would use oxidative phosphorylation. Thus, Warburg’s observation on the origin of cancer cells still retains its validity, even though the molecular basis of the Warburg’s effect has to be reconsidered in the context of a set of concerted changes in energy metabolism and mitochondrial function supporting tumorigenesis. This process is now referred to as metabolic reprogramming and represents an emerging hallmark of cancer cells indicating the importance of deregulated metabolism as a widespread trait of cancer cells, as pointed out by Hanahan and Weinberg [5]. 3
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However, a debate still exists on the role of metabolic reprogramming as a cause of tumorigenesis given the fact that cancer cell metabolic reprogramming is driven by onco- and tumor-suppressor gene mutations affecting core signaling pathways and processes that define
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also the core hallmarks of cancer [6, 7]. From this point of view, the metabolic reprogramming may simply represents another phenotype that is regulated by oncogenes stimulating cell
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proliferation, and thereby has raised doubts on its functional independence from the core cancer
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hallmarks. Recent studies have shown a causal role of mutations in enzymes of the Krebs cycle in the etiology of some cancers, suggesting that, at least in some cancer types, the deregulated
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metabolism may represent a driver of cell malignant transformation [8, 9].
The metabolic shift towards glycolysis, typical of cancer cell metabolic reprogramming,
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is essential to support three basic needs of tumor cells: i) glycolysis would represent the main
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active pathway of ATP production under conditions of oxygen deprivation, i.e. in the hypoxic
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microenvironment of tumor tissues (see par. 3.2); ii) increased biosynthesis of macromolecules (carbohydrates, proteins, lipids, nucleic acids): proliferating tumor cells not only require rapid
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supply of energy, but also building blocks for cell biosynthesis and dividing processes (mitosis). In this context, glycolytic intermediates, particularly glucose-6-phosphate, can be directed to the pentose phosphate pathway to generate ribose-5-phosphate and 3-carbon intermediates important for nucleic acid and lipid biosynthesis, respectively; iii) maintenance of redox homeostasis: NADPH generated through the pentose phosphate pathway would provide reducing equivalents in many biosynthetic reactions. In addition, acting as an antioxidant, NADPH would counteract the detrimental effects of very high levels of ROS produced in rapidly proliferating cancer cells. Indeed, lactate produced through increased fermentation by a population of cancer cells can be used as a fuel substrate by a second population of cancer cells through an active oxidative
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metabolism [10-12]. On the other hand, L- and D-lactate can both enter cells and be formed in the cytosol from glucose via glycolysis and methylglyoxal pathway, respectively. Both the isomers D-lactate
mitochondria by two separate enzymes, namely the mitochondrial
are oxidized inside
L-lactate
dehydrogenase
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can enter mitochondria in a carrier mediated manner; L- and
located in the matrix and the D- lactate dehydrogenase, respectively [13-16]. The mitochondrial
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metabolism of L- and D-lactate in cancer cells is more active than in normal cells and seems to
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account for tricarboxylic acid (TCA) cycle anaplerosis [16-19] contributing to ROS scavenging and fatty acid synthesis, all crucial for cancer cell viability and proliferation and requiring
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NADPH.
Mitochondria are cellular organelles that play a key role in the context of cancer
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metabolic reprogramming because they represent a nodal point where most metabolic and
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signaling pathways converge [20]. They are a central hub for biosynthesis of aminoacids, nucleic
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acids, and lipids and the main providers of NADH and NADPH and thus are important regulators of the redox balance in the cells [21, 22].
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Several evidences [23-25] have shown that mitochondrial dysfunction provides survival advantage to cancer cells, suggesting that mitochondria have a tumor suppressor function. Yet, the mechanisms of such function remain a molecular conundrum. Indeed, mitochondrial dysfunction also occurs en route to most apoptotic pathways [25]. Evasion of apoptosis, the major form of programmed cell death (PCD) in mammals, is a requirement for both neoplastic transformation and sustained growth of tumor cells (reviewed in [5]) as well as a crucial acquired capabilities used by cancer cells to resist to anticancer therapies (reviewed in [26]). With this respect it is of note that in myoblasts mitochondrial genetic stress results in the up-regulation of a number of proapoptotic proteins, including BAD, Bax, and Bid. Yet, this mitochondrial
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dysfunction promotes resistance to apoptosis induction due to mislocalization of the proapoptotic factors and reduced processing of Bid to active tBid (see par. 2.2) [27]. On the other hand, a plethora of genetic and metabolic mitochondrial alterations have been described so far that,
drug sensitivity in cancer cells (for recent reviews see [28, 29]).
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besides affecting the energy metabolism of the cells, are also implicated in the modulation of
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In this review, we will focus on the role of mitochondrial dysfunction in the development
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of chemoresistance and the rationale for targeting cancer cells with mitochondrial dysfunction as a promising pharmacological therapeutic strategy. We will also discuss new studies highlighting
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the importance of the cross-talk between the tumor cell microenvironment and mitochondrial functionality in determining selective response to certain chemotherapeutic drugs as well as the
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use of yeast as a model organism in cancer research.
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2. Mitochondrial genetic and metabolic stress in cancer drug resistance Mitochondria are semi-autonomous organelles which possess their own genome.
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Mitochondrial DNA (mtDNA) is a 16.569 bp circular DNA molecule encoding for 13 proteins belonging to the mitochondrial respiratory chain, 22 tRNAs and 2 rRNAs (Fig. 1). The number of mtDNA molecules per cell varies among cell and tissue types. In addition, within the same cell some mitochondria can have mutation/s of the mtDNA while others have normal mtDNA, a phenomenon termed heteroplasmy. Instead, the term homoplasmy indicates that a cell or a cell population contains either entirely normal or entirely mutant mtDNA molecules. So far, several mutations occurring in nuclear genes encoding for mitochondrial proteins involved in energy metabolism as well as in mtDNA have been identified in different cancer types. For example, mutations in enzymes of the TCA cycle have been linked to tumorigenesis.
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In addition, decreased expression and activity has been described for components of the electron respiratory chain in cancer cells, including complex I [30-32], complex II [33, 34], complex III
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[32-34], complex IV [33, 34] and complex V [33, 35] (Fig. 1).
2.1 Mutations of TCA enzymes
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It has been shown that inactivating-germline mutations in fumarate hydratase (FH) and
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succinate dehydrogenase (SDH), two enzymes of the TCA cycle, are oncogenic because of accumulation of fumarate and succinate [9, 36], small-molecules of the normal metabolism that,
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when abnormally accumulated, cause both metabolic and non-metabolic signaling dysregulation establishing a milieu that initiates malignant transformation, and thereby are termed
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oncometabolites [8, 9]. Loss-of-function mutations in FH and SDH occur in some tumors
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including pheochromocytoma, paraganglioma, renal cell carcinoma, leiomyomas [9] (Table 1).
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The neoplastic effect of fumarate and succinate has been linked to hypoxia-inducible factor 1 (HIF1) stabilization and inhibition of α-ketoglutarate-dependent dioxygenases that regulate DNA
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and histone methylation and, thus, gene expression, thereby promoting epithelial-tomesenchymal transition, angiogenesis, and changes in cell metabolism [37-39]. In FH-deficient kidney mouse cells a metabolic pathway beginning with glutamine uptake and ending with bilirubin excretion, involving the biosynthesis and degradation of haem, has been identified. This pathway enables FH-deficient cells to use the accumulated TCA cycle metabolites and permits partial mitochondrial NADH production. Targeting this pathway would render FH-deficient cells non-viable, while sparing wild-type cells. Thus, inhibition of haem oxygenation has been shown to be synthetically lethal when combined with FH deficiency, providing a new potential target for treating hereditary leiomyomatosis and renal-cell cancer patients [40]. Another
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oncometabolite recently identified is 2-hydroxyglutarate, which accumulates following gain-offunction mutations in another enzyme of the TCA cycle, NADP-dependent isocitrate dehydrogenase (IDH) 1 (cytosolic) and 2 (mitochondrial) in different cancer types, including
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acute myeloid leukemia, glioma, chondrosarcoma and a subtype of breast cancers [41, 42]. Accumulation of 2-hydroxyglutarate causes changes in the activity of DNA demethylases thus
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promoting epigenetic changes [42], a phenotype also observed following loss-of-function
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mutations in FH and SDH (Table 1). While a number of studies have linked mtDNA mutations with changes in cancer cell sensitivity to chemotherapy, the possible role of mutations in the
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TCA cycle enzymes in modulating drug resistance has been poorly investigated.
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2.2 Mitochondrial DNA mutations and retrograde response
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Defects in mtDNA, including a low mtDNA copy number, have been implicated in cancer. In
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particular, a reduction in mtDNA content results in impaired mitochondrial respiration and depolarization of the mitochondrial membrane. Cell response to mitochondrial dysfunction
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occurs via the evolutionally conserved communication pathway from mitochondria to the nucleus, termed retrograde response [43]. The molecular details of retrograde signaling pathway activated in response to mitochondrial dysfunction, including respiratory deficiency due to mtDNA depletion, antimycin A-dependent inhibition of the electron flow along the respiratory chain or carbonyl cyanide m-chlorophenyl hydrazone-dependent uncoupling, have been characterized first in S. cerevisiae [44]. In these cells, retrograde (RTG) signaling allows adaptation to mitochondrial dysfunction by eliciting a metabolic reconfiguration in which specific nuclear genes are up-regulated, including those involved in peroxisomal biogenesis, the glyoxylate cycle and fatty acid oxidation [43]. CIT2, encoding the peroxisomal isoform of citrate
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synthase, is the prototypical target gene of the yeast RTG pathway: its expression is largely increased in cells with mitochondrial dysfunction, including those lacking mtDNA (ρ0). Rtg1p
box) in the promoter region of retrograde-target genes (Fig. 2).
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and Rtg3p form a heterodimeric transcription factor binding the RTG-specific cis-element (R-
The physiological significance of the RTG pathway activation is to allow glutamate
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biosynthesis as nitrogen source for biosynthetic reactions, and to ensure the maintenance of
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mtDNA, through the regulation of ACO1 [45]. Glutamate plays an important role in metabolic reprogramming of cancer cells. Indeed, besides increased glycolysis, metabolic adaptation in
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cancer also includes increased dependence of tumor cells on glutamine, an aminoacid that, upon entry in the cells, can be used as energy source by glutaminolysis as well as a building block in
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anabolic reactions [21]. Glutaminase is responsible for conversion of glutamine to glutamate,
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which, in turn, can be converted either into glutathione (GSH), an abundant antioxidant in the
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cells and a regulator of the cellular redox balance, or into α-ketoglutarate, an intermediate of the TCA cycle. α-Ketoglutarate is in turn reductively carboxylated by the NADPH-linked
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mitochondrial isocitrate-dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate producing ATP and allowing synthesis of other aminoacids and fatty acids. Thus, the high rate of glycolysis, an inefficient way to generate ATP, and glutaminolysis have been proposed to be for cancer as well as for all proliferating cells an adaptive response to facilitate the uptake and incorporation of nutrients into the biomass (e.g. nucleotides, aminoacids, and lipids) needed to generate a new cell [46, 47]. With this respect, retrograde activation of glyoxylate cycle in yeast resembles glutamine-dependent reductive carboxylation of αketoglutarate in tumor cell metabolic reprogramming [48]. The RTG pathway has been shown to promote longevity in yeast [49] and recently to be involved in PCD resistance of yeast cells
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treated with acetic acid [50]. The RTG pathway is linked to other signaling pathways: Target of Rapamycin (TOR) kinase pathway, which inhibits RTG-dependent gene expression [51] in accordance with a causative role for TOR in acetic acid-induced PCD [52] and Ras-cAMP,
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which impacts RTG-dependent lifespan extension [49, 53]. It is of note that Ras-cAMP-protein
function and cell death with nutritional status in yeast [54] (Fig. 2).
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kinase A (PKA) signaling plays a physiological role in coordinating mitochondrial respiratory
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In mammalian cells, the mitochondrial retrograde signaling has been suggested to be an important mechanism for tumorigenesis [55-57] and has been associated with increased cytosolic
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calcium levels [58-60], hypoxic-to-normoxic shift [61], activation of oncogenic signaling molecules/pathways, including PI3-kinase/AKT, JNK, Ras, ERK [60-67] and induction of the
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expression of nuclear genes involved in invasion, including matrix metalloproteinases and
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cathepsin [59], epithelial-to-mesenchymal transition, like Snail, Twist, Slug, TGF-β [63, 65],
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ubiquitination and DNA repair [60, 67], glucose transport, particularly the glucose transporter GLUT1 and GLUT4 that may contribute to the metabolic shift towards a highly glycolytic
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phenotype [64, 68] (Fig. 3). Mutations and/or reduced levels of mtDNA have been reported in several cancer types, including prostate [60, 69-71], kidney [72], breast [73-75], liver [76], colon and rectal cancers [77]. Chemically induced mtDNA depletion has been shown to promote cancer progression to an invasive and apoptosis-resistant phenotype [59, 63, 64, 78-82]. Consistently, in breast and prostate cancer patients, mtDNA mutations and reduced mtDNA copy number are associated with increased metastasis and adverse prognosis [69, 83]. In addition, introduction of homoplasmic pathogenic point mutations in the mtDNA ATP synthase subunit 6 gene in HeLa cells prevented apoptosis and increased their tumorigenic potential in vitro and in vivo [84]. Introduction of the ATP6 T8993G mutation, found in a prostate cancer patient at the
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germline level, into the PC-3 prostate cancer cell line through cybrid transfer also increased tumor growth in vivo as well as ROS levels [85]. Ishikawa et al. [86] showed that presence of two point mutations in the NADH dehydrogenase subunit 6 (ND6), which produced a deficit in
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respiratory complex I activity and ROS overproduction, enhanced the metastatic potential of the cells. Overall, these studies implicate mtDNA mutations in tumorigenesis and cancer
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progression.
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Studies with cells chemically depleted partially (ρ-) or totally (ρ0) of their mtDNA support a role for mitochondrial dysfunction in cancer drug resistance (Fig. 3). It has been
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reported that ρ- or ρ0 prostate cancer cells become less sensitive to the chemotherapeutic drugs
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paclitaxel [78] and N- (4-hydroxyphenyl) retinamide [87]. Naito and coworkers [88] showed that mtDNA depletion promotes resistance to hormone therapy in breast cancer cells. Singh and
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coworkers [89] showed that ρ0 HeLa cells became resistant to adriamycin and photodynamic
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therapy but remained sensitive to γ-rays and DNA alkylating agents. Park and coworkers [90] have shown that hepatoma cells deprived of mtDNA were less sensitive to hydrogen peroxide
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and to ROS-inducing agents, including paraquat, doxorubicin and menadione, and that this drugresistant phenotype was associated with increased expression of the antioxidant enzymes manganese superoxide dismutase and glutathione peroxidase. Consistent with a role of altered redox regulation in the development of resistance to ROS-inducing drugs, cancer cell insensitivity to the chemotherapeutic drugs paclitaxel and cisplatin has been associated with increased antioxidant expression levels [91-93]. However, it should be noted that mtDNA mutations or depletion may exert different effects on drug sensitivity depending on the particular tissue. For example, docetaxel-resistant laryngeal cancer cells exhibit increased mtDNA content [94]. An intriguing phenotype has been recently described in a patient with serous ovarian
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cancer: the presence of a nearly homoplasmic mtDNA mutation in the NADH dehydrogenase subunit 4 (ND4) in the postchemotherapy but absent in the prechemotherapy tissue, which could determine tumor growth arrest [95]. Mutations in mtDNA-encoded proteins belonging to the
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mitochondrial respiratory Complex I, such as those occurring in ND4, have indeed been suggested to play an "oncojanus" role in some tumor tissues: they would promote tumor growth
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when are below a threshold level but would exert the opposite effect when they reach a critical
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mutant load [96].
The molecular mechanism involved in mtDNA-mediated drug resistance is not well
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understood. It has been shown that development of resistance to adriamycin in HeLa ρ0 cells was not due to changes in apoptotic cell death, cell cycle response or to the uptake of adriamycin
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[89]. Instead, since adriamycin activation requires Complex I activity, it has been proposed that
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mtDNA depletion, by impairing the mitochondrial respiratory chain, would prevent its activation
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in the cells thus resulting in cancer resistance to this particular drug [89]. Very recently, it has been demonstrated that depletion of mtDNA in hepatocarcinoma cells results in chemoresistance
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to doxorubicin, cisplatin and SN-38 and that this phenotype is associated with activation of the nuclear factor erythroid 2 [NF-E2]-related factor 2 (NRF-2) signaling pathway and consequent up-regulation of Multi-Drug Resistance gene 1 (MDR1), Multidrug Resistance-associated Protein 1 (MRP1) and 2 (MRP2), proteins notably involved in multidrug resistance [97] (Fig. 3). Increased levels of MDR1 were also observed upon mtDNA depletion in colon cancer cells [98]. Moreover, the ability of doxorubicin, SN-38 and cisplatin to induce proapoptotic signals was weaker in mtDNA-depleted cells, as indicated by increased survivin levels and reduced Bax/Bcl2 expression ratio. On the other hand mtDNA-depleted myoblasts, although showing reduced mitochondrial transmembrane potential, are resistant to staurosporine-mediated apoptosis
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through a mechanism involving a profound modification of the apoptotic machinery, including: i) sequestration of the proapoptotic factors Bid, Bax, BAD in the inner mitochondrial membrane where they cannot interact with Bcl-2; ii) increased expression of the antiapoptotic proteins Bcl-
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2 and Bcl-XL; iii) inability to process Bid into tBid; iv) reduced activation of caspases 3, 9, 8 [27].
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Overall, mtDNA-dependent deregulation of genes involved in apoptosis modulation may
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play a role in drug resistance depending on the cell type and/or the specific drug.
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3. Targeting mitochondria in drug-resistant cancer cell therapy
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3.1 Targeting cancer cells with defective mitochondrial function
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Mitochondrial dysfunction in cancer cells may offer a novel opportunity for selective
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anti-cancer therapy. Indeed, cancer cells with respiration deficiency, though more resistant to conventional chemotherapeutic drugs should be more sensitive than other cancer types to
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inhibitors of the glycolytic pathway because they would rely only on this pathway for energy supply. On the other hand, targeting specific molecular alterations resulting from activation of the mitochondrial retrograde signaling may provide a selective therapeutic approach for cancers with defective mitochondrial respiration.
3.1.1. Glycolysis inhibition To date, several glycolysis inhibitors have been tested as potential anti-cancer drugs. 2Deoxyglucose, an inactive glucose analog that inhibits hexokinase II is actually in clinical trials for cancer therapy [99] (www.clinicaltrials.gov). 2-Deoxyglucose has been reported to promote
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cell death of cancer cells exhibiting mitochondrial defects or in hypoxic conditions [100-102]. 3bromopyruvate, another hexokinase II inhibitor, has shown selective activity in killing colon cancer and lymphoma cells with mitochondrial dysfunction [103]. However, it is not known at
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present whether 3-bromopyruvate will move to clinical trials. Lonidamine, a derivative of indazole-3-carboxylic acid, is another promising glycolytic inhibitor. It prevents tumor growth
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by inhibiting hexokinase II and depleting ATP from the cells [104, 105]. However, it is toxic for
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normal tissues as demonstrated by studies in which lonidamine was administered alone [106] or in combination with the chemotherapeutic drugs doxorubicin, cyclophosphamide or cisplatin in
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animal models [107]. Despite its toxicity, phase II/III clinical trials have showed promising results in the treatment of solid tumors [108, 109]. Thus, lonidamine treatment of tumors with a
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highly glycolytic phenotype, particularly those with mitochondrial defects, may represent a
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promising strategy in the era of personalized medicine.
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Another example of glycolysis inhibitor is dichloroacetate (DCA), a pyruvate mimetic that stimulates pyruvate dehydrogenase by suppressing pyruvate dehydrogenase kinase 1. DCA
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increases mitochondrial pyruvate consumption, lowering the pyruvate pool available for glycolysis. In vitro studies have shown that DCA has selective activity against cells with severe mitochondrial dysfunction, i.e. ρ0 cells, and synergizes with 2-deoxyglucose in p53-/- cells with a deficit in Complex IV activity [110]. DCA synergizes also with antineoplastic agents that cause mtDNA damage, namely cisplatin and topotecan [110]. However, the ability of DCA to selectively target cancer cells with mitochondrial respiratory defects deserves further analysis in preclinical models.
3.1.2 Targeting the mitochondrial retrograde signaling
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Besides inhibiting the glycolytic pathway as a potential effective strategy for cancers with mitochondrial respiratory dysfunction, a recent work has indicated that targeting the molecular alterations resulting from activation of the mitochondrial retrograde signaling pathway may be a
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novel promising anti-cancer strategy. Arbini and coworkers [60] have shown that mtDNA depletion or large deletions activate a calcium-dependent mitochondrial retrograde signaling
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pathway that results in increased levels of the E3 ubiquitin ligase Skp2 mRNA and of miRNA-
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1245, encoded by the nuclear genome (Fig. 3). Skp2 and miR-1245 are negative regulators of the expression of the tumor suppressor protein BRCA2, thus their up-regulation by mitochondrial
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dysfunction promotes reduction of the expression levels of BRCA2 [60]. BRCA2 is a cancer susceptibility gene, i.e. when mutated, results in familial predisposition to several cancers,
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including breast, ovary and prostate cancer [111]. Reduced levels of BRCA2 protein have been
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detected in prostate cancer cell lines derived from sporadic tumors as well as in 70% of sporadic
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prostate cancer specimens in vivo [60, 112]. In addition, loss of nuclear BRCA2 has been reported in about 60% of pancreatic and breast cancer specimens, 80% of high-grade ovarian
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serum carcinomas, 30% of colon cancer samples [113]. On the other side, positive BRCA2 staining in the same cancer types has been correlated with increased overall and disease-free survival [113]. The major function of BRCA2 is to complex with Rad51 and to orchestrate DNA repair through homologous recombination. Several studies have shown that cancer cells lacking BRCA2 are about 1000-fold more sensitive to a novel class of anticancer drugs, namely poly(ADP-ribose) polymerase (PARP) inhibitors [114], that function through the mechanism of synthetic lethality, whereby two defective genes, each having by itself negligible effects on cell viability, become lethal when both are present in the same cells [115]. PARP proteins play a critical role in signaling DNA damage and their inhibition results in accumulation of single- and
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double-strand DNA breaks and apoptosis unless upstream homologous recombination is active and rescues the defect [116]. Thus, tumor cells lacking BRCA2 would undergo apoptosis if PARP is inhibited. This synthetic lethality approach by PARP inhibitors is being proven
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effective in clinical trials for treatment of cancers resulting from inherited mutations in BRCA2 [117]. The finding that mtDNA depletion activates a retrograde signaling pathway that promotes
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reduction of BRCA2 protein levels (Fig. 3) has direct clinical implications because it makes
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tumors featuring mtDNA large deletions or depletion, such as prostate carcinomas [60], promising candidates for tailored and personalized therapeutic regimens encompassing PARP
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inhibitors. Indeed, mtDNA depletion or large deletions might cooperate with PARP inhibition to induce cell death in cancer cells. Phase I/II clinical trials are actually undergoing to assess the
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carcinomas [www.clinicaltrials.gov].
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therapeutic potential of PARP inhibitors in sporadic cancers, including advanced prostate
3.2 Mitochondrial stress response in cancer cell adaptation to tumor microenvironment and
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drug sensitivity
Environmental conditions, such as the concentrations of highly consumed nutrients, represent a critical aspect for supporting the rate of cancer cell proliferation. Thus, for optimal growth, cancer cells must adapt their metabolism to their surrounding tumor microenvironment, which is usually hostile and characterized by acidity, hypoxia and low levels of glucose ([29, 118] and refs therein). As mentioned above, increased fermentation results in high production of lactate causing both tumor microenvironment acidification and possible activation of metalloproteinases and extracellular matrix remodeling enzymes, thus promoting cancer cell invasion [119]. A better understanding of cancer cell adaptations to tumor microenvironment
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might reveal the molecular basis of chemoresistance that can be exploited for targeted-drug development. A number of genes that have been implicated in the metastatic process, involving angiogenesis, survival and growth, have been found to be hypoxia-responsive, although little is
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known about the effects of oxygen level fluctuation over a range of 0 – 2 % on gene expression (for refs see [118]). Due to the complex experimental procedure required to mimic tumor
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microenvironment, few studies have been performed on the effect of glucose deprivation on cell
pathway” in the Web of Science™ yields only 25 articles.
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stress response. Indeed, a search for “low glucose tumor microenvironment cell response
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Making use of a continuous-flow culture apparatus (Nutrostat) for maintaining proliferating cells in low-nutrient media for long periods of time, Sabatini and coworkers
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performed competitive proliferation assays on a pooled collection of barcoded cancer cell lines
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cultured in low-glucose conditions [120]. Their results pinpoint mitochondrial oxidative
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phosphorylation (OXPHOS) as the major pathway required for optimal proliferation in low glucose. Glucose limitation normally causes up-regulation in the OXPHOS genes. Interestingly,
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they found that cell line sensitivity to low glucose was accompanied by defects in the OXPHOS up-regulation as a result of either mtDNA mutations in complex I genes or impaired glucose utilization [120]. These genetic/metabolic defects would predict sensitivity of cancer cells to bioguanides, antidiabetic drugs that inhibit OXPHOS when cancer cells are in the presence of low glucose. Hence, mtDNA mutations and impaired glucose utilization become potential biomarkers for identifying tumors with increased sensitivity to OXPHOS inhibitors. From a general point of view, these results underscore the strict cross-talk between glucose sensing/utilization and mitochondrial function and the importance of considering cell microenvironment when evaluating the sensitivity of cancer cells to certain drugs.
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Recently, in another experimental set-up, an RNA-interference-based transgenic mouse model, that allows tetracyclin-dependent regulation of phosphatase and tensin homolog (PTEN)encoding tumor suppressor gene in a time- and tissue-specific manner, has been generated. Use
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of this model has revealed a complex picture of how PTEN inactivation drives tumor maintenance, showing an interplay between tumor and microenvironment that would not be
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predicted from studies on cultured cells [121]. This produces intratumoral heterogeneity which
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can affect both disease progression and clinical response to molecularly targeted therapies. It is of notice that under similar environmental conditions, unicellular and multicellular
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organisms have similar metabolic phenotypes with an advantage to oxidative metabolism during nutrient limitation and prevalence of non-oxidative metabolism during cell proliferation [47].
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With this respect, the yeast Saccharomyces cerevisiae is an especially suitable model organism:
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it is one of the most thoroughly studied unicellular eukaryotes at the cellular, molecular, and
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genetic level due to its well-known experimental tractability; it may adapt and survive with alternatives in its genome expression and metabolism, allowing to mimic a variety of metabolic
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and (patho)physiological scenarios and to study mitochondrial dysfunctions through the generation of respiratory deficient strains (mutation/deletion of single nuclear genes encoding mitochondrial proteins or via abrogation of mitochondrial DNA) and their effects on cellular processes. Notably, yeast undergoes programmed cell death (PCD) pathways which share most of the morphological and biochemical hallmarks of mammalian apoptosis in response to different stressors [122]. Last, but not least, S. cerevisiae cell growth depends on nutrient availability in its natural environment [123]. Glucose is the preferred carbon source for yeast, regardless the presence of oxygen or other non-fermentable carbon sources. Glucose-grown yeast cells mainly use
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fermentation for carbon and energy metabolism. In contrast, when they grow on a nonfermentable carbon source (i.e., glycerol, ethanol, lactate or acetate), oxidative phosphorylation is the main energy-yielding pathway. The glucose-dependent down-regulation of gluconeogenic
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and oxidative metabolism-related proteins and the enhanced transcription of glycolytic enzymes and glucose transporters is termed carbon catabolite repression pathway [124, 125]. When
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glucose is externally added to an aerobic yeast culture, respiration is immediately arrested and
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glycolysis accelerated at the same time that ethanol is produced. This short-term and reversible transition is termed Crabtree effect. From a metabolic point of view, there are similarities
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between the carbon catabolite repression in yeast and the “Warburg phenotype” of tumor cells; moreover, the Crabtree effect was also observed in tumor cells accounting for cell metabolism
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adaptation to the heterogeneous tumor microenviroment (for extensive review on these topics see
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[126, 127]). Importantly, like oncogenes and tumor suppressors impinge on cellular metabolism
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to support growth and proliferation of cancer cells, similarly yeast homologues of cancer-related genes, such as RAS and SCH9, the yeast homologue of Akt, influence yeast cell metabolism and
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proliferation [123].
Mitochondrial RTG pathway activation of glutamate biosynthesis via the glyoxylate cycle has been suggested to resemble activation of glutaminolysis in tumor cells (see par. 2), yet studies on the role of glutaminolysis during the oxidative and fermentative growth need to be performed in yeast in order to establish a comparison with tumor cell metabolism. In addition, although the accumulation of oncometabolites in mutant SDH- and FH-associated tumors has been proposed to regulate degradation of the HIF-1α transcription factor [36], leading to activation of hypoxia response pathway and increased expression of glycolytic enzymes in cancer cells, yeast cells lack a homologue of HIF-1α. Interestingly enough, in a yeast model it
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has been shown that mitochondrial retrograde response is necessary for maintenance of normal flux through the TCA and glyoxylate cycles in wild type strains and for the oncometabolite fumarate and succinate accumulation in TCA cycle mutants lacking either FH or SDH,
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pinpointing the retrograde pathway as possible target for cancer therapy (see par. 3.1.2) [128]. Thus, notwithstanding these and other limitations, including the fact to be a unicellular
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organism, yeast remains a suitable model organism to study the relations among extracellular
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environment, cellular metabolism and mitochondrial function in tumorigenesis [129-131]. One of the hallmark of cancer cells is their resistance to death, thus the action of many
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anti-cancer drugs occurs through induction of apoptosis. However, the surrounding microenvironment strongly influences the overall cell sensitivity to anti-cancer drugs,
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determining in many cases survival and chemoresistance. Studies on PCD induced by acetic acid
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(AA-PCD) in yeast has provided a comprehensive picture of events, components and signaling
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pathways involved in death execution or inhibition [50, 132] (Fig. 2). Under fermenting growth conditions, i.e. in the presence of glucose as a sole carbon source, acetic acid triggers an early
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burst in ROS production followed by cytochrome c release, mitochondrial dysfunction, DNA fragmentation and ultimately cell death. As mentioned above, in the presence of glucose yeast cells mostly rely on a fermentative metabolism even in the presence of oxygen, with repressed mitochondrial activity due to the carbon catabolite repression. Interestingly, a specific activation of the mitochondrial RTG signaling causes a complete inhibition of AA-PCD under derepressing growth conditions, when raffinose is used as the sole carbon source and mitochondrial oxidative metabolism is stimulated [50] (Fig. 2). These data indicate how the change in cell environment (glucose to raffinose), which causes an increase in respiratory metabolism, can affect mitochondrial stress response pathways leading to cell adaptation. These experimental
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conditions clearly resemble those described above in mammalian cancer cells, in which mitochondrial OXPHOS has been shown to be strictly required for optimal proliferation in low glucose [120]. Thus, yeast is an attractive model to investigate the relations between cell
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microenvironment, mitochondrial dysfunction and adaptation in both physiological and pathological conditions [48, 131].
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Finally, another advantage offered by the yeast model is the possibility of heterologous
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expression of human genes either in the absence or in the presence of obvious yeast homologues [133, 134], including the cancer-susceptibility genes B-cell lymphoma-2 (Bcl2), p53 and
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BRCA1/2 (for refs see [129, 130]). With the aim to investigate the structure/function relationships of human proteins and their role in tumorigenesis, results obtained in the yeast
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model can provide a useful experimental platform also to screen new lead compounds as for their
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anticancer therapeutic efficiency. A successful example of complementation between human and
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yeast has recently revealed a new function of BRCA2 protein as modulator of anoikis sensitivity through an evolutionarily-conserved molecular mechanism involving regulation of ROS
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production and/or detoxification by BRCA2 during PCD processes [135] (Fig. 2). As reported in par. 3.1, in human cancer cells it has been observed that mtDNA depletion negatively affects BRCA2 protein level conferring increased sensitivity to the PARP inhibitor rucaparib [60]. Future studies with the yeast model in this direction may help to gain insight into the role of BRCA2 in cancer cell growth and tumor progression as well as the effects of mitochondrial dysfunctions on drug sensitivity and chemoresistance in BRCA2-related tumors. Thus, although with the aforementioned limitations, yeast could be used to develop a preliminary platform to screen for lead compounds for the designing of new anticancer drugs to be eventually tested in more physiological mammalian cell models.
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4. Conclusions Increasing evidences point to mitochondrial dysfunctions as important determinants in conferring
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cancer cell resistance to certain chemotherapeutic drugs. However, the presence of mitochondrial dysfunctions may also represent the "Achilles’ heel" for cancer cells, as it may provide a
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molecular and biochemical rationale for developing therapeutic strategies to selectively kill
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cancer cells using compounds that target the altered metabolism or signaling pathways. Inhibiting glycolysis may be a possible intervention approach, however, since glycolysis and its
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regulatory machinery is evolutionary conserved and shared with normal cells, glycolysis inhibitors are likely to affect normal cells at various degree. PARP inhibitors may provide a new
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therapeutic opportunity in tumors with mitochondrial dysfunction, e.g. those characterized by a decrease in BRCA2 protein levels. On this basis, it would be important to determine whether
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d
mitochondrial genetic alterations are present in each particular cancer type and whether they are pathogenic, i.e. they impair mitochondrial oxidative metabolism. This screening might be useful
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not only to predict efficacy of current therapeutic strategies but also to design new personalized anti-cancer therapies.
ACKNOWLEDGEMENTS
This work has been funded by grants from project FIRB-Merit RBNE08YFN3_005 to L.M., FIRB-MERIT RBNE08HWLZ and the Italian Ministry of Economy and Finance to the CNR for the Project “FaReBio di Qualità” to S.G.
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Figure legends Figure 1. Schematic representation of mitochondrial structure and oxidative metabolism.
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Mitochondria are cellular organelles composed of a double membrane, the outer and the inner mitochondrial membrane (OMM and IMM, respectively), which delimits the matrix. Glucose is
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metabolized in the cytosol to pyruvate which is transported into mitochondria for further
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catabolism through the TCA cycle coupled with respiration through the electron transport chain. The TCA cycle takes place in the matrix, generating α-ketoglutarate and electron donors (NADH
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and FADH2). The electron transport chain consists of four complexes (I-IV) and mediates transfer of electrons from NADH and FADH2 to the electron acceptor O2. In turn, electron
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transfer through the four respiratory complexes is coupled with proton extrusion out of the matrix toward the intermembrane space (IMS), producing an electrochemical gradient across the
te
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inner membrane. Protons ultimately return to the matrix through the ATP synthase or complex V, using the free energy produced by the electron respiratory chain to synthesize ATP. Essential
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protein components of the mitochondrial respiratory chain are encoded by the mitochondrial DNA (mtDNA), a circular double stranded DNA molecule that, besides containing the genetic information for 13 proteins [NADH dehydrogenase subunits 1, 2, 3, 4, 4L, 5, 6 (ND1, 2, 3, 4, 4L, 5, 6) belonging to Complex I, cytochrome b of Complex III, ATP synthase subunit 6 and 8 (ATPase 6, 8) belonging to Complex V, cytochrome c oxidase I-III (COI-III) belonging to Complex IV], encodes also for two ribosomal RNAs (rRNAs 12S and 16S) and 22 tRNAs (not shown) required for mitochondrial protein synthesis. The displacement loop (D-loop), or noncoding control region, contains sequences for the initiation of both mtDNA replication and transcription. Several molecules of mtDNA are present in each mitochondrion. ACO, aconitase; c, cytochrome c; CS, citrate synthase; FH, fumarate hydratase, IDH2, isocitrate dehydrogenase 2; 23
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KGD, α-ketoglutarate dehydrogenase; MDH, malate dehydrogenase, PDH, pyruvate dehydrogenase, PDK-1, pyruvate dehydrogenase kinase-1, Q, coenzyme Q, SDH, succinate dehydrogenase, SCS, succinyl-CoA synthetase; I-V, mitochondrial respiratory complex I, II, III,
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IV, V.
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Figure 2. Mechanisms of yeast cell death and adaptation in response to acetic acid stress. At
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low extracellular pH undissociated acetic acid enters the cells where it dissociates into acetate and protons causing cytosol acidification. In the presence of glucose as a sole carbon source,
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AA-PCD occurs with an early burst of reactive oxygen species (ROS) followed by release of cytochrome c (c), mitochondrial dysfunction, as revealed by decreased respiratory control index
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(RCI) and cytochrome c oxidase (COX) activity, DNA fragmentation, caspase-like activation
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and complete loss of cell viability. In raffinose grown-cells, following acetic acid treatment a
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lower ROS level is detected, RTG signaling is activated with translocation of Rtg1/3p into the nucleus leading to up-regulation of retrograde-target genes, causing cell resistance to AA-PCD
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(see text for details). RTG signaling is inhibited by TOR. Ras can either impact RTG-dependent lifespan extension or cause mitochondrial dysfunction through cAMP and PKA hyperactivation leading to cell death. Heterologous expression of human BRCA2 gene in yeast potentiates AAPCD through an evolutionarily-conserved mechanism involving regulation of ROS production and/or detoxification. Red, blue and green arrows indicate pro-death, survival and longevity pathways, respectively.
Figure 3. A scheme of mitochondrial stress-mediated pathways involved in cancer progression and chemoresistance. In mammalian cells, mitochondrial dysfunction resulting
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from mtDNA depletion causes impaired mitochondrial respiration and mitochondrial membrane potential depolarization, promoting activation of the retrograde signaling pathway. At variance with yeast cells, the regulation of mitochondria-to-nucleus signaling has not been completely
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characterized. Loss of mitochondrial transmembrane potential promotes increased cytosolic calcium concentration ([Ca2+]c) and activation of calcineurin, thereby eliciting a calcineurin-
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mediated retrograde signaling pathway [136]. The transcription of several target nuclear genes (>
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120 genes identified to date) is up-regulated in response to this calcium/calcineurin-dependent signaling and possibly other mechanisms not yet identified (indicated by a question mark)
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through nuclear translocation of a number of transcription factors (NRF2, NF-κB, CREB and NFAT), and upregulation of the transcriptional coactivator hnRNPA2 [136]. In turn, the genes
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induced by activation of the mitochondrial retrograde pathway can modulate a number of cellular
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functions including: i) apoptosis and multidrug resistance, thereby promoting resistance to
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chemotherapeutic drugs; ii) invasion and epithelial-to-mesenchymal transition, thus sustaining metastatic progression of cancer cells; iii) regulation of protein expression at the post-
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transcriptional level, through upregulation of components of the ubiquitin-proteasome proteolytic pathway (such as Skp-2) and miRNAs (such as miR-1245), thereby modulating the levels of oncosuppressor proteins, like BRCA2 (see the text for details); iv) energy metabolism, through upregulation of glucose transporters and glycolytic enzymes, thus supporting the metabolic shift typical of cancer cells. Overall these modifications can lead to metabolic reprogramming of cancer cells, malignant progression and chemoresistance.
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i cr us
Table 1. Intracellular targets of the oncometabolites succinate, fumarate, and alpha-ketoglutarate Mutated gene Oncometabolite Metabolic changes Molecular targets Cancer type Inhibition of α-KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs; succination of KEAP1 and Aco2
Hereditary skin and uterine leiomyomas and renal cell cancer; renal cysts; breast, bladder and Leydig cell tumors
[36, 38, 40, 137-145]
Decreased flux of metabolites through the Krebs cycle Reduced activity of electron transport chain Impaired oxidative metabolism Increased lactate production
Inhibition of α-KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs
Hereditary paraganglioma, pituitary adenoma and pheochromocytoma; gastrointestinal stromal tumors, renal tumors, thyroid tumors, neuroblastomas, testicular seminomas; childhood T cell acute leukemia
[37, 39, 40, 146-148]
Reduced NADPH levels Reduced glutamate levels Reduced NADPH-dependent reductive carboxylation
Inhibition of α-KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs; inhibition of collagen prolyl and lysyl
Chondrosarcoma, cholangiocarcinoma, glioma, secondary glioblastoma, acute myeloid leukemia, a subset of breast cancers, angioimmunoblastic T-cell lymphoma
[41, 42, 149-155]
Decreased flux of metabolites through the Krebs cycle Impaired oxidative metabolism Increased glutamine uptake and lactate production Accumulation of fumarate-derived argininosuccinate Acquired auxotrophy for arginine Bilirubin excretion
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Fumarate
FH
Succinate
Ac
ce
pt
SDH (loss-of-function)
ed
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(loss-of-function)
IDH1/2 (gain-of-function)
D-2-KG
26
Ref.
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hydroxylases leading to impaired collagen maturation
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ce
pt
ed
M
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α-KG, α-ketoglutarate; Aco2, aconitase 2; FH, fumarate hydratase; HIFs, hypoxia-inducible factors; IDH1/2, isocitrate dehydrogenase 1 and 2; KDMs, Jumonji C domain-containing histone lysine demethylases; KEAP1, Kelch-like ECH-associated protein 1; PHDs, HIF prolyl hydroxylases; SDH, succinate dehydrogenase; TET, ten eleven translocation family of 5-methylcytosine hydroxylases.
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Figure 1
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Figure 2
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Figure 3
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Table 1
Inhibition of -KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs; succination of KEAP1 and Aco2
Hereditary skin and uterine leiomyomas and renal cell cancer; renal cysts; breast, bladder and Leydig cell tumors
[36, 38, 40, 136-144]
Succinate
Decreased flux of metabolites through the Krebs cycle Reduced activity of electron transport chain Impaired oxidative metabolism Increased lactate production
Inhibition of -KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs
Hereditary paraganglioma, pituitary adenoma and pheochromocytoma; gastrointestinal stromal tumors, renal tumors, thyroid tumors, neuroblastomas, testicular seminomas; childhood T cell acute leukemia
[37, 39, 40, 145-147]
Inhibition of -KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs; inhibition of collagen prolyl and lysyl
Chondrosarcoma, cholangiocarcinoma, glioma, secondary glioblastoma, acute myeloid leukemia, a subset of breast cancers, angioimmunoblastic T-cell lymphoma
[41, 42, 148-154]
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Decreased flux of metabolites through the Krebs cycle Impaired oxidative metabolism Increased glutamine uptake and lactate production Accumulation of fumarate-derived argininosuccinate Acquired auxotrophy for arginine Bilirubin excretion
(loss-of-function)
IDH1/2 (gain-of-function)
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SDH (loss-of-function)
Ref.
Fumarate
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FH
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Table 1. Intracellular targets of the oncometabolites succinate, fumarate, and alpha-ketoglutarate Mutated gene Oncometabolite Metabolic changes Molecular targets Cancer type
D-2-KG
Reduced NADPH levels Reduced glutamate levels Reduced NADPH-dependent reductive carboxylation
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hydroxylases leading to impaired collagen maturation
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-KG, -ketoglutarate; Aco2, aconitase 2; FH, fumarate hydratase; HIFs, hypoxia-inducible factors; IDH1/2, isocitrate dehydrogenase 1 and 2; KDMs, Jumonji C domain-containing histone lysine demethylases; KEAP1, Kelch-like ECH-associated protein 1; PHDs, HIF prolyl hydroxylases; SDH, succinate dehydrogenase; TET, ten eleven translocation family of 5-methylcytosine hydroxylases.
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*Graphical Abstract (for review)
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S0006-2952(14)00448-1 http://dx.doi.org/doi:10.1016/j.bcp.2014.07.027 BCP 12043
To appear in:
BCP
Received date: Revised date: Accepted date:
23-6-2014 25-7-2014 28-7-2014
Please cite this article as: Guaragnella N, Giannattasio S, Moro L, Mitochondrial dysfunction in cancer chemoresistance, Biochemical Pharmacology (2014), http://dx.doi.org/10.1016/j.bcp.2014.07.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mitochondrial dysfunction in cancer chemoresistance Nicoletta Guaragnella, Sergio Giannattasio*, Loredana Moro
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Institute of Biomembranes and Bioenergetics, Via Amendola 165/A, 70126 Bari, Italy
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*Corresponding author: Sergio Giannattasio
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Consiglio Nazionale delle Ricerche Istituto di Biomembrane e Bioenergetica
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Via Amendola 165/A
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70126 Bari (ITALY) Email: [email protected]
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Fax +390805443317
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Tel +390805443316
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Abstract
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Mitochondrial dysfunction has been associated with cancer development and progression. Recent evidences suggest that pathogenic mutations or depletion of the mitochondrial genome can
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contribute to development of chemoresistance in malignant tumors. In this review we will
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describe the current knowledge on the role of mitochondrial dysfunction in the development of chemoresistance in cancer. We will also discuss the significance of this research topic in the
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context of development of more effective, targeted therapeutic modalities and diagnostic strategies for cancer patients, with a particular focus on the potential use of PARP inhibitors in
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cancer patients displaying mitochondrial DNA mutations. We will discuss recent studies highlighting the importance of the cross-talk between the tumor microenvironment and
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mitochondrial functionality in determining selective response to certain chemotherapeutic drugs. Finally, owing to the similarities between cancer and yeast cell metabolism, we will point out the
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use of yeast as a model system to study cancer-related genes and for anti-cancer drugs screening.
Keywords: cancer chemoresistance, mitochondrial dysfunction, tumor microenvironment, mitochondrial retrograde signaling, yeast
Chemical compounds cited in this article: 3-bromopyruvate (PubChem CID: 70684 ); 2deoxyglucose (PubChem CID: 439268); dichloroacetate (PubChem CID: 25975); lonidamine (PubChem CID: 39562); rucaparib (PubChem CID: 9931954). 2
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1. Introduction
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“We understand the origin of cancer cells if we know how their large fermentation originates, or, to express it more fully, if we know how the damaged respiration and the
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excessive fermentation of the cancer cells originate” [1]. In this way Warburg described the metabolic feature of tumor cells, which is a metabolic shift from oxidative phosphorylation to
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glycolysis for ATP generation in tumor cells, even in the presence of high oxygen tension,
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termed the "Warburg effect". Originally Warburg proposed that this glycolytic shift was due to defects in mitochondrial function leading to impaired oxidative phosphorylation. It has later been
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appreciated that even tumors with "normal" oxidative phosphorylation and oxygen consumption exhibit increased glycolytic metabolism [2] converting most incoming glucose to lactate rather
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than metabolizing it in the mitochondria through oxidative phosphorylation. New hypothesis
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have been proposed but still there is no definitive conclusion. Zu and Guppy [3] have indicated
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that not all cancer cells exhibit defects in oxidative metabolism. More recently, Moreno-Sanches and co-workers [4] have suggested that cancer cells have a heterogeneous metabolic profile and some cancer cell populations would use glycolysis as main energetic pathway, while others would use oxidative phosphorylation. Thus, Warburg’s observation on the origin of cancer cells still retains its validity, even though the molecular basis of the Warburg’s effect has to be reconsidered in the context of a set of concerted changes in energy metabolism and mitochondrial function supporting tumorigenesis. This process is now referred to as metabolic reprogramming and represents an emerging hallmark of cancer cells indicating the importance of deregulated metabolism as a widespread trait of cancer cells, as pointed out by Hanahan and Weinberg [5]. 3
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However, a debate still exists on the role of metabolic reprogramming as a cause of tumorigenesis given the fact that cancer cell metabolic reprogramming is driven by onco- and tumor-suppressor gene mutations affecting core signaling pathways and processes that define
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also the core hallmarks of cancer [6, 7]. From this point of view, the metabolic reprogramming may simply represents another phenotype that is regulated by oncogenes stimulating cell
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proliferation, and thereby has raised doubts on its functional independence from the core cancer
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hallmarks. Recent studies have shown a causal role of mutations in enzymes of the Krebs cycle in the etiology of some cancers, suggesting that, at least in some cancer types, the deregulated
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metabolism may represent a driver of cell malignant transformation [8, 9].
The metabolic shift towards glycolysis, typical of cancer cell metabolic reprogramming,
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is essential to support three basic needs of tumor cells: i) glycolysis would represent the main
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active pathway of ATP production under conditions of oxygen deprivation, i.e. in the hypoxic
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microenvironment of tumor tissues (see par. 3.2); ii) increased biosynthesis of macromolecules (carbohydrates, proteins, lipids, nucleic acids): proliferating tumor cells not only require rapid
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supply of energy, but also building blocks for cell biosynthesis and dividing processes (mitosis). In this context, glycolytic intermediates, particularly glucose-6-phosphate, can be directed to the pentose phosphate pathway to generate ribose-5-phosphate and 3-carbon intermediates important for nucleic acid and lipid biosynthesis, respectively; iii) maintenance of redox homeostasis: NADPH generated through the pentose phosphate pathway would provide reducing equivalents in many biosynthetic reactions. In addition, acting as an antioxidant, NADPH would counteract the detrimental effects of very high levels of ROS produced in rapidly proliferating cancer cells. Indeed, lactate produced through increased fermentation by a population of cancer cells can be used as a fuel substrate by a second population of cancer cells through an active oxidative
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metabolism [10-12]. On the other hand, L- and D-lactate can both enter cells and be formed in the cytosol from glucose via glycolysis and methylglyoxal pathway, respectively. Both the isomers D-lactate
mitochondria by two separate enzymes, namely the mitochondrial
are oxidized inside
L-lactate
dehydrogenase
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can enter mitochondria in a carrier mediated manner; L- and
located in the matrix and the D- lactate dehydrogenase, respectively [13-16]. The mitochondrial
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metabolism of L- and D-lactate in cancer cells is more active than in normal cells and seems to
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account for tricarboxylic acid (TCA) cycle anaplerosis [16-19] contributing to ROS scavenging and fatty acid synthesis, all crucial for cancer cell viability and proliferation and requiring
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NADPH.
Mitochondria are cellular organelles that play a key role in the context of cancer
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metabolic reprogramming because they represent a nodal point where most metabolic and
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signaling pathways converge [20]. They are a central hub for biosynthesis of aminoacids, nucleic
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acids, and lipids and the main providers of NADH and NADPH and thus are important regulators of the redox balance in the cells [21, 22].
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Several evidences [23-25] have shown that mitochondrial dysfunction provides survival advantage to cancer cells, suggesting that mitochondria have a tumor suppressor function. Yet, the mechanisms of such function remain a molecular conundrum. Indeed, mitochondrial dysfunction also occurs en route to most apoptotic pathways [25]. Evasion of apoptosis, the major form of programmed cell death (PCD) in mammals, is a requirement for both neoplastic transformation and sustained growth of tumor cells (reviewed in [5]) as well as a crucial acquired capabilities used by cancer cells to resist to anticancer therapies (reviewed in [26]). With this respect it is of note that in myoblasts mitochondrial genetic stress results in the up-regulation of a number of proapoptotic proteins, including BAD, Bax, and Bid. Yet, this mitochondrial
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dysfunction promotes resistance to apoptosis induction due to mislocalization of the proapoptotic factors and reduced processing of Bid to active tBid (see par. 2.2) [27]. On the other hand, a plethora of genetic and metabolic mitochondrial alterations have been described so far that,
drug sensitivity in cancer cells (for recent reviews see [28, 29]).
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besides affecting the energy metabolism of the cells, are also implicated in the modulation of
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In this review, we will focus on the role of mitochondrial dysfunction in the development
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of chemoresistance and the rationale for targeting cancer cells with mitochondrial dysfunction as a promising pharmacological therapeutic strategy. We will also discuss new studies highlighting
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the importance of the cross-talk between the tumor cell microenvironment and mitochondrial functionality in determining selective response to certain chemotherapeutic drugs as well as the
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use of yeast as a model organism in cancer research.
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2. Mitochondrial genetic and metabolic stress in cancer drug resistance Mitochondria are semi-autonomous organelles which possess their own genome.
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Mitochondrial DNA (mtDNA) is a 16.569 bp circular DNA molecule encoding for 13 proteins belonging to the mitochondrial respiratory chain, 22 tRNAs and 2 rRNAs (Fig. 1). The number of mtDNA molecules per cell varies among cell and tissue types. In addition, within the same cell some mitochondria can have mutation/s of the mtDNA while others have normal mtDNA, a phenomenon termed heteroplasmy. Instead, the term homoplasmy indicates that a cell or a cell population contains either entirely normal or entirely mutant mtDNA molecules. So far, several mutations occurring in nuclear genes encoding for mitochondrial proteins involved in energy metabolism as well as in mtDNA have been identified in different cancer types. For example, mutations in enzymes of the TCA cycle have been linked to tumorigenesis.
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In addition, decreased expression and activity has been described for components of the electron respiratory chain in cancer cells, including complex I [30-32], complex II [33, 34], complex III
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[32-34], complex IV [33, 34] and complex V [33, 35] (Fig. 1).
2.1 Mutations of TCA enzymes
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It has been shown that inactivating-germline mutations in fumarate hydratase (FH) and
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succinate dehydrogenase (SDH), two enzymes of the TCA cycle, are oncogenic because of accumulation of fumarate and succinate [9, 36], small-molecules of the normal metabolism that,
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when abnormally accumulated, cause both metabolic and non-metabolic signaling dysregulation establishing a milieu that initiates malignant transformation, and thereby are termed
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oncometabolites [8, 9]. Loss-of-function mutations in FH and SDH occur in some tumors
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including pheochromocytoma, paraganglioma, renal cell carcinoma, leiomyomas [9] (Table 1).
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The neoplastic effect of fumarate and succinate has been linked to hypoxia-inducible factor 1 (HIF1) stabilization and inhibition of α-ketoglutarate-dependent dioxygenases that regulate DNA
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and histone methylation and, thus, gene expression, thereby promoting epithelial-tomesenchymal transition, angiogenesis, and changes in cell metabolism [37-39]. In FH-deficient kidney mouse cells a metabolic pathway beginning with glutamine uptake and ending with bilirubin excretion, involving the biosynthesis and degradation of haem, has been identified. This pathway enables FH-deficient cells to use the accumulated TCA cycle metabolites and permits partial mitochondrial NADH production. Targeting this pathway would render FH-deficient cells non-viable, while sparing wild-type cells. Thus, inhibition of haem oxygenation has been shown to be synthetically lethal when combined with FH deficiency, providing a new potential target for treating hereditary leiomyomatosis and renal-cell cancer patients [40]. Another
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oncometabolite recently identified is 2-hydroxyglutarate, which accumulates following gain-offunction mutations in another enzyme of the TCA cycle, NADP-dependent isocitrate dehydrogenase (IDH) 1 (cytosolic) and 2 (mitochondrial) in different cancer types, including
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acute myeloid leukemia, glioma, chondrosarcoma and a subtype of breast cancers [41, 42]. Accumulation of 2-hydroxyglutarate causes changes in the activity of DNA demethylases thus
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promoting epigenetic changes [42], a phenotype also observed following loss-of-function
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mutations in FH and SDH (Table 1). While a number of studies have linked mtDNA mutations with changes in cancer cell sensitivity to chemotherapy, the possible role of mutations in the
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TCA cycle enzymes in modulating drug resistance has been poorly investigated.
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2.2 Mitochondrial DNA mutations and retrograde response
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Defects in mtDNA, including a low mtDNA copy number, have been implicated in cancer. In
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particular, a reduction in mtDNA content results in impaired mitochondrial respiration and depolarization of the mitochondrial membrane. Cell response to mitochondrial dysfunction
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occurs via the evolutionally conserved communication pathway from mitochondria to the nucleus, termed retrograde response [43]. The molecular details of retrograde signaling pathway activated in response to mitochondrial dysfunction, including respiratory deficiency due to mtDNA depletion, antimycin A-dependent inhibition of the electron flow along the respiratory chain or carbonyl cyanide m-chlorophenyl hydrazone-dependent uncoupling, have been characterized first in S. cerevisiae [44]. In these cells, retrograde (RTG) signaling allows adaptation to mitochondrial dysfunction by eliciting a metabolic reconfiguration in which specific nuclear genes are up-regulated, including those involved in peroxisomal biogenesis, the glyoxylate cycle and fatty acid oxidation [43]. CIT2, encoding the peroxisomal isoform of citrate
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synthase, is the prototypical target gene of the yeast RTG pathway: its expression is largely increased in cells with mitochondrial dysfunction, including those lacking mtDNA (ρ0). Rtg1p
box) in the promoter region of retrograde-target genes (Fig. 2).
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and Rtg3p form a heterodimeric transcription factor binding the RTG-specific cis-element (R-
The physiological significance of the RTG pathway activation is to allow glutamate
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biosynthesis as nitrogen source for biosynthetic reactions, and to ensure the maintenance of
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mtDNA, through the regulation of ACO1 [45]. Glutamate plays an important role in metabolic reprogramming of cancer cells. Indeed, besides increased glycolysis, metabolic adaptation in
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cancer also includes increased dependence of tumor cells on glutamine, an aminoacid that, upon entry in the cells, can be used as energy source by glutaminolysis as well as a building block in
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anabolic reactions [21]. Glutaminase is responsible for conversion of glutamine to glutamate,
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which, in turn, can be converted either into glutathione (GSH), an abundant antioxidant in the
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cells and a regulator of the cellular redox balance, or into α-ketoglutarate, an intermediate of the TCA cycle. α-Ketoglutarate is in turn reductively carboxylated by the NADPH-linked
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mitochondrial isocitrate-dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate producing ATP and allowing synthesis of other aminoacids and fatty acids. Thus, the high rate of glycolysis, an inefficient way to generate ATP, and glutaminolysis have been proposed to be for cancer as well as for all proliferating cells an adaptive response to facilitate the uptake and incorporation of nutrients into the biomass (e.g. nucleotides, aminoacids, and lipids) needed to generate a new cell [46, 47]. With this respect, retrograde activation of glyoxylate cycle in yeast resembles glutamine-dependent reductive carboxylation of αketoglutarate in tumor cell metabolic reprogramming [48]. The RTG pathway has been shown to promote longevity in yeast [49] and recently to be involved in PCD resistance of yeast cells
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treated with acetic acid [50]. The RTG pathway is linked to other signaling pathways: Target of Rapamycin (TOR) kinase pathway, which inhibits RTG-dependent gene expression [51] in accordance with a causative role for TOR in acetic acid-induced PCD [52] and Ras-cAMP,
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which impacts RTG-dependent lifespan extension [49, 53]. It is of note that Ras-cAMP-protein
function and cell death with nutritional status in yeast [54] (Fig. 2).
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kinase A (PKA) signaling plays a physiological role in coordinating mitochondrial respiratory
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In mammalian cells, the mitochondrial retrograde signaling has been suggested to be an important mechanism for tumorigenesis [55-57] and has been associated with increased cytosolic
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calcium levels [58-60], hypoxic-to-normoxic shift [61], activation of oncogenic signaling molecules/pathways, including PI3-kinase/AKT, JNK, Ras, ERK [60-67] and induction of the
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expression of nuclear genes involved in invasion, including matrix metalloproteinases and
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cathepsin [59], epithelial-to-mesenchymal transition, like Snail, Twist, Slug, TGF-β [63, 65],
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ubiquitination and DNA repair [60, 67], glucose transport, particularly the glucose transporter GLUT1 and GLUT4 that may contribute to the metabolic shift towards a highly glycolytic
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phenotype [64, 68] (Fig. 3). Mutations and/or reduced levels of mtDNA have been reported in several cancer types, including prostate [60, 69-71], kidney [72], breast [73-75], liver [76], colon and rectal cancers [77]. Chemically induced mtDNA depletion has been shown to promote cancer progression to an invasive and apoptosis-resistant phenotype [59, 63, 64, 78-82]. Consistently, in breast and prostate cancer patients, mtDNA mutations and reduced mtDNA copy number are associated with increased metastasis and adverse prognosis [69, 83]. In addition, introduction of homoplasmic pathogenic point mutations in the mtDNA ATP synthase subunit 6 gene in HeLa cells prevented apoptosis and increased their tumorigenic potential in vitro and in vivo [84]. Introduction of the ATP6 T8993G mutation, found in a prostate cancer patient at the
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germline level, into the PC-3 prostate cancer cell line through cybrid transfer also increased tumor growth in vivo as well as ROS levels [85]. Ishikawa et al. [86] showed that presence of two point mutations in the NADH dehydrogenase subunit 6 (ND6), which produced a deficit in
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respiratory complex I activity and ROS overproduction, enhanced the metastatic potential of the cells. Overall, these studies implicate mtDNA mutations in tumorigenesis and cancer
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progression.
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Studies with cells chemically depleted partially (ρ-) or totally (ρ0) of their mtDNA support a role for mitochondrial dysfunction in cancer drug resistance (Fig. 3). It has been
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reported that ρ- or ρ0 prostate cancer cells become less sensitive to the chemotherapeutic drugs
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paclitaxel [78] and N- (4-hydroxyphenyl) retinamide [87]. Naito and coworkers [88] showed that mtDNA depletion promotes resistance to hormone therapy in breast cancer cells. Singh and
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coworkers [89] showed that ρ0 HeLa cells became resistant to adriamycin and photodynamic
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therapy but remained sensitive to γ-rays and DNA alkylating agents. Park and coworkers [90] have shown that hepatoma cells deprived of mtDNA were less sensitive to hydrogen peroxide
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and to ROS-inducing agents, including paraquat, doxorubicin and menadione, and that this drugresistant phenotype was associated with increased expression of the antioxidant enzymes manganese superoxide dismutase and glutathione peroxidase. Consistent with a role of altered redox regulation in the development of resistance to ROS-inducing drugs, cancer cell insensitivity to the chemotherapeutic drugs paclitaxel and cisplatin has been associated with increased antioxidant expression levels [91-93]. However, it should be noted that mtDNA mutations or depletion may exert different effects on drug sensitivity depending on the particular tissue. For example, docetaxel-resistant laryngeal cancer cells exhibit increased mtDNA content [94]. An intriguing phenotype has been recently described in a patient with serous ovarian
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cancer: the presence of a nearly homoplasmic mtDNA mutation in the NADH dehydrogenase subunit 4 (ND4) in the postchemotherapy but absent in the prechemotherapy tissue, which could determine tumor growth arrest [95]. Mutations in mtDNA-encoded proteins belonging to the
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mitochondrial respiratory Complex I, such as those occurring in ND4, have indeed been suggested to play an "oncojanus" role in some tumor tissues: they would promote tumor growth
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when are below a threshold level but would exert the opposite effect when they reach a critical
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mutant load [96].
The molecular mechanism involved in mtDNA-mediated drug resistance is not well
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understood. It has been shown that development of resistance to adriamycin in HeLa ρ0 cells was not due to changes in apoptotic cell death, cell cycle response or to the uptake of adriamycin
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[89]. Instead, since adriamycin activation requires Complex I activity, it has been proposed that
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mtDNA depletion, by impairing the mitochondrial respiratory chain, would prevent its activation
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in the cells thus resulting in cancer resistance to this particular drug [89]. Very recently, it has been demonstrated that depletion of mtDNA in hepatocarcinoma cells results in chemoresistance
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to doxorubicin, cisplatin and SN-38 and that this phenotype is associated with activation of the nuclear factor erythroid 2 [NF-E2]-related factor 2 (NRF-2) signaling pathway and consequent up-regulation of Multi-Drug Resistance gene 1 (MDR1), Multidrug Resistance-associated Protein 1 (MRP1) and 2 (MRP2), proteins notably involved in multidrug resistance [97] (Fig. 3). Increased levels of MDR1 were also observed upon mtDNA depletion in colon cancer cells [98]. Moreover, the ability of doxorubicin, SN-38 and cisplatin to induce proapoptotic signals was weaker in mtDNA-depleted cells, as indicated by increased survivin levels and reduced Bax/Bcl2 expression ratio. On the other hand mtDNA-depleted myoblasts, although showing reduced mitochondrial transmembrane potential, are resistant to staurosporine-mediated apoptosis
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through a mechanism involving a profound modification of the apoptotic machinery, including: i) sequestration of the proapoptotic factors Bid, Bax, BAD in the inner mitochondrial membrane where they cannot interact with Bcl-2; ii) increased expression of the antiapoptotic proteins Bcl-
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2 and Bcl-XL; iii) inability to process Bid into tBid; iv) reduced activation of caspases 3, 9, 8 [27].
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Overall, mtDNA-dependent deregulation of genes involved in apoptosis modulation may
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play a role in drug resistance depending on the cell type and/or the specific drug.
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3. Targeting mitochondria in drug-resistant cancer cell therapy
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3.1 Targeting cancer cells with defective mitochondrial function
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Mitochondrial dysfunction in cancer cells may offer a novel opportunity for selective
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anti-cancer therapy. Indeed, cancer cells with respiration deficiency, though more resistant to conventional chemotherapeutic drugs should be more sensitive than other cancer types to
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inhibitors of the glycolytic pathway because they would rely only on this pathway for energy supply. On the other hand, targeting specific molecular alterations resulting from activation of the mitochondrial retrograde signaling may provide a selective therapeutic approach for cancers with defective mitochondrial respiration.
3.1.1. Glycolysis inhibition To date, several glycolysis inhibitors have been tested as potential anti-cancer drugs. 2Deoxyglucose, an inactive glucose analog that inhibits hexokinase II is actually in clinical trials for cancer therapy [99] (www.clinicaltrials.gov). 2-Deoxyglucose has been reported to promote
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cell death of cancer cells exhibiting mitochondrial defects or in hypoxic conditions [100-102]. 3bromopyruvate, another hexokinase II inhibitor, has shown selective activity in killing colon cancer and lymphoma cells with mitochondrial dysfunction [103]. However, it is not known at
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present whether 3-bromopyruvate will move to clinical trials. Lonidamine, a derivative of indazole-3-carboxylic acid, is another promising glycolytic inhibitor. It prevents tumor growth
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by inhibiting hexokinase II and depleting ATP from the cells [104, 105]. However, it is toxic for
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normal tissues as demonstrated by studies in which lonidamine was administered alone [106] or in combination with the chemotherapeutic drugs doxorubicin, cyclophosphamide or cisplatin in
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animal models [107]. Despite its toxicity, phase II/III clinical trials have showed promising results in the treatment of solid tumors [108, 109]. Thus, lonidamine treatment of tumors with a
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highly glycolytic phenotype, particularly those with mitochondrial defects, may represent a
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promising strategy in the era of personalized medicine.
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Another example of glycolysis inhibitor is dichloroacetate (DCA), a pyruvate mimetic that stimulates pyruvate dehydrogenase by suppressing pyruvate dehydrogenase kinase 1. DCA
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increases mitochondrial pyruvate consumption, lowering the pyruvate pool available for glycolysis. In vitro studies have shown that DCA has selective activity against cells with severe mitochondrial dysfunction, i.e. ρ0 cells, and synergizes with 2-deoxyglucose in p53-/- cells with a deficit in Complex IV activity [110]. DCA synergizes also with antineoplastic agents that cause mtDNA damage, namely cisplatin and topotecan [110]. However, the ability of DCA to selectively target cancer cells with mitochondrial respiratory defects deserves further analysis in preclinical models.
3.1.2 Targeting the mitochondrial retrograde signaling
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Besides inhibiting the glycolytic pathway as a potential effective strategy for cancers with mitochondrial respiratory dysfunction, a recent work has indicated that targeting the molecular alterations resulting from activation of the mitochondrial retrograde signaling pathway may be a
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novel promising anti-cancer strategy. Arbini and coworkers [60] have shown that mtDNA depletion or large deletions activate a calcium-dependent mitochondrial retrograde signaling
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pathway that results in increased levels of the E3 ubiquitin ligase Skp2 mRNA and of miRNA-
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1245, encoded by the nuclear genome (Fig. 3). Skp2 and miR-1245 are negative regulators of the expression of the tumor suppressor protein BRCA2, thus their up-regulation by mitochondrial
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dysfunction promotes reduction of the expression levels of BRCA2 [60]. BRCA2 is a cancer susceptibility gene, i.e. when mutated, results in familial predisposition to several cancers,
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including breast, ovary and prostate cancer [111]. Reduced levels of BRCA2 protein have been
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detected in prostate cancer cell lines derived from sporadic tumors as well as in 70% of sporadic
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prostate cancer specimens in vivo [60, 112]. In addition, loss of nuclear BRCA2 has been reported in about 60% of pancreatic and breast cancer specimens, 80% of high-grade ovarian
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serum carcinomas, 30% of colon cancer samples [113]. On the other side, positive BRCA2 staining in the same cancer types has been correlated with increased overall and disease-free survival [113]. The major function of BRCA2 is to complex with Rad51 and to orchestrate DNA repair through homologous recombination. Several studies have shown that cancer cells lacking BRCA2 are about 1000-fold more sensitive to a novel class of anticancer drugs, namely poly(ADP-ribose) polymerase (PARP) inhibitors [114], that function through the mechanism of synthetic lethality, whereby two defective genes, each having by itself negligible effects on cell viability, become lethal when both are present in the same cells [115]. PARP proteins play a critical role in signaling DNA damage and their inhibition results in accumulation of single- and
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double-strand DNA breaks and apoptosis unless upstream homologous recombination is active and rescues the defect [116]. Thus, tumor cells lacking BRCA2 would undergo apoptosis if PARP is inhibited. This synthetic lethality approach by PARP inhibitors is being proven
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effective in clinical trials for treatment of cancers resulting from inherited mutations in BRCA2 [117]. The finding that mtDNA depletion activates a retrograde signaling pathway that promotes
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reduction of BRCA2 protein levels (Fig. 3) has direct clinical implications because it makes
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tumors featuring mtDNA large deletions or depletion, such as prostate carcinomas [60], promising candidates for tailored and personalized therapeutic regimens encompassing PARP
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inhibitors. Indeed, mtDNA depletion or large deletions might cooperate with PARP inhibition to induce cell death in cancer cells. Phase I/II clinical trials are actually undergoing to assess the
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carcinomas [www.clinicaltrials.gov].
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therapeutic potential of PARP inhibitors in sporadic cancers, including advanced prostate
3.2 Mitochondrial stress response in cancer cell adaptation to tumor microenvironment and
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drug sensitivity
Environmental conditions, such as the concentrations of highly consumed nutrients, represent a critical aspect for supporting the rate of cancer cell proliferation. Thus, for optimal growth, cancer cells must adapt their metabolism to their surrounding tumor microenvironment, which is usually hostile and characterized by acidity, hypoxia and low levels of glucose ([29, 118] and refs therein). As mentioned above, increased fermentation results in high production of lactate causing both tumor microenvironment acidification and possible activation of metalloproteinases and extracellular matrix remodeling enzymes, thus promoting cancer cell invasion [119]. A better understanding of cancer cell adaptations to tumor microenvironment
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might reveal the molecular basis of chemoresistance that can be exploited for targeted-drug development. A number of genes that have been implicated in the metastatic process, involving angiogenesis, survival and growth, have been found to be hypoxia-responsive, although little is
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known about the effects of oxygen level fluctuation over a range of 0 – 2 % on gene expression (for refs see [118]). Due to the complex experimental procedure required to mimic tumor
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microenvironment, few studies have been performed on the effect of glucose deprivation on cell
pathway” in the Web of Science™ yields only 25 articles.
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stress response. Indeed, a search for “low glucose tumor microenvironment cell response
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Making use of a continuous-flow culture apparatus (Nutrostat) for maintaining proliferating cells in low-nutrient media for long periods of time, Sabatini and coworkers
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performed competitive proliferation assays on a pooled collection of barcoded cancer cell lines
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cultured in low-glucose conditions [120]. Their results pinpoint mitochondrial oxidative
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phosphorylation (OXPHOS) as the major pathway required for optimal proliferation in low glucose. Glucose limitation normally causes up-regulation in the OXPHOS genes. Interestingly,
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they found that cell line sensitivity to low glucose was accompanied by defects in the OXPHOS up-regulation as a result of either mtDNA mutations in complex I genes or impaired glucose utilization [120]. These genetic/metabolic defects would predict sensitivity of cancer cells to bioguanides, antidiabetic drugs that inhibit OXPHOS when cancer cells are in the presence of low glucose. Hence, mtDNA mutations and impaired glucose utilization become potential biomarkers for identifying tumors with increased sensitivity to OXPHOS inhibitors. From a general point of view, these results underscore the strict cross-talk between glucose sensing/utilization and mitochondrial function and the importance of considering cell microenvironment when evaluating the sensitivity of cancer cells to certain drugs.
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Recently, in another experimental set-up, an RNA-interference-based transgenic mouse model, that allows tetracyclin-dependent regulation of phosphatase and tensin homolog (PTEN)encoding tumor suppressor gene in a time- and tissue-specific manner, has been generated. Use
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of this model has revealed a complex picture of how PTEN inactivation drives tumor maintenance, showing an interplay between tumor and microenvironment that would not be
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predicted from studies on cultured cells [121]. This produces intratumoral heterogeneity which
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can affect both disease progression and clinical response to molecularly targeted therapies. It is of notice that under similar environmental conditions, unicellular and multicellular
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organisms have similar metabolic phenotypes with an advantage to oxidative metabolism during nutrient limitation and prevalence of non-oxidative metabolism during cell proliferation [47].
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With this respect, the yeast Saccharomyces cerevisiae is an especially suitable model organism:
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it is one of the most thoroughly studied unicellular eukaryotes at the cellular, molecular, and
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genetic level due to its well-known experimental tractability; it may adapt and survive with alternatives in its genome expression and metabolism, allowing to mimic a variety of metabolic
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and (patho)physiological scenarios and to study mitochondrial dysfunctions through the generation of respiratory deficient strains (mutation/deletion of single nuclear genes encoding mitochondrial proteins or via abrogation of mitochondrial DNA) and their effects on cellular processes. Notably, yeast undergoes programmed cell death (PCD) pathways which share most of the morphological and biochemical hallmarks of mammalian apoptosis in response to different stressors [122]. Last, but not least, S. cerevisiae cell growth depends on nutrient availability in its natural environment [123]. Glucose is the preferred carbon source for yeast, regardless the presence of oxygen or other non-fermentable carbon sources. Glucose-grown yeast cells mainly use
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fermentation for carbon and energy metabolism. In contrast, when they grow on a nonfermentable carbon source (i.e., glycerol, ethanol, lactate or acetate), oxidative phosphorylation is the main energy-yielding pathway. The glucose-dependent down-regulation of gluconeogenic
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and oxidative metabolism-related proteins and the enhanced transcription of glycolytic enzymes and glucose transporters is termed carbon catabolite repression pathway [124, 125]. When
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glucose is externally added to an aerobic yeast culture, respiration is immediately arrested and
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glycolysis accelerated at the same time that ethanol is produced. This short-term and reversible transition is termed Crabtree effect. From a metabolic point of view, there are similarities
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between the carbon catabolite repression in yeast and the “Warburg phenotype” of tumor cells; moreover, the Crabtree effect was also observed in tumor cells accounting for cell metabolism
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adaptation to the heterogeneous tumor microenviroment (for extensive review on these topics see
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[126, 127]). Importantly, like oncogenes and tumor suppressors impinge on cellular metabolism
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to support growth and proliferation of cancer cells, similarly yeast homologues of cancer-related genes, such as RAS and SCH9, the yeast homologue of Akt, influence yeast cell metabolism and
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proliferation [123].
Mitochondrial RTG pathway activation of glutamate biosynthesis via the glyoxylate cycle has been suggested to resemble activation of glutaminolysis in tumor cells (see par. 2), yet studies on the role of glutaminolysis during the oxidative and fermentative growth need to be performed in yeast in order to establish a comparison with tumor cell metabolism. In addition, although the accumulation of oncometabolites in mutant SDH- and FH-associated tumors has been proposed to regulate degradation of the HIF-1α transcription factor [36], leading to activation of hypoxia response pathway and increased expression of glycolytic enzymes in cancer cells, yeast cells lack a homologue of HIF-1α. Interestingly enough, in a yeast model it
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has been shown that mitochondrial retrograde response is necessary for maintenance of normal flux through the TCA and glyoxylate cycles in wild type strains and for the oncometabolite fumarate and succinate accumulation in TCA cycle mutants lacking either FH or SDH,
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pinpointing the retrograde pathway as possible target for cancer therapy (see par. 3.1.2) [128]. Thus, notwithstanding these and other limitations, including the fact to be a unicellular
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organism, yeast remains a suitable model organism to study the relations among extracellular
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environment, cellular metabolism and mitochondrial function in tumorigenesis [129-131]. One of the hallmark of cancer cells is their resistance to death, thus the action of many
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anti-cancer drugs occurs through induction of apoptosis. However, the surrounding microenvironment strongly influences the overall cell sensitivity to anti-cancer drugs,
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determining in many cases survival and chemoresistance. Studies on PCD induced by acetic acid
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(AA-PCD) in yeast has provided a comprehensive picture of events, components and signaling
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pathways involved in death execution or inhibition [50, 132] (Fig. 2). Under fermenting growth conditions, i.e. in the presence of glucose as a sole carbon source, acetic acid triggers an early
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burst in ROS production followed by cytochrome c release, mitochondrial dysfunction, DNA fragmentation and ultimately cell death. As mentioned above, in the presence of glucose yeast cells mostly rely on a fermentative metabolism even in the presence of oxygen, with repressed mitochondrial activity due to the carbon catabolite repression. Interestingly, a specific activation of the mitochondrial RTG signaling causes a complete inhibition of AA-PCD under derepressing growth conditions, when raffinose is used as the sole carbon source and mitochondrial oxidative metabolism is stimulated [50] (Fig. 2). These data indicate how the change in cell environment (glucose to raffinose), which causes an increase in respiratory metabolism, can affect mitochondrial stress response pathways leading to cell adaptation. These experimental
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conditions clearly resemble those described above in mammalian cancer cells, in which mitochondrial OXPHOS has been shown to be strictly required for optimal proliferation in low glucose [120]. Thus, yeast is an attractive model to investigate the relations between cell
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microenvironment, mitochondrial dysfunction and adaptation in both physiological and pathological conditions [48, 131].
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Finally, another advantage offered by the yeast model is the possibility of heterologous
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expression of human genes either in the absence or in the presence of obvious yeast homologues [133, 134], including the cancer-susceptibility genes B-cell lymphoma-2 (Bcl2), p53 and
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BRCA1/2 (for refs see [129, 130]). With the aim to investigate the structure/function relationships of human proteins and their role in tumorigenesis, results obtained in the yeast
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model can provide a useful experimental platform also to screen new lead compounds as for their
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anticancer therapeutic efficiency. A successful example of complementation between human and
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yeast has recently revealed a new function of BRCA2 protein as modulator of anoikis sensitivity through an evolutionarily-conserved molecular mechanism involving regulation of ROS
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production and/or detoxification by BRCA2 during PCD processes [135] (Fig. 2). As reported in par. 3.1, in human cancer cells it has been observed that mtDNA depletion negatively affects BRCA2 protein level conferring increased sensitivity to the PARP inhibitor rucaparib [60]. Future studies with the yeast model in this direction may help to gain insight into the role of BRCA2 in cancer cell growth and tumor progression as well as the effects of mitochondrial dysfunctions on drug sensitivity and chemoresistance in BRCA2-related tumors. Thus, although with the aforementioned limitations, yeast could be used to develop a preliminary platform to screen for lead compounds for the designing of new anticancer drugs to be eventually tested in more physiological mammalian cell models.
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4. Conclusions Increasing evidences point to mitochondrial dysfunctions as important determinants in conferring
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cancer cell resistance to certain chemotherapeutic drugs. However, the presence of mitochondrial dysfunctions may also represent the "Achilles’ heel" for cancer cells, as it may provide a
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molecular and biochemical rationale for developing therapeutic strategies to selectively kill
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cancer cells using compounds that target the altered metabolism or signaling pathways. Inhibiting glycolysis may be a possible intervention approach, however, since glycolysis and its
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regulatory machinery is evolutionary conserved and shared with normal cells, glycolysis inhibitors are likely to affect normal cells at various degree. PARP inhibitors may provide a new
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therapeutic opportunity in tumors with mitochondrial dysfunction, e.g. those characterized by a decrease in BRCA2 protein levels. On this basis, it would be important to determine whether
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mitochondrial genetic alterations are present in each particular cancer type and whether they are pathogenic, i.e. they impair mitochondrial oxidative metabolism. This screening might be useful
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not only to predict efficacy of current therapeutic strategies but also to design new personalized anti-cancer therapies.
ACKNOWLEDGEMENTS
This work has been funded by grants from project FIRB-Merit RBNE08YFN3_005 to L.M., FIRB-MERIT RBNE08HWLZ and the Italian Ministry of Economy and Finance to the CNR for the Project “FaReBio di Qualità” to S.G.
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Figure legends Figure 1. Schematic representation of mitochondrial structure and oxidative metabolism.
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Mitochondria are cellular organelles composed of a double membrane, the outer and the inner mitochondrial membrane (OMM and IMM, respectively), which delimits the matrix. Glucose is
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metabolized in the cytosol to pyruvate which is transported into mitochondria for further
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catabolism through the TCA cycle coupled with respiration through the electron transport chain. The TCA cycle takes place in the matrix, generating α-ketoglutarate and electron donors (NADH
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and FADH2). The electron transport chain consists of four complexes (I-IV) and mediates transfer of electrons from NADH and FADH2 to the electron acceptor O2. In turn, electron
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transfer through the four respiratory complexes is coupled with proton extrusion out of the matrix toward the intermembrane space (IMS), producing an electrochemical gradient across the
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inner membrane. Protons ultimately return to the matrix through the ATP synthase or complex V, using the free energy produced by the electron respiratory chain to synthesize ATP. Essential
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protein components of the mitochondrial respiratory chain are encoded by the mitochondrial DNA (mtDNA), a circular double stranded DNA molecule that, besides containing the genetic information for 13 proteins [NADH dehydrogenase subunits 1, 2, 3, 4, 4L, 5, 6 (ND1, 2, 3, 4, 4L, 5, 6) belonging to Complex I, cytochrome b of Complex III, ATP synthase subunit 6 and 8 (ATPase 6, 8) belonging to Complex V, cytochrome c oxidase I-III (COI-III) belonging to Complex IV], encodes also for two ribosomal RNAs (rRNAs 12S and 16S) and 22 tRNAs (not shown) required for mitochondrial protein synthesis. The displacement loop (D-loop), or noncoding control region, contains sequences for the initiation of both mtDNA replication and transcription. Several molecules of mtDNA are present in each mitochondrion. ACO, aconitase; c, cytochrome c; CS, citrate synthase; FH, fumarate hydratase, IDH2, isocitrate dehydrogenase 2; 23
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KGD, α-ketoglutarate dehydrogenase; MDH, malate dehydrogenase, PDH, pyruvate dehydrogenase, PDK-1, pyruvate dehydrogenase kinase-1, Q, coenzyme Q, SDH, succinate dehydrogenase, SCS, succinyl-CoA synthetase; I-V, mitochondrial respiratory complex I, II, III,
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IV, V.
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Figure 2. Mechanisms of yeast cell death and adaptation in response to acetic acid stress. At
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low extracellular pH undissociated acetic acid enters the cells where it dissociates into acetate and protons causing cytosol acidification. In the presence of glucose as a sole carbon source,
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AA-PCD occurs with an early burst of reactive oxygen species (ROS) followed by release of cytochrome c (c), mitochondrial dysfunction, as revealed by decreased respiratory control index
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(RCI) and cytochrome c oxidase (COX) activity, DNA fragmentation, caspase-like activation
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and complete loss of cell viability. In raffinose grown-cells, following acetic acid treatment a
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lower ROS level is detected, RTG signaling is activated with translocation of Rtg1/3p into the nucleus leading to up-regulation of retrograde-target genes, causing cell resistance to AA-PCD
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(see text for details). RTG signaling is inhibited by TOR. Ras can either impact RTG-dependent lifespan extension or cause mitochondrial dysfunction through cAMP and PKA hyperactivation leading to cell death. Heterologous expression of human BRCA2 gene in yeast potentiates AAPCD through an evolutionarily-conserved mechanism involving regulation of ROS production and/or detoxification. Red, blue and green arrows indicate pro-death, survival and longevity pathways, respectively.
Figure 3. A scheme of mitochondrial stress-mediated pathways involved in cancer progression and chemoresistance. In mammalian cells, mitochondrial dysfunction resulting
24
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from mtDNA depletion causes impaired mitochondrial respiration and mitochondrial membrane potential depolarization, promoting activation of the retrograde signaling pathway. At variance with yeast cells, the regulation of mitochondria-to-nucleus signaling has not been completely
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characterized. Loss of mitochondrial transmembrane potential promotes increased cytosolic calcium concentration ([Ca2+]c) and activation of calcineurin, thereby eliciting a calcineurin-
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mediated retrograde signaling pathway [136]. The transcription of several target nuclear genes (>
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120 genes identified to date) is up-regulated in response to this calcium/calcineurin-dependent signaling and possibly other mechanisms not yet identified (indicated by a question mark)
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through nuclear translocation of a number of transcription factors (NRF2, NF-κB, CREB and NFAT), and upregulation of the transcriptional coactivator hnRNPA2 [136]. In turn, the genes
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induced by activation of the mitochondrial retrograde pathway can modulate a number of cellular
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functions including: i) apoptosis and multidrug resistance, thereby promoting resistance to
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chemotherapeutic drugs; ii) invasion and epithelial-to-mesenchymal transition, thus sustaining metastatic progression of cancer cells; iii) regulation of protein expression at the post-
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transcriptional level, through upregulation of components of the ubiquitin-proteasome proteolytic pathway (such as Skp-2) and miRNAs (such as miR-1245), thereby modulating the levels of oncosuppressor proteins, like BRCA2 (see the text for details); iv) energy metabolism, through upregulation of glucose transporters and glycolytic enzymes, thus supporting the metabolic shift typical of cancer cells. Overall these modifications can lead to metabolic reprogramming of cancer cells, malignant progression and chemoresistance.
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i cr us
Table 1. Intracellular targets of the oncometabolites succinate, fumarate, and alpha-ketoglutarate Mutated gene Oncometabolite Metabolic changes Molecular targets Cancer type Inhibition of α-KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs; succination of KEAP1 and Aco2
Hereditary skin and uterine leiomyomas and renal cell cancer; renal cysts; breast, bladder and Leydig cell tumors
[36, 38, 40, 137-145]
Decreased flux of metabolites through the Krebs cycle Reduced activity of electron transport chain Impaired oxidative metabolism Increased lactate production
Inhibition of α-KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs
Hereditary paraganglioma, pituitary adenoma and pheochromocytoma; gastrointestinal stromal tumors, renal tumors, thyroid tumors, neuroblastomas, testicular seminomas; childhood T cell acute leukemia
[37, 39, 40, 146-148]
Reduced NADPH levels Reduced glutamate levels Reduced NADPH-dependent reductive carboxylation
Inhibition of α-KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs; inhibition of collagen prolyl and lysyl
Chondrosarcoma, cholangiocarcinoma, glioma, secondary glioblastoma, acute myeloid leukemia, a subset of breast cancers, angioimmunoblastic T-cell lymphoma
[41, 42, 149-155]
Decreased flux of metabolites through the Krebs cycle Impaired oxidative metabolism Increased glutamine uptake and lactate production Accumulation of fumarate-derived argininosuccinate Acquired auxotrophy for arginine Bilirubin excretion
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Fumarate
FH
Succinate
Ac
ce
pt
SDH (loss-of-function)
ed
M
(loss-of-function)
IDH1/2 (gain-of-function)
D-2-KG
26
Ref.
Page 26 of 52
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hydroxylases leading to impaired collagen maturation
Ac
ce
pt
ed
M
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α-KG, α-ketoglutarate; Aco2, aconitase 2; FH, fumarate hydratase; HIFs, hypoxia-inducible factors; IDH1/2, isocitrate dehydrogenase 1 and 2; KDMs, Jumonji C domain-containing histone lysine demethylases; KEAP1, Kelch-like ECH-associated protein 1; PHDs, HIF prolyl hydroxylases; SDH, succinate dehydrogenase; TET, ten eleven translocation family of 5-methylcytosine hydroxylases.
27
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Figure 2
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Figure 3
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Table 1
Inhibition of -KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs; succination of KEAP1 and Aco2
Hereditary skin and uterine leiomyomas and renal cell cancer; renal cysts; breast, bladder and Leydig cell tumors
[36, 38, 40, 136-144]
Succinate
Decreased flux of metabolites through the Krebs cycle Reduced activity of electron transport chain Impaired oxidative metabolism Increased lactate production
Inhibition of -KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs
Hereditary paraganglioma, pituitary adenoma and pheochromocytoma; gastrointestinal stromal tumors, renal tumors, thyroid tumors, neuroblastomas, testicular seminomas; childhood T cell acute leukemia
[37, 39, 40, 145-147]
Inhibition of -KGdependent dioxygenases (KDMs, TET) modulating DNA and histone methylation; activation of HIFs through allosteric inhibition of PHDs; inhibition of collagen prolyl and lysyl
Chondrosarcoma, cholangiocarcinoma, glioma, secondary glioblastoma, acute myeloid leukemia, a subset of breast cancers, angioimmunoblastic T-cell lymphoma
[41, 42, 148-154]
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Decreased flux of metabolites through the Krebs cycle Impaired oxidative metabolism Increased glutamine uptake and lactate production Accumulation of fumarate-derived argininosuccinate Acquired auxotrophy for arginine Bilirubin excretion
(loss-of-function)
IDH1/2 (gain-of-function)
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SDH (loss-of-function)
Ref.
Fumarate
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Table 1. Intracellular targets of the oncometabolites succinate, fumarate, and alpha-ketoglutarate Mutated gene Oncometabolite Metabolic changes Molecular targets Cancer type
D-2-KG
Reduced NADPH levels Reduced glutamate levels Reduced NADPH-dependent reductive carboxylation
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hydroxylases leading to impaired collagen maturation
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-KG, -ketoglutarate; Aco2, aconitase 2; FH, fumarate hydratase; HIFs, hypoxia-inducible factors; IDH1/2, isocitrate dehydrogenase 1 and 2; KDMs, Jumonji C domain-containing histone lysine demethylases; KEAP1, Kelch-like ECH-associated protein 1; PHDs, HIF prolyl hydroxylases; SDH, succinate dehydrogenase; TET, ten eleven translocation family of 5-methylcytosine hydroxylases.
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*Graphical Abstract (for review)
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