Targeting cell cycle regulation in cancer therapy

Pharmacology & Therapeutics 138 (2013) 255–271

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Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate Editor: B. Teicher

Targeting cell cycle regulation in cancer therapy Santiago Diaz-Moralli, Míriam Tarrado-Castellarnau, Anibal Miranda, Marta Cascante ⁎ Faculty of Biology, Department of Biochemistry and Molecular Biology, Universitat de Barcelona, Barcelona, Spain

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Keywords: Cell cycle Cancer CDK–cyclin complexes Metabolism ROS

a b s t r a c t Cell proliferation is an essential mechanism for growth, development and regeneration of eukaryotic organisms; however, it is also the cause of one of the most devastating diseases of our era: cancer. Given the relevance of the processes in which cell proliferation is involved, its regulation is of paramount importance for multicellular organisms. Cell division is orchestrated by a complex network of interactions between proteins, metabolism and microenvironment including several signaling pathways and mechanisms of control aiming to enable cell proliferation only in response to specific stimuli and under adequate conditions. Three main players have been identified in the coordinated variation of the many molecules that play a role in cell cycle: i) The cell cycle protein machinery including cyclin-dependent kinases (CDK)–cyclin complexes and related kinases, ii) The metabolic enzymes and related metabolites and iii) The reactive-oxygen species (ROS) and cellular redox status. The role of these key players and the interaction between oscillatory and non-oscillatory species have proved essential for driving the cell cycle. Moreover, cancer development has been associated to defects in all of them. Here, we provide an overview on the role of CDK–cyclin complexes, metabolic adaptations and oxidative stress in regulating progression through each cell cycle phase and transitions between them. Thus, new approaches for the design of innovative cancer therapies targeting crosstalk between cell cycle simultaneous events are proposed. © 2013 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . 2. Canonical cell cycle regulation . . . 3. Metabolic control of cell cycle . . . 4. Redox status in cell cycle regulation 5. Cell cycle defects and cancer . . . . 6. Conclusions . . . . . . . . . . . . Conflict of interest statement . . . . . . Acknowledgments . . . . . . . . . . . References . . . . . . . . . . . . . . .

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1. Introduction The cell cycle is a set of organized and monitored events responsible of proper cell division into two daughter cells. It is a high energy demanding process that requires an encompassed and

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ordered series of events to guarantee the correct duplication and segregation of the genome. This process involves four sequential phases that go from quiescence (G0 phase) to proliferation (G1, S, G2, and M phases) and back to quiescence (Norbury & Nurse, 1992).

Abbreviations: APC/C, anaphase-promoting complex/cyclosome; ATM, ataxia-telangiectasia-mutated; β-TrCP, protein-β-transduction repeat-containing protein; CAK, CDK activating kinase; CDK, cyclin dependent kinases; CKI, cyclin-dependent kinase inhibitors; CTD, carboxy-terminal domain; DHFR, dihydrofolate reductase; ETC, electron transfer chain; F2,6P2, fructose-2,6-bisphosphate; F6P, fructose-6-phosphate; FAS, fatty acid synthase; FOXO, forkhead box O; G6PD, glucose-6-phosphate dehydrogenase; GLS1, glutaminase 1; GLUT, glucose transporter; HK, hexokinase; LDH, lactate dehydrogenase; MAPK, mitogen-activated protein kinases; NIMA, never-in-mitosis Aspergillus; NO, nitric oxide; NOX, NADPH oxidase; PFK/FB3, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3; PFK1, 6-phosphofructo-1-kinase; PK, pyruvate kinase; PLK, polo-like kinase; PP2A, protein phosphatase 2A; PPP, pentose phosphate pathway; PUMA, p53 upregulated modulator of apoptosis; R5P, ribose-5-phosphate; RB, retinoblastoma; ROS, reactive oxygen species; SAC, spindle assembly checkpoint; SCF, Skp1/cullin/F-box; TKT, transketolase; TKTL1, transketolase-like protein 1. ⁎ Corresponding author at: Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Univ. of Barcelona, Av Diagonal 645 Annex Bldg., floor -2, 08028 Barcelona, Spain. Tel.: +34 93 4021593; fax: +34 4021559. E-mail address: [email protected] (M. Cascante). 0163-7258/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pharmthera.2013.01.011

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Cells can enter the first gap phase (G1) from the quiescent state G0 or, if they are proliferating, after completing cytokinesis. The progression through G1 is mitogen dependent up to the restriction point (R) (Pardee, 1974), after which cells can proliferate independently of mitogenic stimuli and are committed to enter the synthesis (S) phase, when DNA replication occurs. Progression through the mammalian cell cycle requires the accurate orchestration of a sequence of events. Among the countless elements taking part in this process, the sequential activation of heterodimeric CDK–cyclin complexes (cyclins and their counterpart cyclin-dependent kinases (CDKs)) has been described as the key regulatory events. The kinase activity of CDKs is tightly regulated by the binding to cyclins, the activating subunits which are expressed in an oscillatory way, the binding to negative regulators (CDK inhibitors, CKI) and phosphorylation/dephosphorylation events (Manchado et al., 2012). Progression through each cell cycle phase and transition from one phase to the next are monitored by sensor mechanisms, called checkpoints, which maintain the accurate sequence of events (Hartwell & Weinert, 1989). Cell cycle checkpoints prevent the transition to the next phase until the previous one is fully completed. The ultimate goal is to ensure the detection and repair of genetic damage, prevent the uncontrolled cell division and guarantee that the two daughter cells inherit a complete and faithful copy of the genome. Checkpoints are activated when problems are detected inducing cell cycle arrest until the problem is solved, if possible, or else, if the repair is not successful, driving cell to senescence or apoptosis (Viallard et al., 2001). Once the chromosomes are correctly duplicated, cells can enter G2, another gap phase to prepare for the mitosis (M), where the cell generates two genomically stable daughter cells. Abrogation of cell cycle checkpoints can result in many diseases including cancer. Since cell cycle entry is an energetically demanding process that requires high metabolic activity to fuel rapid increase of the cell mass, cells have to assess whether there is an adequate metabolic status to initiate and complete cell cycle properly before division (Buchakjian & Kornbluth, 2010). In fact, as early as in 1974 it was established that the availability of nutrients is a key factor for cell proliferation and it was described that glucose availability is a metabolic checkpoint in cell cycle linked to the progression of the cell from G1- to S-phase in which most biosynthetic reactions occur (Pardee, 1974). However, even though the knowledge on the cell cycle key players and regulatory mechanisms has advanced dramatically in the last 40 years, the links between the cell cycle machinery and the mechanisms that ensure the supply of the necessary intermediates for de novo biosynthesis of the macromolecules to accomplish the task have remained obscure (Morrish et al., 2009; Moncada et al., 2012).It has been only recently, with the finding that the activities of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase, isoform 3 (PFK/FB3) and glutaminase 1 (GLS1) are regulated by an ubiquitin ligase (Anaphase-promoting complex/cyclosome (APC/C)-CDH1), that has become clear that the process of ubiquitylation not only orchestrate the periodic transcriptional regulation of cyclins and other cell cycle regulators, but also is crucial in the metabolic regulation of the cell cycle (Almeida et al., 2010; Bolanos et al., 2010; Colombo et al., 2010; Moncada et al., 2012). Interestingly, it has been also reported that the induction of mitochondria hyperfusion can trigger the expression of cyclin E, the cyclin responsible for G1-toS-phase progression. Given the significant energy required for synthesizing all the macromolecules necessary for cell division, there is a need for further investigation of the connection between mitochondrial energetic status and cell cycle progression to fully understand the coordination between the consumer and supplier of ATP (Finkel & Hwang, 2009; Mitra et al., 2009).In addition, the widely recognized role of ROS as intracellular messengers that are able to modulate cell signaling pathways, and its direct relation with mitochondrial activity highlight the possible existence of new regulatory mechanisms of the cell cycle (Sauer et al., 2001; Jones, 2008; Burhans & Heintz, 2009). This hypothesis is also backed by the reported oscillatory variations of ROS during cell cycle (Tu et al., 2005; Burhans & Heintz, 2009)

and the fact that its physiological effects are mediated not only by the reactive species themselves, but also by the alteration of the global redox status of the cell (D'Autreaux & Toledano, 2007; Veal et al., 2007; Jones, 2008). Thus, it is of great interest to elucidate whether metabolic enzymes and cellular redox potential have a cyclic regulation, and what role they play in driving the cell cycle to ensure the efficient use of energy and substrates. 2. Canonical cell cycle regulation Cyclin-dependent kinases (CDK) are a family of mammalian heterodimeric serine/threonine protein kinases composed of two subunits, the catalytic one known as CDK and the regulatory one known as cyclin (Shapiro, 2006; Malumbres & Barbacid, 2007). This family of proteins is divided into two groups according to their role in eukaryotic cell cycle progression or in transcriptional regulation (Meyerson et al., 1992; Sausville, 2002). CDK activation is always dependent on the binding to specific cyclins and is regulated by the union to inhibitors and by positive or negative phosphorylation events (Sherr, 1994; Morgan, 1997; Nurse, 2000). However, since CDK subunits are constitutively expressed, and each of them only binds to its specific cyclins, the activation of the CDK–cyclin complexes governing the transitions between cell cycle phases depends on the availability of the regulatory subunits. Thereby, cell machinery regulates cyclin oscillatory changes by controlling their synthesis and degradation at specific times, leading to the orchestrated progression of the cell cycle (Nurse, 2000). In quiescent cells, cyclins are subjected to ubiquitylation and proteasome degradation (Glotzer et al., 1991). However, mitogenic stimuli inactivate the E3 ubiquitin ligases that target cyclins for degradation, leading to their accumulation, activating cell division. There are two E3 ubiquitin ligase complexes, APC/C (anaphase-promoting complex/cyclosome) and SCF (Skp1/cullin/F-box), that distinctively recognize different cyclins and other cell-cycle proteins and regulate their sequential degradation due to specific domains in their nucleotidic sequences allowing them to drive the cell cycle by peaking at different phases (Vodermaier, 2004). CDKs that modulate transcriptional regulation are also dependent on the interaction between the catalytic and the cyclin regulatory subunits. However, in contrast to the ones involved in cell cycle progression, these cyclins do not cycle. The main role of these CDK complexes is to promote RNA transcription activating RNA polymerase II by phosphorylating its carboxy-terminal domain (CTD) and favoring initiation and elongation of nascent RNA transcripts (Prelich, 2002; Palancade & Bensaude, 2003; Meinhart et al., 2005). In total, around twenty different CDKs (CDK1–20) have been identified in mammals. All of them have been reported to interact with specific cyclins (CDK1–14) or include cycling-binding domains in their structure (CDK15–20) (Malumbres et al., 2009). CDK1, 2, 4 and 6 have been proven to drive cell cycle events and CDK3 and CDK5 may also be involved in this process in certain cell lines (Ye et al., 2001; Ren & Rollins, 2004; Maestre et al., 2008; Zhang et al., 2008). Other CDKs play a significant role in transcriptional regulation with some implications in cell cycle control (CDK7–11) or alternative splicing (CDK12 and 13) (Chen et al., 2006, 2007). The role of the rest of CDKs requires further investigation in order to determine if they may be part of one of the abovementioned groups or make a new one (Malumbres & Barbacid, 2005; Malumbres et al., 2009). The accurately orchestrated sequence of events that lead to cell division is regulated by different checkpoints. The first of them is situated in the mid-to-late G1-phase and is considered the initial control and the entrance to the cycle of division. It is also known as the restriction point (R) due to the importance for cell proliferation that this sensitive period shows (Strauss et al., 1995; Sherr, 1996). CDK4 and CDK6 are the two interphase cyclin dependent kinases that control cell cycle entry and progression through G1-phase. They are activated by D-type cyclins (D1, D2 and D3) forming

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CDK4/6-cyclin D complexes. In response to mitogenic signaling through pathways like the RAS/RAF/MAPK cells synthesize D-type cyclins. These include characteristic amino acid sequences such as destruction (D) or KEN (lysine, glutamate, asparagine) boxes that are recognized by APC/C-CDH1 which ubiquitylates them and leads to its proteasome degradation (Glotzer et al., 1991; Arellano & Moreno, 1997; Fang et al., 1998; Pfleger & Kirschner, 2000; Burton & Solomon, 2001; Peters, 2002; Vodermaier, 2004; Peters, 2006; Barford, 2011). Therefore, in order to effectively activate cell cycle progression, mitogenic stimuli not only have to induce the synthesis of cyclins but also have to lead to the inactivation of the APC/C-CDH1 complex by CDH1 phosphorylation (Kramer et al., 2000; Peters, 2002). Once the cell reaches sufficient levels of D-type cyclins, these bind CDK4 and CDK6, becoming active complexes with capacity to phosphorylate and partially inactivate the members of the retinoblastoma (RB) protein family including pRB, p107 (RBL1) and p130 (RBL2) (Malumbres & Barbacid, 2001, 2009). RB members are transcriptional regulators that repress transcription of several genes involved in DNA replication by the binding and inactivation of transcription factors such as the E2F family, and the recruitment of repressor complexes such as histone deacetylases and chromosomal remodeling SWI/SNF complexes (Harbour & Dean, 2000; Malumbres & Barbacid, 2001). The protein complexes E2F are heterodimers composed of an E2F protein (E2F-1 to 6) and a protein from the DP family (DP-1 and DP-2) (Wu et al., 1995; La Thangue, 1996). Therefore, the phosphorylation of RB proteins by CDK4/6-cyclin D complexes leads to their partial inactivation and relieves the transcriptional repression mediated by the RB–E2F complex allowing both the transcription of E-type cyclins (E1 and E2), which bind and activate CDK2, and surpassing the restriction point (Ortega et al., 2002). Immediately, CDK2–cyclin E complexes completely inactivate RB proteins by hyperphosphorylation, allowing the E2F activation, that directs the transient transcription of genes required for entering the S-phase. Active CDK2–cyclin E also phosphorylates proteins involved in histone modification, DNA replication and repair, (Sherr & Roberts, 1999) centrosome duplication and maturation, and its own inhibitor p27Kip1 (Malumbres & Barbacid, 2005; Shapiro, 2006). CDK3 might also participate in inactivation of pRB, this kinase is highly related to CDK2 and CDK1 and interacts with A-type and E-type cyclins, being involved in G1 progression, transition to S-phase and DNA repair (Malumbres, 2011). CDK5 is activated by p35 and p39 although can also bind D-type and E-type cyclins, forming complexes whose activity is still unclear (Malumbres, 2011). Once in S-phase, in order to avoid the re-replication of DNA, the CDK2–cyclin E complexes are inactivated due to the degradation of cyclin E. This process is mediated by SCF-related complexes, which recognize cyclin E targeting it for degradation in the proteasome (Vodermaier, 2004; Zhang & Koepp, 2006). At the same time, the full inactivation of pRB permits the transcription of A-type (A1 and A2) and B-type (B1, B2 and B3) cyclins. Free CDK2 binds to cyclin A resulting in active CDK2–cyclin A complexes which phosphorylate E2F and DP, releasing the dimer from the DNA (Kitagawa et al., 1995) and consequently, downregulating E2F activity to allow S-phase transition to G2. At the end of the S phase, the mitotic CDK1 is associated with cyclin A and phosphorylates a wide range of proteins (Malumbres & Barbacid, 2005) including E2F, thus promoting the formation of RB–E2F complexes. During G2, A-type cyclins are degraded by ubiquitin-mediated proteolysis while B-type cyclins are actively synthesized and able to bind free CDK1. CDK1–cyclin B complexes are essential for initiating mitosis (M) and can phosphorylate a broad spectrum of proteins involved in regulatory and structural processes required for mitosis such as nuclear envelope breakdown, chromosomal condensation, fragmentation of the Golgi apparatus, formation of the spindle and attachment of chromosomes to it (Malumbres & Barbacid, 2005; Malumbres, 2011). Exiting mitosis requires the inactivation of CDK1–cyclin B, which is carried out by the ubiquitin-dependent proteolysis of B-type cyclins by the APC/C-CDC20 complex (Harper et al., 2002; Peters, 2002).

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The kinase activity of CDKs as mentioned above is regulated by the interaction with activating subunits (cyclins), the binding to negative regulators (CDK inhibitors, CKI) and phosphorylation/ dephosphorylation events. There are two families of CKI: INK4 proteins (p16INK4a, p15INK4b, p18INK4c and p19INK4d) that bind specifically to CDK4 and CDK6 but not to other CDKs preventing their union to D-type cyclins, and the Cip/Kip family (p21Cip1, p27Kip1 and p57Kip2) which forms heterotrimeric complexes with the cyclin D-, cyclin E- and cyclin A-dependent kinases complexes (Sherr & Roberts, 1999). CDK4/6-cyclin D complexes facilitate G1 progression not only by partial phosphorylation of pRB but also by sequestrating p21Cip1 and p27Kip1, releasing CDK2–cyclin E from these inhibitors and promoting CDK2 kinase activity (Sherr & Roberts, 1999; Blain, 2008). Moreover, the active CDK2–cyclin E complexes can phosphorylate their own inhibitor p27Kip1 triggering its degradation by the SCF ubiquitin ligase and their consequential self-activation (Sheaff et al., 1997; Massague, 2004). It has been reported that p27Kip1 binding to CDK4/6-cyclin D complexes is essential for their formation, and that this association does not affect their kinase activity (Sherr & Roberts, 1999) except in quiescent cells (G0), where p27Kip1 union to CDK4/6-cyclin D is in an inhibitory mode (Blain, 2008). Cell cycle arrest is commanded by anti-mitogenic signals, such as TGF-β, resulting in D-type cyclin synthesis stop, INK4 binding to CDK4/6 and, as a result, forcing the redistribution of p27Kip1 to CDK2–cyclin E, precluding pRB phosphorylation thus repressing transcription (Blain, 2008). DNA damage and metabolic stress activate p53 which induces p21Cip1 transcription causing the inhibition of both CDK2 and CDK4 activities, resulting in G1 arrest in mammalian cells (He et al., 2005; Cazzalini et al., 2010; Hoeferlin et al., 2011; Lee et al., 2012). Phosphorylation and dephosphorylation events also control the CDK activity as binding to cyclins is not enough to activate CDK–cyclin complexes. The resulting formation exposes the threonine residue in the T-loop of the CDK subunit to the CDK7-cyclin H-Mat1 complex (CDK Activating Kinase, CAK) allowing its phosphorylation (Morgan, 1997; Viallard et al., 2001). The role of CAK is to activate CDK complexes and it does not require phosphorylation to be active. CAK is inhibited by phosphorylation of cyclin H by CDK8-cyclin C (Malumbres & Barbacid, 2005). In addition, CAK is involved in promoter clearance and progression of transcription as it is part of the general transcription factor TFIIH (Malumbres, 2011). Once a CDK–cyclin complex is formed, it can be activated by CAK or inactivated by the WEE1 and MYT1 kinases that phosphorylate adjacent threonine and tyrosine residues (Thr14/Tyr15 in CDK1) in the CDK subunit. The inhibitory phosphorylations can be reversed by CDC25 phosphatases (CDC25A, CDC25B and CDC25C) (Malumbres & Barbacid, 2005). In order to have a complete view of the involvement of kinases within the cell cycle, it is necessary to portray the other kinase families with important roles in cell cycle apart from CDKs such as Aurora kinases, Polo-like kinases, SAC kinases, NIMA-related kinases and Checkpoint kinases CHK1 and CHK2. Aurora kinases (Auroras A, B and C) contribute to mitosis entry by assisting the establishment of the proper chromosome structures, building of the bipolar mitotic spindle, centrosome separation and microtubule dynamics, ensuring an accurate cell division. Polo-like kinases (PLK1–5) are less studied. PLK1 plays a significant role in entering mitosis, with major functions in centrosome maturation and cytokinesis while PLK4 is essential in centriole duplication. PLK1 is the most studied member of the family and it is only found (along with PLK4) in proliferating cells whereas PLK2, 3 and 5 are also expressed in non-dividing cells. Noteworthy, Aurora A and PLK1 expression is also regulated by the APC/C-CDH1 complex (Harper et al., 2002; Castro et al., 2005). The Spindle Assembly Checkpoint (SAC) is a signaling pathway that inhibits anaphase start until chromosomes are correctly attached to the mitotic spindle, ensuring accurate segregation of sister chromatids. The SAC kinases BUB1 and BUBR1 participate in this proper chromosome-spindle

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attachment, while the SAC kinase MPS1 is involved in the regulation of APC/C, the centrosome duplication and cytokinesis (Palframan et al., 2006). NIMA (Never-in-mitosis Aspergillus)-related kinases (NEK) is a family of eleven serine/threonine kinases (NEK1–11) generally implicated in microtubule organization (Malumbres & Barbacid, 2007; Malumbres, 2011). Checkpoint kinases CHK1 and CHK2 are key signal transducers of the genome integrity checkpoints that are activated by genotoxic insults. Once active, CHK1 and CHK2 phosphorylate downstream effectors which further propagate the checkpoint signaling leading to one response mechanism such as switching to damage-induced transcription, DNA repair, cell cycle arrest, apoptosis or chromatin remodeling (Bartek & Lukas, 2003; Kastan & Bartek, 2004). As mentioned above, proper transition between cell cycle phases require the strictly regulated degradation of cell cycle progression proteins. The activity of the ubiquitin ligase complexes controlling sequential degradation of cell-cycle proteins, APC/C and SCF, is precisely regulated ensuring the coordinate degradation of the key cell cycle regulators based on signaling stimuli (Harper et al., 2002). SCF is activated from late G1 to early M-phase by SKP2, β-TrCP and Fbw7, whereas APC/C is functional from mid M to G1 by the action of CDC20 (this association requires the phosphorylation of APC/C by CDKs) and CDH1 (its phosphorylation prevents its union with APC/ C) (Vodermaier, 2004; Nakayama & Nakayama, 2006; Tudzarova et al., 2011). Both complexes play an essential role in cell cycle progression modulating its specificity through the binding to different activators as it has been described. SCF degradation of its substrates (p21Cip1, p27Kip1, p57Kip2, cyclin E, WEE1 and EMI1) allows overcoming the blocking activities of cell cycle progression (Harper et al., 2002; Vodermaier, 2004; Nakayama & Nakayama, 2006). However, to complete the cycle of division, exit mitosis and return to the initial situation, the activation of APC/C complexes is required. On the first hand, APC/C leads to the degradation of the mitotic cyclins enabling the beginning of telophase (Nakayama & Nakayama, 2006). And on the other hand, this complex also degrades most cell cycle progression activators (PLK1, E2-C, SKP2, CDC25A, CDK1, Tome-1, Hsl1, Aurora kinases A and B, TPX2, FOXM1, CDC6 and germinin), proteins related to chromosome segregation (NEK2, cyclin A, securin, cyclin B) and eventually its own activators CDC20 and CDH1 for preparing the next cycle (Harper et al., 2002; Vodermaier, 2004; Palframan et al., 2006; Peters, 2006; Malumbres & Barbacid, 2009). It is noteworthy that the classic consideration that each cell cycle phase is driven by specific CDKs has been challenged by some genetic studies in mice that reveal that interphase CDKs (CDK2, CDK4 and CDK6) are not essential for the mammalian cell cycle of most cell types (Barbacid et al., 2005; Malumbres & Barbacid, 2005, 2009). Mice lacking CDK4, CDK6 or CDK2 are viable, suggesting a possible compensatory role between cyclin dependent kinases (Barriere et al., 2007). In fact, the knockout of CDK loci in the mouse germline showed that interphase CDKs are only required for the proliferation of specific cell types. For instance, CDK4 is essential for postnatal proliferation of specialized endocrine cell types such as pancreatic β-cells and pituitary lactotrophs (Barriere et al., 2007). It has been also reported defects in the erythroid lineage in CDK6-deficient and double mutant lacking CDK4 and CDK6 mice (Malumbres et al., 2004). CDK2 is necessary for completion of prophase I during meiotic cell division in both male and female germ cells but is not required for proliferation in mitotic cells (Berthet et al., 2003; Ortega et al., 2003) and cell cycle regulation mediated by p21Cip1 and p27Kip1 (Martin et al., 2005). On the other hand, CDK1 mutant mice embryos are not able to develop beyond the two-cell stage, indicating that the mitotic kinase CDK1 is essential for cell division in the embryo and that interphase CDKs cannot compensate for its absence (Malumbres & Barbacid, 2009). In addition, CDK1 is sufficient to drive the mammalian cell cycle in all cell types, undergoing organogenesis and developing to midgestation (Santamaria et al., 2007). All these data show the complexity of the CDK-dependent regulation of the cell cycle and the

variability that exists between different cell lines depending on its origin. Thus, when cell proliferation is not an option but an obligation, like in embryonic development, interphase CDKs are less important, whereas in situations where proliferation has to be only a response to specific mitogenic stimuli these CDKs play their canonical role governing the entry in the cycle and the progression through G1-phase. 3. Metabolic control of cell cycle As it has been mentioned above, cell division is a finely regulated mechanism involving several signaling pathways controlling expression, degradation, activation and inhibition of different cyclins and CDKs. This has led to propound the balance of these proteins as the key factor in the government of the normal orderly progression through the cell cycle (Meyerson et al., 1992; Sherr, 1994; Sausville, 2002). However, given that from a metabolic point of view cell cycle progression is a process demanding high doses of energy and substrates that support doubling of cell mass (Jones et al., 2005), the availability and ordered metabolism of exogenous nutrients may also play a main role in the regulation of the cycle in order to guarantee the successful division of the cells. In this regard, a plethora of metabolic processes that are essential for the activation of proliferation have been described in the last years. These processes are discussed below. As described in the previous section, initiation of the cell cycle involves growth factor mitogenic stimuli that lead to an eventual decrease in the E3 ubiquitin ligase APC/C-CDH1 activity. Since APC/C-CDH1 induces ubiquitylation and degradation of proteins containing D or KEN boxes by silencing the CDH1 activator subunit, proteins containing these sequences are accumulated. Among these proteins worth mentioning there is cyclin D1, a component of D-type cyclins which play a main role in progression through G1-phase, and metabolic enzymes such as 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFK/FB3) and glutaminase 1 (GLS1), all of which include KEN boxes in their sequences (Herrero-Mendez et al., 2009; Colombo et al., 2010). Thus, through APC/C-CDH1 inactivation cell cycle regulators promote the accumulation of D-type cyclins, PFK/FB3 and GLS1 in late G1 (Pesin & Orr-Weaver, 2008; Herrero-Mendez et al., 2009; Bolanos et al., 2010; Colombo et al., 2010). Interestingly, apart from the direct effect played by APC/C-CDH1 complex over the metabolic enzymes PFK/FB3 and GLS1, D-type cyclins have also been described as critical targets in metabolism-cell cycle crosstalk (Sakamaki et al., 2006; Bienvenu et al., 2010; Buchakjian & Kornbluth, 2010; Hanse et al., 2012). PFK/FB3 is one of the four different isoforms of the PFK/FB protein. All of them are bifunctional enzymes that can both catalyze the phosphorylation of fructose-6-phosphate (F6P) to fructose-2,6-bisphosphate (F2,6P2) and the reverse reaction of dephosphorylation of F2,6P2 to F6P. However, PFK/FB3 is also known as PFK-2 since this is the isoform with the highest kinase-to-phosphatase ratio (710:1) (Sakakibara et al., 1997; Okar et al., 2001; Yalcin et al., 2009). Given that F2,6P2 is a powerful allosteric activator of one of the main regulators of the glycolytic pathway (6-phosphafructo-1-kinase, PFK1) the accumulation of PFK/FB3 leads to the activation of glycolysis and an increase in lactate production. The other metabolic enzyme accumulated in response to CDH1 downregulation is GLS1, the first enzyme in the glutaminolytic pathway that also contributes to the increase in lactate production by stimulating glutamine degradation. This accumulation of both PFKFB3 and GLS1 and the increase in lactate production have been shown essential for cell cycle progression from G1 to S (Almeida et al., 2010; Colombo et al., 2011; Tudzarova et al., 2011). Moreover cyclin D1 is able to downregulate the expression of lipogenic enzymes such as acetylCoA carboxylase or fatty acid synthase, preventing pyruvate consumption in lipogenesis and contributing to lactate formation (Sakamaki et al., 2006; Buchakjian & Kornbluth, 2010; Hanse et al., 2012).

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Cell cycle progression beyond the restriction point involves further CDK–cyclin regulation and metabolic adaptations that allow the cell to fulfill mitotic requirements. Among these metabolic adaptations, the association between stimulation of cell growth and activation of the pentose phosphate pathway (PPP) is widely recognized (Sochor et al., 1986; Kletzien et al., 1994; Tian et al., 1998; Li et al., 2009; Jiang et al., 2011). Moreover it has been also reported that the well known tumor suppressor p53, which invokes anti-proliferative processes like cell cycle arrest or apoptosis (Vogelstein et al., 2000; Vousden & Prives, 2009), is also a direct inhibitor of the PPP (Jiang et al., 2011). The PPP is divided into two parts known as the oxidative and the non-oxidative branches. The oxidative branch catalyzes the irreversible transformation of glucose-6-phosphate into ribose-5-phosphate (R5P). During this process important amounts of NADPH are produced. The non-oxidative branch is a bidirectional pathway that interconverts R5P and glycolytic intermediaries. The enzymes that mainly regulate the PPP are glucose6-phosphate dehydrogenase (G6PD) in the oxidative branch and transketolase (TKT) in the non-oxidative branch (Boros et al., 1997; Comin-Anduix et al., 2001; Boren et al., 2002). As we previously reported, proliferating cells increase G6PD activity during late G1- and S-phases (Vizan et al., 2009). Moreover, during S-phase the activation of the SCF ubiquitin ligase by its interaction with the protein-βtransudction repeat-containing protein (β-TrCP) allows the recognition of the DSG box of the PFKFB3 leading to its proteasome degradation (Cardozo & Pagano, 2004; Tudzarova et al., 2011). Through these mechanisms cells redirect the glucose flux from the direct glycolytic pathway to the PPP allowing the formation of NADPH and R5P. NADPH plays an important role in lipid synthesis and redox balance maintenance, and the R5P is a building block for nucleotides for DNA duplication, which is the main characteristic process in S-phase. The temporary coincidence between G6PD activity increase and the accumulation of cyclin E that lead to the formation of the CDK2–cyclin E complex and the entrance in S-phase suggest an association between G6PD and cyclin E regulation. In fact, G6PD regulation seems to be more related to transcriptional CDK–cyclin complexes (CDK7–cyclin H and CDK9–cyclin T) than to the oscillatory cell cycle regulatory CDK–cyclin complexes. As it has been reported, transcriptional CDKs induce MDM2 transcription by activating RNA polymerase II through phosphorylation of its CTD carboxy-terminal domain (Palancade & Bensaude, 2003; Meinhart et al., 2005). Subsequently MDM2 promotes p53 degradation (Momand et al., 2000; Bond et al., 2004) leading to an increase in G6PD activity (Jiang et al., 2011). It is also noteworthy that cyclin E accumulation necessary for G1 to S transition is also inversely dependent of p53 activation. Specifically, it has been shown that under metabolic stress conditions p53 is activated causing transcriptional up-regulation of F-box proteins responsible for cyclin E ubiquitylation and proteolysis (Mandal et al., 2010). Therefore, p53 may be the key that coordinates cyclin signaling with the metabolic condition in G1 to S transition. The hyper fusion of mitochondria recently reported may be another mechanism coordinating metabolic signaling and cyclin E accumulation during G1 to S transition (Finkel & Hwang, 2009; Mitra et al., 2009). It has been demonstrated that during G1 to S transition mitochondria coalesce into a single, giant tubular network electrically continuous and unusually hyperpolarized. This unique morphological mitochondrial network shows the highest ATP producing capacity described for mitochondria to date. Furthermore, it has been associated to the induction of the expression of cyclin E. An increase in transketolase activity was also detected in late G1and S-phases (Vizan et al., 2009). However, even though G6PD activity increased constantly during S-phase, transketolase activity showed an acute increase in late S (unpublished data, Fig. 1). This shift allows an accumulation in the R5P pool in late G1- and S-phases in order to sustain the nucleotide synthesis thanks to G6PD activation, and recycling the excess of R5P back to glycolysis in late S- and G2-phases, when lipid synthesis and mass doubling are the highly substrate-demanding processes. Moreover, the drop in cyclin D1 levels in S-phase may enable

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Fig. 1. Modulation of the activities of the enzymes that control the pentose phosphate pathway (PPP) flux. Glucose-6-phosphate dehydrogenase controls the flux through the oxidative branch of the PPP leading to the formation of the nucleotide precursor ribose-5-phosphate and NADPH, whereas transketolases (TKT and TKTL1) regulate the bidirectional non-oxidative branch balancing the flux between ribose-5-phosphate formation to fuel nucleotide synthesis and its reincorporation to the glycolytic pathway to supply substrates to lipogenesis.

the activation of transcription of lipogenic enzymes contributing to this process (Yang et al., 2006). Thus by increasing transketolase activity over G6PD levels, but maintaining the latter also active and by degrading cyclin D1, proliferating cells recycle R5P to the glycolytic pathway channeling carbon substrates into lipid biosynthesis without decreasing NADPH production. Interestingly, transketolase activity determinations include the effect of two different isoenzymes, TKT itself and transketolase-like protein 1 (TKTL1). Thus, when an increase in transketolase activity is detected it could be due to two independent metabolic entities. However in highly proliferating cells and tissues it has been reported that the main responsible for transketolase activity is TKTL1 (Coy et al., 2005; Langbein et al., 2006; Zhang et al., 2007; Xu et al., 2009). Therefore TKTL1 is proposed as the protein that may catalyze transketolase activity in S- and G2- phases in proliferating cells. This hypothesis is backed by the fact that TKTL1, unlike TKT contains a putative destruction D-box in its amino acid sequence (Q5TYJ8 UniProt, position 79) analog to that described in cyclin A: RxxLxxxxN (den Elzen & Pines, 2001). Since cyclin A accumulates simultaneously with the transketolase activity increases, a common mechanism for regulation of cyclin A and TKTL1 may involve this element. Differences may arise in protein degradation inasmuch as the proteolysis of cyclin A in M-phase is dependent on a D-box adjacent lysine-rich region lacking in TKTL1. In short, cell cycle progression involves not only oscillatory changes in CDK–cyclin complexes but also metabolic sequential adaptations. First, the overcoming of the G1 restriction point mediated by CDK4/ 6-cyclin D complexes formation requires the activation of the glycolytic and glutaminolytic metabolism through PFKFB3 and GLS1 accumulation leading to a peak in the production of lactate and probably also in ATP synthesis. In this point the role of D-type cyclins on the regulation of (lipogenic) metabolism is also important. Subsequently, the entrance to S-phase induced by CDK2–cyclin E correlates with mitochondrial hyper fusion and a simultaneous increase in G6PD and transketolase activities that allow redirecting the glycolytic flux to the PPP, leading to the accumulation of R5P necessary for nucleotide synthesis and NADPH essential in the maintenance of the redox balance. This step appears to be coordinated by p53 and hyperfused mitochondria effects over cyclin E and metabolism. Later, the successful completion of S- and G2- phases involves the accumulation of cyclin A and an even higher activation of transketolase activity probably mediated by TKTL1. The new metabolic change allows the recycling of carbon substrates to the glycolytic

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pathway generating acetyl-CoA and keeping the NADPH synthesis active in order to supply the lipogenic demands of dividing cells. Common regulation of D-type cyclin components, PFKFB3 and GLS1 thanks to APC/ C-CDH1 ubiquitin ligase function ensures the simultaneity of cyclin and metabolic effects on the restriction point. Similarly, cyclin A regulation may be coordinated with TKTL1 expression, maybe through elements like the D-box, in order to allow the smooth running of the cycle. Worth to note that while transketolase activity seems to cycle, once G6PD activity is activated, maintains its levels. This may be due to the differences in the regulation of both enzymes. TKTL1 may be regulated by activation and inhibition of its proteolysis thanks to its D-box like other cycling proteins; whereas G6PD is regulated in response to p53 degradation similarly to transcriptional CDKs, being activated in response to induction of proliferation and remaining active until cell cycle inhibitory signals arise. Through these finely orchestrated mechanisms cells coordinately regulate the progression of the cell cycle and are able to sustain the synthesis of the metabolic substrates necessary for DNA and biomass duplication. 4. Redox status in cell cycle regulation One of the main roles of mitochondria in eukaryotic cells is the transference of electrons from NADH to oxygen through the electron transfer chain (ETC) in order to generate the gradient of protons that allows the ATP synthase to synthesize ATP. This mechanism, which enables the catabolic metabolism of nutrients with a high energetic yield, comes also with other metabolic and physiologic implications. The terminal enzyme of the ETC, the cytochrome c oxidase catalyzes the transference of the electrons to O2, producing H2O. However, different modulators such as nitric oxide (NO) can affect oxygen uptake reducing O2 consumption and leading to ROS formation, process elegantly reviewed elsewhere (Antico Arciuch et al., 2012). ROS are a group of molecules that include species that possess an increased reactivity compared to that of molecular oxygen, and include superoxide (O2−), hydrogen peroxide (H2O2), hydroxyl radical (•OH) and singlet oxygen (1O2). Endogenous production of ROS does not only arise from the mitochondrial metabolism, but can also come from other organelles such as the peroxisome or through the NADPH oxidase (NOX) complex (Kobayashi & Suda, 2012; Liu & Phang, 2012). The role of ROS as intracellular messengers regulating intensity and duration of cell signaling pathways is widely recognized (Sauer et al., 2001; Jones, 2008; Burhans & Heintz, 2009). Nonradical oxidants such as H2O2, unlike more reactive free radicals such as O2−, •OH or 1O2, modulate the redox status of cysteine residues in kinases, phosphatases and other regulatory factors controlling redox-dependent signal transduction (D'Autreaux & Toledano, 2007; Veal et al., 2007; Jones, 2008). Aerobic metabolism has led to mechanisms for protection against ROS generation, such as enzymes like glutathione peroxidase, peroxiredoxin, superoxide dismutase or catalase, and to the use of ROS in signal transduction pathways that regulate the respiratory state and cell fate: apoptosis, cell growth and proliferation among others (Chen & Pervaiz, 2007). This is possible because ROS formation is closely related to cellular redox state. Thus, these species are used by the cell to sense the oxidative stress and mediate the adequate responses. However, some controversy seems to be on the effect of ROS on the progression of cell cycle. For instance, H2O2 can both halt and promote the cell cycle, while O2− has been shown to provoke or prevent apoptosis, depending on the cell type (Caputo et al., 2012). These discrepancies may arise from the dual role of ROS that regulate contradictory mechanisms in a dose-dependent manner. High ROS levels mean high risk of DNA and cellular damage, so the cell response is the suppression of DNA replication, arresting cell cycle and after prolonged arrest, triggering apoptosis (Burch et al., 2004; Bajad et al., 2006; Hwang et al., 2012). On the other hand, it has been also reported that moderate increases in ROS are required for cell cycle entry after quiescence and for G1-phase progression (Burch & Heintz, 2005; Havens et al., 2006; Burhans & Heintz, 2009).

Therefore, when redox-dependent signaling effects are addressed, fine evaluation of ROS concentration is required in order to be able to describe its role unequivocally. Intracellular ROS levels show a cyclic distribution through the cell cycle. Even in yeast a respiratory cycle regulating oxidative metabolism has been described to correlate with the division cycle (Tu et al., 2005). However, in eukaryotic cells the network of metabolic and signaling pathways modulated by ROS that finely control the cell cycle progression is much more complex. In the first instance the G0 to G1 transition that leads the cells from the quiescence to division is the only cell cycle transition that is independent of CDK–cyclin complexes; it is rather regulated by redox-dependent signaling (Burhans & Heintz, 2009). Growth factor induction of proliferation requires H2O2 formation to activate SOS–Ras–Raf–ERK and PI3K/AKT kinase cascades. Through these pathways, and mainly through modification of subcellular localization of ERK, H2O2 modulates G0 to G1 transition (Burch et al., 2004). Moreover, it has been described that redox-dependent signaling pathways mediating G0 to G1 transition converge on regulators of G1 CDKs such as p16, p27 and cyclin D1 (Gartel & Radhakrishnan, 2005; Borriello et al., 2007; Fujikawa et al., 2007; Blain, 2008). High oxidative stress induces p21 activation and p16 and p27 accumulation through p38 MAPK activation and forkhead box O (FOXO) transcription factors, respectively. These proteins lead to CDK–cyclin D sequestration and cell cycle arrest and subsequently to senescence. However, antioxidant treatment of proliferating cells reduces ROS levels to a minimum and leads to a decrease in cyclin D1 levels, an accumulation of p27 and the hypophosphorylation of pRB that also ends in cell cycle arrest in G1-phase (Menon et al., 2003; Havens et al., 2006). Therefore, moderate ROS formation is essential for G1 progression and cell proliferation, but higher levels lead to the opposite effect. In fact ROS act over p16, p21, p27 and p53 regulating transcription and activity of D-type cyclins, which are essential for G1 progression (Burch & Heintz, 2005; Burhans & Heintz, 2009). Moreover, moderate ROS levels have been described to favor cyclin accumulation by inducing APC/C-CDH1 dissociation which also enables cell cycle progression (Havens et al., 2006). The key event regulating G1 to S transition is pRB hyperphosphorylation, mediated by CDK–cyclin complexes in response to growth stimuli and ROS. Hyperphosphorylated pRB allows DNA synthesis and S to G2 progression. ROS levels play a key role in this process, since it is only possible when ROS is depleted. Thus, when intracellular redox potential is higher than −207 mV (the redox potential of the reaction of phosphorylation of the pRB) the protein is dephosphorylated and cell cycle is arrested in S-phase (Hoffman et al., 2008). Finally, hyperphosphorylated pRB enables S-phase progression by binding and inhibiting protein phosphatase 2A (PP2A) (Ranjan & Heintz, 2006). The process that leads to ROS depletion in S-phase for preventing pRB dephosphorylation remains unclear. However, the abovementioned activation of G6PD activity described in late G1- and S-phases leads to NADPH formation, which may play a role in this process by regenerating cellular antioxidants such as reduced glutathione (GSH). During G2/M a new peak in ROS levels has been described (Havens et al., 2006; Burhans & Heintz, 2009). This shift in the cellular redox potential may induce pRB dephosphorylation and PP2A release activating its function as transcription factor mediator. Since it has been described that PP2A plays a role in the activation of the expression of proteins that have a carbohydrate-response element in their regulatory sequence such as fatty acid synthase (FAS) or liver-pyruvate kinase (L-PK), ROS levels may also regulate metabolic adaptations characteristic of G2-phase through this mechanism (Kabashima et al., 2003; Diaz-Moralli et al., 2012). In addition, the peak of ROS in G2/M may enhance cyclin B expression via ERK1/2 and through the FOXO transcription factor FOXM1 (Laoukili et al., 2007). However, given that the proteins of the CDC25 phosphatase family remove inhibitory phosphates from CDK1–cyclin B complex, the increase in the oxidative levels in G2-phase may also be moderated in order to prevent the oxidation and inhibition of their active

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sites (Nilsson & Hoffmann, 2000). Thus, redox-dependent signaling allows cells to complete its cycle of division. 5. Cell cycle defects and cancer During the last years many authors have arrived to the conclusion that cancer can be considered a disease of the cell cycle. This assessment is supported by the fact that while normal cells only proliferate in response to growth stimuli and specific mitogenic signals, cancer cells proliferate in an unregulated way. Moreover, new links between alterations in the cell cycle regulatory machinery and tumorigenesis are being constantly reported and virtually all molecular species involved in regulating cell proliferation described in the literature have been related to malignant transformation. As it has been described, cell cycle progression in mammalian cells is regulated by different mechanisms: the CDK-dependent pathways, which include the vast number of proteins that modulate the activity of CDK complexes and directly act over the cycle machinery, the metabolic adaptations and the redox-dependent signaling. All of them enable cell cycle initiation, progression and completion by establishing a complex network of interactions. For instance, the study of cell cycle regulation led to the development of new models for the characterization of CDK-dependent control of the cell cycle (Ingolia, 2005; Gerard & Goldbeter, 2011; Uhlmann et al., 2011). Given that the main characteristic of tumor cells is its uncontrolled proliferation it is not surprising that key players of all the abovementioned mechanisms are frequently altered in cancer. This has led to propose and test most of them as anti-cancer targets with different success (Box 1). Interestingly, inhibition not only of oncogenes but also of well-known tumor suppressor genes such as checkpoint kinases has shown good results in impairing cancer cell proliferation (Malumbres, 2011). Overexpression of CDKs and cyclins and loss of CKI and pRB expression are often reported in human neoplasias (Malumbres & Barbacid, 2001; Shapiro, 2006). These tumor-associated modifications commonly result from chromosome alterations (amplifications and translocations of oncogenes and deletions of tumor suppressors) or epigenetic inactivation (methylation of tumor suppressor promoters). Misregulated CDKs can induce constitutive mitogenic signaling and defective responses to anti-mitogenic signals, leading to unscheduled proliferation and genomic and chromosomal instability (Massague, 2004; Malumbres & Barbacid, 2009). As it has been reported most of human tumors show defects in G1 to S transition control. Accordingly, the CDKs involved in this transition, CDK4, 6 and 2 are altered in more than 80–90% of tumors, being the misregulation of their activity a selective step during tumor development (Malumbres & Barbacid, 2001; Tetsu & McCormick, 2003; Malumbres, 2011). The high percentage of tumors including altered CDK activity led researchers to postulate therapeutic strategies against cancer directly targeting these proteins (Box 1). In this regard, preclinical data indicate that the inhibition of cyclin D-dependent kinase activity may have therapeutic benefits (Ortega et al., 2002; Ely et al., 2005; Landis et al., 2006; Malumbres & Barbacid, 2006; Shapiro, 2006; Yu et al., 2006; Molenaar et al., 2008; Graf et al., 2009). In addition, metabolic studies reported that inhibition of CDK4 and CDK6 disturbs the balance between the oxidative and the non-oxidative branches of the pentose phosphate pathway, which has been previously described as one of the most robust tumor metabolic adaptations (Zanuy et al., 2012). It has also been demonstrated that mice expressing a mutant form of cyclin D1 that bound to its kinase targets CDK4 and CDK6 without activating their catalytic activity were resistant to mammary tumor development induced by the Erbb2 oncogene (Landis et al., 2006; Yu et al., 2006). Accordingly, downregulation of CDK4 expression in Erbb2-induced mammary tumor cells abrogates tumor formation when reinoculated into mammary fat pads (Yu et al., 2006). Moreover, CDK2-null mice and those with hemizygous disruption of Cdc25A are also protected from Erbb2

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induced mammary tumorigenesis (Ray et al., 2007, 2011), indicating that CDK2 and active CDK4-cyclin D1 complexes are required for Erbb2-driven mammary oncogenesis. However, cyclin D1 ablation has no effect on breast tumor development induced by Myc or Wnt1 oncogenes (Yu et al., 2001). This fact along with the results that demonstrate that CDK1 is sufficient to drive the mammalian cell cycle (Santamaria et al., 2007) and those that suggest that cells lacking interphase CDKs are able to normally complete the proliferation cycle, point out the compensatory role that other kinases may play to enable cell cycle progression (Berthet et al., 2003; Martin et al., 2005; Barriere et al., 2007). Therefore, it is essential to accurately depict cancer cell characteristics to choose the most suitable therapy for each kind of tumor. In ERBB2-positive breast tumors pharmacological specific inhibition of interphase CDKs could be a good strategy (Malumbres & Barbacid, 2006), while in RAS induced tumors CDK4 seems to be the best target and in MYC induced tumors the inactivation of CDK2 activity may possibly be a good approach for therapy (Campaner et al., 2010; Hydbring et al., 2010; Malumbres, 2011). Moreover, in cells with compromised p53 activity the aurora kinases have been proposed as suitable targets (Ruchaud et al., 2007). There is a wide range of drugs frequently used in chemotherapy targeting CDK activities. These molecules target both cell cycle machinery and transcriptional CDKs inducing cell cycle arrest and cell death (Box 1). For a detailed review see Shapiro (2006). Other strategies aiming cancer cell proliferation impairment are focused on the inhibition of the proteins that collaborate with CDK in the regulation of the progression through the different phases of the cycle. In this regard, the key role played by CDC7 kinase on the regulation of S-phase progression has led in the last years most major pharmaceutical companies worldwide such as Pfizer, Roche, Novartis, Sanofi-Aventis, Nerviano Medical Sciences or Bristol Myers Squibb (BMS) to design CDC7 inhibitors (Swords et al., 2010). These inhibitors showed antitumor activity in pre-clinical trials, thus Nerviano (NMS-1116354) and BMS (XL-413/BMS-863233) compounds entered phase I–II clinical trials from 2009. Both molecules are ATP-competitor inhibitors of CDC7 and are showing promising effects on the treatment of solid tumors impairing DNA replication and inducing apoptosis without causing significant toxicity (Montagnoli et al., 2010; Swords et al., 2010). Mutations in DNA damage checkpoint, mainly in the ATM (ataxiatelangiectasia-mutated)-CHK2-p53 pathway, can result in CDK hyperactivity, cell cycle progression in presence of damaged DNA, genomic instability and ultimately, cancer (Kastan & Bartek, 2004; Malumbres & Barbacid, 2009). In fact, genome instability has been reported as an enabling characteristic that facilitates the acquisition of the hallmarks of cancer stressing its significance in cancer development (Hanahan & Weinberg, 2011). The mitotic checkpoint prevents chromosome missegregation, aneuploidy and genome instability, which are common characteristics of many human cancers (Kops et al., 2005). Either defects in this checkpoint, in the mitotic kinases (CDK1, Aurora and PLK kinases) or in the spindle assembly checkpoint (SAC) pathway can end in apoptosis or in abnormal chromosome content. Therefore novel anticancer strategies propose taking advantage of the characteristic genome instability of tumor cells to activate their apoptosis under conditions that do not affect normal cells (Kops et al., 2005; Holland & Cleveland, 2009). Aurora kinase inhibitors have been used in clinical trials for the treatment of solid tumors and hematologic malignancies as leukemia due to their ability to affect chromosome segregation and arrest cell cycle (Box 1). Several molecules have been designed to inhibit aurora kinases, some of them are selective for aurora A or aurora B, but most of them are active not only against aurora kinases but also against many other kinases. Phase I and II clinical trials evaluating aurora kinases inhibitors are being conducted. ENMD-2076, a synthetic molecule designed for selectively inhibiting aurora A and not aurora B that also affects other kinase families, has been tested with promising results both in phase I clinical trials to treat hematologic malignancies and myeloma and in phase II clinical trials in patients with platinum-resistant

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Box 1 Targeting the cell cycle in cancer therapy. The main role played by the deregulation of the cell cycle in tumorigenesis has led to the development of several strategies that target different components of the cell cycle machinery for cancer therapy. Given the importance of CDK–cyclin complexes in conducting the cycle, their inhibition has been addressed as a promising strategy to fight cancer (Davies et al., 2002; Collins & Garrett, 2005; Lapenna & Giordano, 2009; Cicenas & Valius, 2011) leading to the description of a wide number of drugs targeting CDK inhibition. The structural knowledge of cell cycle kinases has allowed the development of inhibitors of CDKs with potential therapeutic effects that have been used in clinical trials. Unfortunately, hitherto none of these molecules have been approved for its commercial use as anticancer drugs. CDK small-molecule inhibitors developed to date can be divided into two groups, the broad-range inhibitors or pan-CDK inhibitors (flavopiridol, olomoucine, R-roscovitine, kenpaullone, SNS-032, AT7519, AG-024322, R547, ZK 304709 and BAY 1000394) and the highly selective CDK inhibitors (Fascaplysin, Ryuvidine, Purvalanol A, NU2058, BML-259, SU 9516, PD-0332991, P276-00, Dinaciclib and AZD5438). The first generation of pan-CDK inhibitors such as flavopiridol, olomoucine or R-roscovitine, despite showing good results in pre-clinical tests (Meijer et al., 1997; Alessi et al., 1998; Sedlacek, 2001; Siemeister et al., 2006; McClue & Stuart, 2008), did not meet expectations displaying low activity and/or toxicity in clinical trials (Lapenna & Giordano, 2009; Lin et al., 2009; Le Tourneau et al., 2010; Cicenas & Valius, 2011; Garrofe-Ochoa et al., 2011; Bible et al., 2012; Luke et al., 2012). These results reinforced the interest in searching for new compounds targeting CDKs more specifically and led to a second generation of more potent CDK inhibitors that mainly interact with the catalytic active site of the kinases competing with ATP or blocking its binding (Johnson et al., 2010, 2012; Shafiq et al., 2012; Shirsath et al., 2012). However, some clinical trials with promising pre-clinical results (for example AG-024322 or AZD5438) were discontinued after phase I due to the inability of the compounds to effectively discriminate from other treatment options or due to poor clinical tolerance (Graf et al., 2011). Also, it is important to bear in mind that certain tumor types might display a different sensitivity to CDK inhibition depending on its pathogenic spectrum of mutations. This consideration can be of greater importance when evaluating a new CDK inhibitor undergoing clinical trials (Malumbres & Barbacid, 2009). CDK–cyclin complexes are not the only cell cycle-related targets addressed by antitumoral therapies. The effect of taxanes and vinca alkaloids on microtubule dynamics and their use to treat patients with breast and ovarian cancers (Jordan & Wilson, 2004; Kops et al., 2005) is widely known. Inhibitors of aurora kinases such as ENMD-2076, Barasertib, Danusertibor PF-03814735 or inhibitors of polo-like kinases such as GSK461364, BI2536, rigosertibin or TAK-960 have shown its efficacy in blocking G2/M transition inducing significant tumor cell death. These results have been validated by clinical evaluation in patients (Jani et al., 2010; Lowenberg et al., 2011; Olmos et al., 2011; Arkenau et al., 2012; Frost et al., 2012; Hikichi et al., 2012; How & Yee, 2012; W. Ma et al., 2012; Matulonis et al., 2013; Meulenbeld et al., 2012). Furthermore, drugs targeting CHK1 and CHK2 such as XL-844 or AZD7762 have shown their capacity to sensitize tumor cells to radiation in pre-clinical studies (Riesterer et al., 2011; Z. Ma et al., 2012). On the other hand, promising results have been obtained for the treatment of cancer with drugs targeting the inhibition of the proteasomedependent proteolysis. Bortezomib is a proteasome inhibitor used for the treatment of relapsed multiple myeloma, among other cancers, that has shown favorable results (Ling et al., 2003; Ludwig et al., 2005; Nakayama & Nakayama, 2006). The efficacy of bortezomib has been corroborated in many clinical trials supporting its suitability for cancer treatment. However, due to the intolerance or resistance usually developed to bortezomib, this drug is currently being tested in combination with radiotherapy and with several other drugs such as the histone deacetylase inhibitor vorinostat (MK-0683), or the cytochrome P450 3A4 (CYP3A4) inducers rifampicin and dexamethasone among others, to take advantage of its capacity to sensitize cancer cells to cytotoxic chemotherapy (search for bortezomib or NCT00011778, NCT00773747 and NCT00608907 at www.clinicaltrials.gov/ct2/search). Moreover, the imidazoline scaffold TCH-013 has been described as a new generation proteasome inhibitor that overcomes the resistance to bortezomib and should offer better results in the treatment of cancer patients (Lansdell et al., in press). Other drugs have been described inhibiting proteolysis by different ways. For instance small molecules blocking the MDM2-p53 interaction lead to the stabilization and reactivation of the tumor suppressor protein p53. Bortezomib has been proposed to inhibit p53 degradation in human non-small cell lung cancer cell line H460 (Ling et al., 2003), however more specific antagonists of MDM2such as nutlins have been described as potential chemotherapeutics (Vassilev et al., 2004; Nakayama & Nakayama, 2006)and subsequent studies have supported its use in cancer therapy (for a review see (Shangary & Wang, 2009)). These results indicated that specific inhibition of the SCF or the APC/C complexes could be effective against cancer and have led to the research of molecules targeting the ubiquitin ligases. An example of these drugs is TAME (tosyl-L-arginine methyl ester), a small molecule that in pre-clinical studies has shown efficient activity preventing APC/C activation by CDC20 and CDH1, leading to arrest of tumor cells in mitosis and triggering tumor cell death (Zeng et al., 2010). Less exploited is the use of therapies against metabolic targets for cancer treatment. Pre-clinical studies have focused mainly in blocking the synthesis of fatty acid by treatment with fatty acid synthase inhibitors such as cerulenin and its chemical analog C75, orlistat or triclosan and with ATP-citrate lyase inhibitors such as SB-204990 (Liu et al., 2002; Kridel et al., 2004; Hatzivassiliou et al., 2005; Menendez et al., 2005; Swinnen et al., 2006; Deepa et al., 2012; Seguin et al., 2012; Zaidi et al., 2012). Clinical trials have been mainly focused in nutrient interventions helping to prevent cancer development and in testing the use of orlistat for treatment of obesity and polycystic ovaries syndrome for instance, but not for antitumoral therapy. Lipid synthesis has been also targeted by activating the metabolic regulator AMPK with acadesine (AICAR,5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside) or rosiglitazone. Since these molecules have shown lipid synthesis-blocking capacity and are able to impair tumor growth, they have entered clinical trials to test their suitability as antitumoral drugs (Xiang et al., 2004; Rattan et al., 2005; Swinnen et al., 2005; Van Den Neste et al., 2010) (for more information regarding clinical trials search for NCT00559624, NCT00369174 and NCT00182052 at www.clinicaltrials.gov/ct2/ search). The other metabolic pathway proposed as potential target against cancer is the pentose phosphate pathway. Inhibitors of the two main regulatory activities of the pathway have been reported as potential antitumorals, dehydroepiandrosterone for G6PD and oxythiamine for TKT activities (Boros et al., 1997; Rais et al., 1999; Vizan et al., 2009; Buchakjian & Kornbluth, 2010). Dehydroepiandrosterone entered several clinical trials in order to test its efficacy against different diseases and in the last years it is also being tested inbreast cancer patients (search for NCT00972023 at www.clinicaltrials.gov/ct2/search).

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ovarian cancer (How & Yee, 2012; Matulonis et al., 2013). On the other hand, Barasertib (AZD1152) is a selective inhibitor of aurora B that has been used in phase I and II clinical trials to treat patients with advanced acute myeloid leukemia with a response rate of 25% and manageable adverse effects (Lowenberg et al., 2011). Moreover, Danusertib (PHA-739358) and AT9283 are inhibitors of both aurora A and B kinases that have also entered clinical trials. The latter has only followed to date a phase I dose escalation study whereas the former has been tested in phase II trials for the treatment of different tumors (Arkenau et al., 2012; Meulenbeld et al., 2012). PLK1 has been also widely accepted as a feasible antitumor target, however since the inhibition of the other polo-like kinases (PLK2–5) may lead to tumor development, the specificity of PLK1 inhibitors is of paramount importance. It has been tested several PLK1 inhibitors such as GSK461364, BI2536 or rigosertibin phase I trials and the obtained results have led to the proposal of further studies in the future (Olmos et al., 2011; Frost et al., 2012; W. Ma et al., 2012). As mentioned above, genome instability and accumulation of mutations are enabling characteristics for the acquisition of the malignant phenotype. They are enhanced by defects in the genome maintenance mechanisms such as DNA repair and cell cycle checkpoint pathways. Consequently, the checkpoint kinase CHK2 is a candidate tumor suppressor that is found altered in some types of cancer, like colon or breast cancer (Bartek & Lukas, 2003). Mutations in CHK2 can lead to abnormal activation of CHK2 and increased levels of the proapoptotic E2F1 transcription factor. The suprathreshold stabilization of E2F1 using β-lapachone results in selectively cancer cell death (Li et al., 2003). Also, recent studies show that β-lapachone and several derivatives mediate cancer cell toxicity through ROS formation and DNA damage, which demonstrate the difficulty of isolating a single target for a drug effect. Targeting not only CHK2 but also the other checkpoint regulator CHK1 and E3 ubiquitin ligases involved in mediating cyclin proteasome degradation have been also proposed as relevant anticancer therapeutic approaches. Preclinical studies have shown that CHK1 and CHK2 inhibitors such as XL-844 or AZD7762 sensitize different cancer cells to radiation via inhibition of DNA repair and induction of mitotic catastrophe (Riesterer et al., 2011; Z. Ma et al., 2012). On the other hand, blocking mitotic exit by affecting CDC20 has been proposed as a cancer therapeutic strategy that might circumvent resistances that have arisen in response to therapies targeting checkpoint machinery (Huang et al., 2009; Manchado et al., 2010). Both strategies are in pre-clinical studies and require further research but may be useful to develop new anticancer therapies targeting cell cycle defects in the future. Development of malignancy comes along with a metabolic reprogramming closely related to the acquisition of most of cancer hallmarks (Kroemer & Pouyssegur, 2008; Hanahan & Weinberg, 2011). One of these metabolic alterations associated to cell malignant transformation is the aerobic glycolysis, consisting in an increased metabolism of glucose to lactate even in presence of oxygen, which is commonly referred to as the “Warburg effect” due to Otto Warburg who was the first to describe this phenomenon in the 1920s (Warburg et al., 1924, 1927). Metabolic adaptations and redox signaling as key players in the regulation of proliferation are also involved in different therapeutic approaches for cancer therapy. Briefly, the metabolic pathways that enable cell transformation are commonly glycolysis, PPP, nucleic acid synthesis and lipogenesis. Glycolysis supplies the energy and carbon sources for biosynthesis and cell duplication leading to the formation of lactate, PPP regulates the flux of carbons between nucleic acid synthesis and lipogenesis and the latter two generate the elemental units for genetic material and cell membranes duplication. Moreover, pathways like glutaminolysis have been described essential for many tumors, thus confirming the importance of their role in controlling proliferation. The acquisition of most of cancer hallmarks has been reported to be accompanied of a complete metabolic reprogramming (Kroemer & Pouyssegur, 2008). Interestingly, there is a close relationship between metabolism and redox balance

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mediated not only by mitochondrial activity but also to a lesser extent by all NAD(P) +/NAD(P)H-dependent reactions. The importance of metabolism in cancer is illustrated by the fact that the first chemotherapeutical agents used in the mid-twentieth century in cancer treatment were antimetabolites that blocked precisely the nucleotide synthesis: antifolates (aminopterin and methotrexate) and 5-fluorouracil (Farber & Diamond, 1948; Heidelberger et al., 1957). Methotrexate allosterically inhibits dihydrofolate reductase (DHFR), an enzyme that participates in the tetrahydrofolate synthesis, necessary for the synthesis of nucleotides. Whereas 5-fluorouracil is able to irreversibly interact with thymidylate synthase, which is essential for thymidine synthesis. From then on several metabolic pathways and enzymes have been proposed and successfully used as anticancer targets. Given the fact that one of the main metabolic features of cancer cells is the high production of lactate from glucose even in the presence of oxygen, the Warburg effect, other body of research has been conducted to inhibit this pathway by blocking the enzymes that control it, such as pyruvate kinase (Chaneton & Gottlieb, 2012; Sun et al., 2012; Zhou et al., 2012). Metabolites have been also used as activators of CKI in certain tumors highlighting the interconnection existing between different kinds of cell cycle regulators. For instance, CKI p21 that activates apoptosis in response to cisplatin in human ovarian adenocarcinoma cells SKOV3 (Lincet et al., 2000), responds to sodium butyrate inducing programmed cell death in human breast cancer MCF-7 cell line (Chopin et al., 2004), and to c6-ceramide leading to apoptosis hepatoma cells (Kang et al., 1999). Contrary to p21-dependent apoptosis induced by metabolites in these cell lines, in human colon carcinoma HCT116 and in human lung adenocarcinoma A549 cell lines treatment with antimetabolites of folate or folate synthesis blockers induce apoptosis due to mutations in p21. These treatments cause a strong depletion of the purine and ATP pools resulting in metabolic stress. Under these conditions, p53 activates both p21 which mediates a metabolic arrest effect and PUMA which triggers apoptosis in the absence of p21. Therefore, in p21 active cell lines, metabolite deprivation induces p21-dependent cell cycle arrest and in absence of DNA damage an antiapoptotic response, however when p21 is inactivated apoptosis is induced by PUMA (Hoeferlin et al., 2011). Mutations in p21 have also been reported to mediate an apoptotic effect in response to methotrexate (Kraljevic Pavelic et al., 2008). In addition, metabolic stress caused by nutrient withdrawal can lead to the autophagy gene product ATG7 activation and p53-mediated p21 activation, which induce cell cycle arrest in G1-phase. In absence of ATG7, starved cells fail to undergo cell cycle arrest and there is no accumulation of p21. With prolonged nutrient deprivation, the lack of ATG7 causes ROS and DNA damage increase, which in turn activates CHK2 and p53-mediated transcription of proapoptotic genes (Lee et al., 2012). 6. Conclusions Since the middle of the last century different drugs have been used in cancer therapy. These molecules in the vanguard of chemotherapy were antimetabolites such as aminopterin, methotrexate or 5-fluorouracil, aiming to impair nucleotide synthesis and DNA replication as one of the essential processes for cell proliferation (Farber & Diamond, 1948; Heidelberger et al., 1957). However, it was not until decades later that the Nobel laureates Leland H. Hartwell, Timothy Hunt and Paul M. Nurse began to describe the role of CDK and cyclins in driving the cell cycle opening new avenues in cancer therapy research. More recently the role of ROS in the regulation of cell cycle progression has led to a complex view of the proliferation control where the protein machinery, metabolism and redox state interact to enable or restrict cell division. In the last years a revolutionary milestone in the description of the cell cycle control mechanisms has been reached; the characterization of the oscillatory variations of metabolic enzyme activities and ROS

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levels and its coordination with cyclins and cell cycle phases (Tu et al., 2005; Burhans & Heintz, 2009; Vizan et al., 2009; Almeida et al., 2010; Colombo et al., 2010). In this review, we have put together all this information about the species that cycle during cell division, correlating their function and highlighting its role in controlling cell cycle progression. This knowledge has to lead to a better understanding of the crosstalk between signaling and metabolic processes underlying the cell cycle regulation and to the proposal of new combinatory approaches in cancer therapy. As it has been also reported cancer cells accumulate modifications in a wide range of the mechanisms controlling its cell cycle. However, it is not yet fully understood which of these modifications are cause of cancer onset and which of them are the effect of the progression of the disease. Interestingly, it has been reported that mutations in the proto-oncogen c-myc are able to induce rapid cell cycle entry due to its dual effect over cell cycle machinery and metabolism (Morrish et al., 2008, 2009). However, similar results backed the role of p53 in controlling the activation of cell proliferation (Vousden & Ryan, 2009; Puzio-Kuter, 2011). These data suggest that induction of proliferation and malignant transformation is not the result of a single pathway, but the integrative response to different sets of events. One of the main objectives aimed by cancer researchers is to accurately identify the main differences existing between normal proliferating cells and cancer cells. This is essential but not easy given that cell division of non tumor cells is a perfectly orchestrated mechanism and its deregulation leads to a very similar but uncontrolled process in cancer cells. In this regard it is necessary to differentiate two independent although related processes. The first of them is the G0 to G1 transition that allows cells to leave quiescence and enter the cell cycle. The other is the progression through the different phases of the cycle controlled primarily by the so-called restriction point and subsequently by the rest of the checkpoints. This differentiation makes sense because alterations in the mechanisms that regulate G0 to G1 transition may be involved in allowing the initial transformation of cells but not necessarily in the later tumor growth. Therefore, by targeting mechanisms preventing entrance to the cell cycle it is possible to avoid cancer onset but probably it is not effective in treating tumors. It is relevant for the research of products with preventive effect against cancer such as antioxidants that can be introduced as dietary supplements. On the other hand, therapies addressed to hinder cell cycle progression by impairing biosynthetic pathways or activating proapoptotic/inhibiting promitotic signaling may show its efficacy on the treatment of established tumors. However, since proliferating cells in late M-phase have to decide whether entering G0-phase or continuing with a new cycle of division, therapies that induce cancer cells to enter quiescence after mitosis can also be combined with therapies that prevent new G0 to G1 transitions in quiescent cells. In summary cell cycle progression is controlled not only by a mechanism of coordinated expression and degradation of proteins but also by many other players (Fig. 2). Progression through the G1-phase requires sequential activation of CDK4/6 by binding D-type cyclins. The mechanism regulating cyclin ubiquitylation and its proteasomal degradation also allows activation of glycolysis and glutaminolysis during G1-phase through accumulation of PFKFB3 and GLS1 (Fig. 2 B) (Almeida et al., 2010; Colombo et al., 2010, 2011). Thus in mid to late G1 an increase in lactate and ATP production is reached (Fig. 2 B). It has been also reported that a mechanism of hyper fusion of mitochondrion may play a role in the peak in ATP synthesis (Mitra et al., 2009). More controversial is the peak in ROS levels also described in G1 (Burch & Heintz, 2005; Burhans & Heintz, 2009). Anyway the peak in

ATP, lactate and/or ROS may be interpreted by the cells as a signal indicating that available metabolic sources are sufficient to sustain the cycle of division allowing R overcoming and activating proliferation (Fig. 2 B). The transition between G1- and S-phases requires changes in proteins, metabolism and ROS levels (Fig. 2 C). On the first hand, D-type cyclins are depleted, probably by a mechanism mediated by ROS (Havens et al., 2006), and CDK2 is activated thanks to cyclin E accumulation in a process where mitochondria hyper fusion has been reported to also play a role (Mitra et al., 2009). On the other hand, in order to supply the precursors for DNA replication in S-phase cells trigger ribose synthesis by activating G6PD, this leads to an increase in NADPH synthesis that may also help to deplete ROS once in S-phase in order to prevent DNA damage (Fig. 2 C-D). It has been reported that G6PD activation may depend on p53 signaling (Jiang et al., 2011), which in addition may induce a new change in cyclin expression leading to proteasomedependent cyclin E degradation and the CDK2–cyclin A activation characteristic of S-phase (Mandal et al., 2010). During S-phase progression metabolism is re-adapted enabling DNA replication and preparing the cell for G2. Transketolase activity is enhanced allowing recycling the excess of riboses to biosynthetic pathways without dropping the oxidative PPP flux in order to provide lipogenic pathways with NADPH and keep low oxidative levels. Furthermore, the cyclin D1 drop preceding S-phase entrance enhances the expression of lipogenic enzymes which will play a role during G2 (Fig. 2 E). In fact, the physiologic significance of G2-phase is the duplication of membranes and cell mass to prepare mitosis, thus the combination of transketolase and lipogenic enzymes activity allow enhancing lipogenesis and fulfilling the metabolic needs of proliferating cells. Once cells are ready for division the activation of the CDK1–cyclin B complex and a new peak in ROS levels are responsible for the activation of mitosis (Fig. 2 F) (Havens et al., 2006). Cell division leads to two new cells in G1-phase where CDK complexes, metabolic activities and ROS levels must come to initial state (Fig. 2 G-H). To date, cancer patients have been treated with therapies targeting cell cycle progression (Table 1), most of them used in combination to achieve the higher effect over cancer cells with the minimum impact over normal proliferating cells. However, in the light of the coordinative mechanisms described above it is possible to suggest new approaches for cancer therapy that may boost synergies. The key is to target events occurring simultaneously in the cell cycle to increase the efficacy of the arrest and its consequences. For example, one strategy for drugs aiming to prevent the overcoming of the restriction point, could be aiming for glycolysis inhibition, ATP depletion, cyclin E downregulation and ROS scavenging. In the case of drugs affecting S-phase progression such as antifolates, they could be combined with inhibitors of cyclin E degradation or prooxidant molecules. Moreover, the accurate characterization of tumors is essential in order to determine where the defects on the cell cycle control are accumulated and which of the phases should be targeted to ensure therapeutic success. Combination of chemotherapeutic agents affecting mechanisms regulating the same cell cycle phase should improve cytostatic and even cytotoxic efficacy of cancer treatments but may not lead to higher specificity due to the similarity between normal and cancer cell proliferation. However, given that it has been reported the involvement of tumor specific enzymes, not active in normal proliferating cells, in malignant transformation this problem could be solved targeting these isoenzymes. These tumor-related isoenzymes are glucose transporter 1 (GLUT1), hexokinase 2 (HK-2), lactate dehydrogenase A (LDH-A) (Herling et al., 2011; Wolf et al., 2011) and TKTL1 (Langbein et al., 2006; Zhang et al., 2007; Xu et al., 2009). Pyruvate kinase M2 (PKM2) has been also reported as a tumor specific isoenzyme (Zhou et al., 2012), however discrepancies

Fig. 2. Schematic representation of the key players in the cell cycle progression and the main metabolic changes that accompany every step. Displayed are the state of cellular quiescence (A), the G1 initiation as consequence of mitogenic stimuli (B), the G1/S transition (C), the S phase progress (D), S/G2 transition (E), the G2 phase (F) and finally the progress to mitosis (G) and back to G0/G1 phases (H). APC/C, anaphase-promoting complex/cyclosome; CDK, cyclin dependent kinases; FA, fatty acids; G6PDH, glucose-6-phosphate dehydrogenase; Glc, glucose; Gln, glutamine; GLS1, glutaminase 1; Lac, lactate; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; R5P, ribose-5-phosphate; ROS, reactive oxygen species; SCF, Skp, Cullin, F-box containing complex; TKT, transketolase; TKTL1, transketolase-like protein 1; β-TrCP, protein- -transduction repeat-containing protein.

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Table 1 When the drugs affect more than one CDK bolded ones are the most specific targets. Drug

Specific target (Impaired Mechanism)

Studies

References

Taxanes & Vinca alkaloids

Microtubule dynamics (Cytoskeleton)

Clinical trials and therapy

Flavopiridol

CDK1, CDK2, CDK4, CDK6, CDK7, CDK9, GSK3β (Canonical Cell Cycle Regulation)

Clinical Trials

Olomoucine

CDK1, CDK2, CDK5; ERK1 (Canonical Cell Cycle Regulation) CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK9 (Canonical Cell Cycle Regulation) CDK1, CDK2, CDK5, GSK3β (Canonical Cell Cycle Regulation) CDK2, CDK7, CDK9 (Canonical Cell Cycle Regulation) CDK1, CDK2, CDK4, CDK5, CDK6, CDK9, GSK3β (Canonical Cell Cycle Regulation) CDK1, CDK2, CDK4 (Canonical Cell Cycle Regulation) CDK1, CDK2, CDK4 (Canonical Cell Cycle Regulation) CDK1, CDK2, CDK4, CDK7, CDK9 (Canonical Cell Cycle Regulation) CDK1, CDK2, CDK4, CDK9 (Canonical Cell Cycle Regulation) CDK4, CDK6 (Canonical Cell Cycle Regulation) CDK4 (Canonical Cell Cycle Regulation) CDK2, CDK4, CDK5 (Canonical Cell Cycle Regulation) CDK1, CDK2 (Canonical Cell Cycle Regulation) CDK2, CDK5 (Canonical Cell Cycle Regulation) CDK1, CDK2, CDK4, PKC, p38, PDGFR, EGFR (Canonical Cell Cycle Regulation) CDK4, CDK6 (Canonical Cell Cycle Regulation)

Pre-clinical

Jordan & Wilson, 2004 Kops et al., 2005 Lin et al., 2009 Luke et al., 2012 Bible et al., 2012 Cicenas & Valius, 2011 Garrofe-Ochoa et al., 2011 Le Tourneau et al., 2010 Garrofe-Ochoa et al. 2011 Rivest et al., 2011

Seliciclib Kenpaullone SNS-032 AT7519 AG-024322 R547 ZK 304709 BAY 1000394 Fascaplysin Ryuvidine Purvalanol A NU2058 BML-259 SU 9516 PD-0332991

P276-00

Clinical trials Pre-clinical Clinical trials Clinical trials Clinical trials discontinued (2007)

Clinical trials

Zhong et al., 2009 Lapenna & Giordano, 2009 DePinto et al., 2006 Berkofsky-Fessler et al., 2009 Graham et al., 2008 Scott et al., 2009 Siemeister et al., 2012

Pre-clinical

Shafiq et al., 2012

Pre-clinical Pre-clinical

Ryu et al., 2000 Cicenas & Valius, 2011 Hikita et al., 2010

Pre-clinical

Johnson et al., 2010

Pre-clinical

Kazmierczak et al., 2011

Pre-clinical

Uchiyama et al., 2010

Clinical trials

Schwartz et al., 2011 Flaherty et al., 2012 Leonard et al., 2012 Cicenas & Valius, 2011 Shirsath et al., 2012 Gorlick et al., 2012 Zhang et al., 2012 Boss et al., 2010

Clinical trials Clinical trials

CDK1, CDK2, CDK4 (Canonical Cell Cycle Regulation) CDK1, CDK2, CDK5, CDK9 (Canonical Cell Cycle Regulation) CDK1, CDK2, CDK9 (Canonical Cell Cycle Regulation) CDK1, CDK2, CDK4, CDK5, CDK7, TRKA (Canonical Cell Cycle Regulation)

Clinical trials

Terameprocol

CDK1, survivin, VEGFRs (Canonical Cell Cycle Regulation)

Clinical trials

Indisulam

Cyclin E (Canonical Cell Cycle Regulation)

Clinical trials

NMS-1116354 XL-413/BMS-863233 PF-03814735 Danusertib AT9283 ENMD-2076

CDC7 Kinase (Canonical Cell Cycle Regulation) Aurora kinases (Canonical Cell Cycle Regulation)

Clinical trials

Aurora kinase A (Canonical Cell Cycle Regulation) Aurora Kinase B (Canonical Cell Cycle Regulation) PLK1 (Canonical Cell Cycle Regulation)

Clinical trials

CHK 1/2 (Canonical Cell Cycle Regulation) Proteasome (Proteolysis) Proteasome (Proteolysis) MDM2-p53 (Ubiquitin Ligases-Proteolysis)

Pre-clinical

Dinaciclib AZD5438 PHA-848125

Barasertib GSK461364 BI2536 Rigosertibin TAK-960 XL-844 AZD7762 Bortezomib TCH-013 Nutlins

Heath et al., 2008 Tong et al., 2010 Mahadevan et al., 2011

Clinical trials Clinical trials discontinued (2009) Clinical trials

Clinical trials

Clinical trials Clinical trials

Brasca et al., 2009 Albanese et al., 2010 Caporali et al., 2012 Khanna et al., 2008 Lapenna & Giordano, 2009 Grossman et al., 2012 Terret et al., 2003 Talbot et al., 2007 Lapenna & Giordano, 2009 Montagnoli et al., 2010 Swords et al., 2010 Jani et al., 2010 Meulenbeld et al., 2012 Arkenau et al., 2012 How & Yee, 2012 Matulonis et al., 2013 Lowenberg et al., 2011

Clinical trials and therapy

Olmos et al., 2011 Frost et al., 2012 W. Ma et al., 2012 Hikichi et al., 2012 Riesterer et al., 2011 Z. Ma et al., 2012 Ludwig et al. 2005

Pre-clinical

Lansdell et al., in press

Pre-clinical Clinical trials

Vassilev et al., 2004 Shangary & Wang, 2009

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Table 1 (continued) Drug

Specific target (Impaired Mechanism)

Studies

References

TAME

APC/C (Ubiquitin Ligases-Proteolysis) FAS (Metabolic Enzymes) FAS (Metabolic Enzymes)

Pre-clinical

Zeng et al., 2010

Pre-clinical

ATP-Citrate Lyase (Metabolic Enzymes) AMPK activation (Metabolic Enzymes)

Pre-clinical

Deepa et al., 2012 Seguin et al., 2012 Menendez et al., 2005 Deepa et al., 2012 Seguin et al., 2012 Zaidi et al., (2012)

G6PD (Metabolic Enzymes) TKT (Metabolic Enzymes)

Clinical trials

Cerulenin/C75 Triclosan Orlistat

SB-204990 Acadesine Rosiglitazone

Dehydroepiandrosterone Oxythiamine a

Pre-clinical Clinical trials and therapya

Clinical trials

Pre-clinical

Swinnen et al., 2005 Rattan et al., 2005 Van Den Neste et al., 2010 Xiang et al., 2004 Boros et al., 1997 Rais et al., 1999 Boros et al., 1997 Rais et al., 1999

Used in diseases different from cancer.

have arisen regarding this issue (Bluemlein et al., 2011; Chaneton & Gottlieb, 2012). Interestingly, most of the isoenzymes are mediating glycolysis and only TKTL1 is involved in PPP control. Thus, specific therapies against GLUT1, HK-2, LDH-A or PKM2 should be combined with drugs of the G1-phase, whereas therapies targeting TKTL1 should be used with anti-S-phase agents in order to obtain both specificity and efficacy. Unfortunately, the design of isoform-specific inhibitors is a very complicated task and there is still a long way to go. However, despite chemotherapy against specific isoenzymes seems chimeric nowadays, further studies aiming the identification of new tumor specific isoenzymes is required to better characterize the cell cycle and understand the differences existing between normal and malignant proliferating cells since this information will lead to the design of innovative anticancer therapies in the future. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the Spanish Government and the European Union FEDER funds (SAF2011-25726), Generalitat de Catalunya-AGAUR (2009SGR1308) and “ICREA Academia prize” (MC). References Albanese, C., Alzani, R., Amboldi, N., Avanzi, N., Ballinari, D., Brasca, M. G., et al. (2010). Dual targeting of CDK and tropomyosin receptor kinase families by the oral inhibitor PHA-848125, an agent with broad-spectrum antitumor efficacy. Mol Cancer Ther 9(8), 2243–2254. Alessi, F., Quarta, S., Savio, M., Riva, F., Rossi, L., Stivala, L. A., et al. (1998). The cyclin-dependent kinase inhibitors olomoucine and roscovitine arrest human fibroblasts in G1 phase by specific inhibition of CDK2 kinase activity. Exp Cell Res 245(1), 8–18. Almeida, A., Bolanos, J. P., & Moncada, S. (2010). E3 ubiquitin ligase APC/C-Cdh1 accounts for the Warburg effect by linking glycolysis to cell proliferation. Proc Natl Acad Sci U S A 107(2), 738–741. Antico Arciuch, V. G., Elguero, M. E., Poderoso, J. J., & Carreras, M. C. (2012). Mitochondrial regulation of cell cycle and proliferation. Antioxid Redox Signal 16(10), 1150–1180. Arellano, M., & Moreno, S. (1997). Regulation of CDK/cyclin complexes during the cell cycle. Int J Biochem Cell Biol 29(4), 559–573. Arkenau, H. T., Plummer, R., Molife, L. R., Olmos, D., Yap, T. A., Squires, M., et al. (2012). A phase I dose escalation study of AT9283, a small molecule inhibitor of aurora kinases, in patients with advanced solid malignancies. Ann Oncol 23(5), 1307–1313. Bajad, S. U., Lu, W., Kimball, E. H., Yuan, J., Peterson, C., & Rabinowitz, J. D. (2006). Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry. J Chromatogr A 1125(1), 76–88.

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