Cancer cell cycle

Nuno Cerveira1, Susana Bizarro1, and Manuel R Teixeira1,2,* ABSTRACT

At the cell level, cancer is a genetic disease that is characterized by genetic changes, including in oncogenes and tumor suppressor genes. Abnormal cell proliferation, a common feature of human cancer cells, is usually the result of cell cycle deregulation, which in turns associates with genomic and chromosomal instability. Orderly transition between cell cycle phases requires the scheduled activity of cyclin-dependent kinases (CDKs), regulated in part by their associations with cyclins and CDK inhibitors. In cancer, these processes are deregulated, with tumor-associated cell cycle defects being often mediated by alterations both in CDKs and/or their regulators. We here review the mechanisms involved in cell cycle deregulation in cancer, as well as attempts to target CDKs in novel cancer therapies.
1

Department of Genetics, Portuguese Oncology Institute, Rua Dr. Antnio BernardiAbel Salazar Biomedical Sciences Institute (ICBAS), University of Porto, Largo Prof.

no de Almeida, 4200-072 Porto, Portugal;

2

Abel Salazar, 2, 4099-003 Porto, Portugal.

Corresponding authors contact: Department of Genetics, Portuguese Oncology Institute, Rua Dr. Antnio Bernardino de Almeida, 4200-072 Porto, Portugal; tel: +351 225084000; fax: +351 225084016; e-mail: manuel.teixeira@ipoporto.min-saude.pt

INTRODUCTION Cancer is the second leading cause of death in Western countries, surpassed only by heart disease. It strikes people of all ages, and one out of three people will experience a cancer diagnosis sometime in his or her lifetime. Evidence accumulated during the last 30 years demonstrates that cancer is a genetic disease, with changes in oncogenes and tumor suppressor genes leading to abnormal cell differentiation and proliferation. Indeed, unscheduled proliferation, as a result of cell cycle deregulation, is a common feature of human cancer cells, which in turn is associated with genomic and chromosomal instability. In this review, we will provide a broad perspective on how the misregulation of cell cycle control can contribute to cancer. In particular, we will focus in a subfamily of cyclin-dependent kinases (CDKs), their activators (cyclins), and their inhibitors (CKIs), and how deregulation of their activity in cancer cells, as a result of genetic or epigenetic abnormalities, is associated with tumorigenesis.

REGULATION OF THE EUKARYOTIC CELL CYCLE Cell cycle consists of two consecutive periods, mainly characterized by DNA replication and segregation of replicated chromosomes into two separate daughter cells. The cell cycle can be divided into ve phases: G0, G1, S, G2, and M. Cell division, or cytokinesis, occurs during the M (mitosis) phase, and is preceded by a preparative phase, or interphase, that includes G0, G1, S and G2 (Fig. 1). DNA replication occurs in a specic part of the interphase, the S (synthesis) phase, which is preceded by a gap called G1, where the cell prepares for DNA synthesis, and followed by G2, during which the cell prepares for mitosis. The vast majority of the cells are able to divide but do so only when appropriate to replace damaged or dead cells. Indeed, cells in G0 account for the major part of the nongrowing, non-proliferating compartment of the human body, being essentially arrested in their growth. Cells in G0 can often be stimulated to enter the cell cycle by external growth signals such as growth factors and

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The transition between cell cycle phases occurs in an orderly fashion, being tightly regulated by different cellular proteins. Indeed, appropriate progression through the cell cycle is monitored in checkpoints that sense possible defects during DNA synthesis and chromosome segregation. Activation of these checkpoints induces a temporary halt in cell cycle progression until a set of conditions is met, namely accurate repair of DNA and chromosomal errors, thus preventing their transmission to the daughter cells. The key regulatory proteins are a family of serine/threonine protein kinases that are activated at specic points of the cell cycle. These proteins, known as cyclin-dependent kinases (CDKs) are heterodimeric protein kinases composed of a catalytic subunit known as CDK and a regulatory subunit known as cyclin [1, 2]. Cyclins are synthesized and degraded at specic times during the cell cycle, thus regulating kinase activity in a timely fashion [2]. In addition to cyclin binding, CDK activity is also regulated by phosphorylation on conserved threonine and tyrosine residues. These modications induce conformational changes and enhance cyclin binding [3, 4]. Multiple loci encoding both CDKs and cyclins can be found in human cells, however only a small subset of CDK-cyclin complexes is directly involved in driving
Figure 1: In the eukaryotic cell cycle the interphase comprises three phases: G1, S, and G2. Orderly transition between cell cycle phases requires the scheduled activity of cyclin-dependent kinases (CDKs), regulated in part by their associations with cyclins and CDK inhibitors. External growth signals activate multiple signal transduction pathways, including those involving the MYC and RAS genes, which lead to synthesis and stabilization of cyclin D that binds and activates CDK4. Throughout the G1 phase, the RB1 protein becomes phosphorylated by the CDK4/cyclin D1 complex. Phosphorylated RB1 releases its bound regulatory proteins, such as E2F1, which can now induce the expression of cyclin E, and more than 30 other genes, whose products are required for the G1-S transition. The cell cycle can be blocked by the Cip and Kip inhibitors (CDKN1A and CDKN1B) and by the INK4 inhibitors, such as CDKN2A (p14, p16). Cell-cycle arrest in response to DNA damage is mediated through TP53, whose levels are under negative regulation by MDM2, through a feedback loop that is inhibited by CDKN2A.

the cell cycle [1, 5]. These include the interphase CDKs CDK2, CDK4, and CDK6, the mitotic CDK CDK1, and ten regulatory cyclins belonging to four different classes, known as the A, B, D, and E type cyclins [1, 5]. The three D type cyclins - cyclin D1 (CCND1), cyclin D2 (CCND2), and cyclin D3 (CCND3) - bind and activate CDK4 and CDK6, with the resulting complexes being

hormones that bind to cell surface receptors and then convey the signal from the

plasma membrane to the nucleus, a process known as signal transduction.

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CDKs, or their regulatory subunits cyclins, which when hyperactive may favor tumor development by inducing unscheduled cell division. In the second group, we can nd regulators of CDK activity, CDK inhibitors (CKIs) that belong to two families: INK4 proteins, including CDKN2A (p14, ARF, p16, INK4A), CDKN2B (p15, INK4B), CDKN2C essential to G1 phase entry [6]. Activation of these complexes leads to partial inactivation of the pocket proteins RB1 (retinoblastoma), RBL1 (also known as p107) and RBL2 (also known as p130) allowing the expression of the E-type cyclins (CCNE1 and CCNE2), which in turn bind and activate CDK2, thus regulating progression from G1 into S phase [7, 8]. The CDK2cyclin E complexes further phosphorylate these pocket proteins, leading to their complete inactivation [8, 9]. Subsequently, CDK2 is activated by cyclin A2 (CCNA2) during the late stages of DNA replication, driving the transition from S phase to mitosis [10, 11]. Finally, at the end of interphase, in late G2 and early mitosis, cyclin A complexes with CDK1, leading to its activation, to facilitate the onset of mitosis. Following nuclear envelope breakdown, A-type cyclins are degraded, leading to the formation of the CDK1cyclin B complexes responsible for driving cells through mitosis [1]. (p18, INK4C), and CDKN2D (p19, INK4D); and the Cip and Kip family, composed of CDKN1A (p21, Cip1), CDKN1B (p27, Kip1), and CDKN1C (p57, Kip2) [12]. Constitutive and deregulated CDK activation may contribute not only to unscheduled proliferation but also to genomic (defective DNA repair and DNA damage checkpoints) and chromosomal instability (defective mitotic checkpoints and chromosomal segregation) in cancer cells [12]. The alteration of the DNA damage and mitotic checkpoints frequently results in increased CDK activity that drives tumor cell cycle [12]. In addition, many molecules involved in signal transduction pathways, including cell membrane receptors and intermediate proteins, are prone to genetic abnormalities. Indeed, mutations in genes that encode these signaling proteins can make the proteins hyperactive, allowing them to relay proliferation signals to the nucleus even in the absence of signals from the extracellular matrix or from neighboring cells. This uncontrolled signaling can lead to loss of cell cycle regulation and as a CELL-CYCLE DEREGULATION IN CANCER One of the fundamental abnormalities in all cancer cells is a loss of control over cell proliferation. In normal cells, the growth and differentiation must be strictly regulated, otherwise the functional integrity of tissues, organs, organ systems, and ultimately, the person, would be compromised by inappropriate types and quantities of cells. Both cell-cycle checkpoints and cell-cycle control molecules are regulated by interplay of genes whose products either promote or suppress cell division. These two types of genes are proto-oncogenes and tumor suppressor genes, which promote or inhibit cell progression trough the cell cycle, respectively. In the rst group we include ONCOGENES AND THE CELL CYCLE Proto-oncogenes encode transcription factors that regulate the expression of other genes, signal transduction proteins that stimulate cell division, and cell cycle regulators that push the cell trough the cell cycle. They are crucial players in normal cell functions but can be converted into oncogenes by gain-of-function mutations. In this case, only one allele of a proto-oncogene needs to be altered in order to trigger uncontrolled growth; hence, oncogenes confer a dominant cancer phenotype. Gainconsequence to unrestrained cell division, a hallmark of cancer cells.

of-function mutations can modify the function of the proteins that promote passage through the cell cycle in two different ways. One way involves the alteration of the gene sequence, which leads to a change in the structure of the protein it codes for, so that the function of the protein is altered, becoming hyperactive. Alternatively, gain-offunction mutations can result in increased gene expression, resulting in abnormal levels of a structurally normal protein, which also result in enhancement of protein function. When normal cells become quiescent and cease division they repress the expression or activity of most proto-oncogenic proteins. However, in cancer cells one or more proto-oncogenes are altered in such a way that their activity is not tightly controlled. As examples of oncogene activation of cell cycle regulators are abnormalities affecting both CDKs and their regulatory subunits, the cyclins. Deregulation of CDKs, such as CDK4 and CDK6, have been implicated in a wide range of tumors [13-19]. CDK4 is mutated in a small subset of melanoma patients in which a germline mutation, and subsequent protein dysfunction, causes hereditary melanoma [13-15]. Mutation of CDK4 blocks binding of INK4 inhibitors, thus providing a pathway to uncontrolled cell division [13-15]. In addition, abnormalities of gene expression have also been associated with deregulation of the cell cycle. Indeed, CDK2, CDK4 and CDK6 were reported to be overexpressed in several malignancies, namely sarcoma, neuroblastoma, breast cancer, lymphoma, melanoma and colorectal cancer [16-18]. Abnormalities of CDK regulators, such as cyclins, have also been associated with human neoplasia. Indeed, misregulation of Dtype cyclins is a common feature of several tumor types [19]. Cyclin D acts as a growth sensor, providing a link between mitogenic stimuli and cell cycle entry. Cyclin D1 (CCND1) binds to CDK4 and CDK6, being essential to G1 phase entry. Aberrant CCND1 expression, as a result of overexpression or amplication, has been reported in many

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human cancers. For instance, the characteristic chromosomal translocation t(11;14) (q13;q32) present in mantle cell lymphoma juxtaposes the CCND1 gene to the immunoglobulin heavy chain locus, leading to CCND1 overexpression in mantle cell lymphoma [20]. In addition, CCND1 overexpression, as a result of gene amplication, was described in parathyroid adenoma, esophageal cancer, bladder cancer, breast cancer, colon cancer, melanoma and prostate cancer [21-26]. Cyclins A, D2, D3, and E have also been reported to be overexpressed in some human tumors. Indeed, both cyclin A and E are overexpressed in lung carcinoma, and cyclin E has been found to be amplied, overexpressed or both in cases of breast and colon cancer and in acute myeloblastic or lymphoblastic leukemia [27-30].

be altered for control over cell proliferation to be lost; hence, tumor suppressor genes confer a recessive cancer phenotype. Since these genes usually exert their activity at cell cycle checkpoints, they are also known as gatekeepers, and the proteins encoded by them can prevent a cell from proceeding further through the cell cycle unless specic conditions have been met. Indeed, when tumor suppressor genes are mutated or inactivated, cells are unable to respond normally to cell-cycle checkpoints, or are unable to undergo programmed cell death if DNA damage is extensive. This can lead to a further increase in mutations and to the inability of the affected cell to leave the cell cycle when it should become quiescent and eventually cells may become tumorigenic. As examples of inactivation of tumor suppressor genes involved in cell cycle regulation are abnormalities affecting CDK inhibiThe RB1 protein is not only the target of CKIs but can also by itself act as a tumor suppressor gene. The RB1 gene was originally identied in patients with hereditary retinoblastoma, an inherited disorder in which tumors develop in the eyes of young children [37]. All somatic cells of patients with hereditary retinoblastoma contain one mutated allele of the RB1 gene, however, for retinoblastoma to develop, the second normal allele of the RB1 gene must be lost or mutated [38]. As mentioned above, the RB1 protein is involved in the regulation of the G1-S transition. When cells are in the G0 phase of the cell cycle, the RB1 protein is non-phosphorylated and binds to transcription factors such as E2F1 (also known as E2F), leading to their inactivation (Fig. 1) [39]. When the cell is stimulated to proliferate, it enters G1 and approaches S phase. Throughout the G1 phase, the RB1 protein becomes phosphorylated by the CDK4/cyclin D1 complex. Phosphorylated RB1 releases its bound regulatory proteins, such as E2F1, that can now induce the expression of over 30 genes whose products are required for the G1-S transition [39, 40]. After mitosis, RB1 reverts to a nonphosphorylated state, binds again to regulatory proteins, and keeps them sequestered until required for the next cell cycle, preventing S phase entry [39, 40]. In many tumors, including retinoblastoma, osteosarcoma, and small cell lung cancer, as well as at a lower frequency in other types of cancer, both copies of the RB1 gene are defective, inactive, or absent, and progression through the cell cycle is not regulated [40]. Another frequently mutated gene involved in the regulation of checkpoint transition is the tumor suppressor gene TP53. The TP53 protein is a sequence-specic DNA-

TUMOR SUPRESSOR GENES AND THE CELL CYCLE Since proto-oncogenic proteins are involved in the promotion of cell proliferation and survival, there is a need to counteract their activity with proteins that inhibit cell proliferation or induce cell death. The proteins that carry out these functions are coded by tumor suppressor genes that halt progress through cell cycle in response to DNA damage or growth-suppression signals from the extracellular environment. Just as gain-of-function mutations convert proto-oncogenes to oncogenes leading to cell cycle deregulation, a similar consequence occurs when tumor suppressor genes are affected by loss-of-function mutations. In opposition to gain-of-function mutations, this type of mutations can alter the structure of protein in a way that it reduces, or completely abolishes, its activity. In alternative, loss-of-function mutations can cause a decrease in the expression of a gene yielding lower levels of functional protein. Either way, loss-of-function mutations in tumor suppressor genes involved in cell cycle can result in a loss of control over cell proliferation. In this case, both alleles of a tumor suppressor gene need to

tors, or checkpoint regulators such as the RB1 and TP53 (p53) proteins. The inhibitory activity of CKIs results in growth suppression through activation of the RB1 protein, reecting the tumor suppression function of the CKIs. For instance, the CDKN2A gene is altered in a high percentage of human cancers and can be inactivated by several mechanisms, including deletion, point mutations, and epigenetic modifying through hypermethylation [31, 32]. As examples we have CDKN2A inactivation by mutation or deletion in inherited melanoma, pancreatic adenocarcinoma, and esophageal cancer [33-36]. As previously described, the CDKN2A protein, is a specic inhibitor of the CDK-cyclin D complexes, preventing phosphorylation-dependent degradation of the RB1 protein and G1-S transition. As a consequence, cells with an abnormal CDKN2A protein will be unrestrained to proceed trough G1, providing a means to unchecked cell proliferation. Another example of CKI inactivation is the loss of CDKN1B in several human cancers. Indeed, loss of CDKN1B expression has been reported for a number of human neoplasias, including breast, ovarian, and bladder cancer [32].

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CDKS AS TARGETS FOR THERAPEUTIC INTERVENTION IN CANCER Nowadays, based on the recent advances in molecular oncology and the increasing understanding of relevant pathways in tumorigenesis and cancer progression, the process of identifying new drugs for killing tumor cells has switched to a more mechanistic binding protein that is able to induce either cell cycle arrest or apoptosis at cell cycle checkpoints in damaged or transformed cells [41, 42]. Indeed, the TP53 protein is a major player in the cellular events that occur following DNA damage induced by exposure to ultraviolet (UV) radiation. Following exposure to UV radiation and DNA damage, cells increase production of TP53 leading to cell-cycle arrest, DNA repair, and, if the damage is too extensive to be corrected, apoptosis [42]. This is accomplished trough upregulation of the expression of several genes, including the CDK inhibitor CDKN1A (Fig. 1) [42]. The binding of the CDKN1A protein to a CDK inhibits the CDK ability to phosphorylate several target proteins, ultimately resulting in cell-cycle arrest, and ensuring that the cell is blocked in G1, until the damage is repaired [42]. In this way, a loss-offunction mutation in the TP53 gene would result in absence of CDKN1A protein synthesis in response to DNA damage and, as a consequence, inappropriate expression of S-phase genes. This, in turn, can lead to replication of damaged genes or chromosomes, a hallmark of cancer cells [43]. It is estimated that more than 50% of all cancers possess mutations in the TP53 gene, making it the most frequently mutated gene in human cancers [43, 44]. Indeed, the frequency of TP53 mutation varies from nearly 10% in hematopoietic malignancies to 50 70% in ovarian cancer, colorectal cancer, and head and neck tumors [45]. In addition, germline mutation of the TP53 gene causes the Li-Fraumeni syndrome, a familial cancer syndrome characterized by the development of several malignancies, including breast cancer, soft tissue sarcomas, and several other types of cancer [46]. strategy, trying to act on molecular targets that underlie cell transformation. Due to their critical role in cell cycle progression, as well as the association of their activities with the processes of differentiation and apoptosis, the CDKs comprise an attractive set of targets for novel anti-cancer drugs. Thus, the evidence that CDKs, their regulators and substrates are targets of genetic alteration in different types of human cancer has prompted the search for synthetic CDK inhibitors. Drugs targeting cell cycle regulatory proteins, such as CDKs, have been studied since the mid 90s, however, in the clinical setting CDK inhibitory molecules demonstrated modest activity and high toxicity, demonstrating somehow disappointing results [47]. The less-than-optimal preclinical optimization of rst generation compounds, combined with the not expected off-target effects, as well as the reappraisal of the role that CDKs play during the cell cycle in both normal and genetically compromised tumor cells, were responsible for the delay in the development of these molecules [47-49]. Theoretically, CDK activity can be modulated by targeting the major regulators of CDK activity (indirect strategy) or by inhibiting the catalytic activity of the CDKs (direct strategy) [50]. Approaches for the indirect strategy include overexpression of CKIs, synthesis of peptides mimicking the effects of CKIs, decrease of cyclin levels, modulation of the proteasomal machinery, modulation of the phosphorylated state of CDKs and of the enzymes responsible for that regulation [32]. Direct inhibition has been the most successful strategy for the development of potent cell cycle inhibitors. Indeed, more than 50 small-molecule compounds have been identied so far (Table 1) [48]. These compounds

compete to target the ATP binding pocket of the catalytic site of CDKs [32, 48]. An important question regarding these agents is whether promiscuous CDK inhibition is preferable to selective CDK inhibition [48]. Three different groups of CDK inhibitors have entered clinical trials so far: Group I includes compounds with a broad inhibition prole (also called pan-CDK inhibitors); Group II includes compounds that exclusively or preferentially inhibit either CDK4/CDK6 or CDK2 activity; and Group III includes those molecules inhibiting CDKs and additional kinase targets of interest in oncology [47, 49]. Some of these inhibitors are currently undergoing clinical trials, and are described in more detail in Table 1. Of these, and as an example, Alvocidib (avopiridol), a semi-synthetic avonoid derived from rohitukine (an alkaloid isolated from the stem bark of Dysoxylum binectariferum, a plant native to India) was the rst CKI to enter clinical trials, having been tested so far in more than 50 clinical trials [49, 51-53]. Preclinical data indicated that avopiridol could block the proliferation of neoplastic cells and induce programmed cell death as a single agent [49, 51-53]. Flavopiridol is the most potent known inhibitor of CDK9. CDK9 is a transcription elongation factor that regulates RNA Polymerase II (RNAPII) activity following transcriptional initiation, playing also an important role in co-transcriptional histone modication and mRNA processing events such as splicing and 3 end processing [54]. Flavopiridol blocks cell cycle progression at G1/S and G2/M boundaries and it down-regulates cyclin D1, cyclin D3, MYC and the apoptosis regulators MCL1 and XIAP [49, 51-53]. This drug also activates TP53 through the down-regulation of MDM2 thereby promoting apoptosis [48]. This compound was granted Orphan Drug status by the EMEA (European Medicines Agency) in 2007 for chronic lymphocytic leukemia (CLL), a disease where tumor cells survive on account of persistent expression of CDK9-dependent anti-apoptotic proteins. However, clinical studies in a number of other malignan-

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cies have not revealed signicant efcacy at the doses and administration schedules evaluated [47, 48]. Recent emerging data revealed that avopiridol can potentiate, in a dose- and sequence-dependent manner, the anti-tumor effects of many established chemotherapeutic agents [48, 55]. Taking into account that the results of these clinical trials are not as good as expected, the study of the therapeutic value of these

small molecule inhibitors deserves more detailed evaluation. Above all it is essential to better understand why normal and tumor cells have specic requirements for individual interphase CDKs.

GLOSSARY
Oncogene A gene with the capacity, when activated, to transform a cell, eventually leading to the development of cancer. Oncogenes are generally mutated forms of normal cellular genes (proto-oncogenes).

Table 1: Examples of CDK inhibitors undergoing clinical trials. NA - not available.

Company NCI Name Alvocidib (avopiridol) Seliciclib (R-roscovitine) SNS-032 (BMS-387032) Class of Inhibitor Group I Group I Group I II (2007) I/II (2007) Clinical phase (last reported date) III (2008) Applications Chronic lymphocytic leukemia NA Advanced breast cancer, non-small cell lung cancer, B-cell malignancies Advanced or metastatic solid tumors, refractory nonHodgkins lymphoma NA NA Advanced solid tumors Non-Hodgkins lymphoma, multiple myeloma Advanced refractory neoplasms, multiple myeloma Non-Hodgkins lymphoma NA Relapsed/refractory solid tumors Relapsed/refractory solid tumors Multiple myeloma Myelodysplastic syndrome References 49, 51-53

Cyclacel Sunesis

49, 51, 52 49, 51

Astex

AT-7519

Group I

I/IIa (2007)

49, 51, 52

Astex NMS Hoffmann-LaRoche Schering-Plough

AT-9311 PHA-793887 R-547 (Ro-4584820) SCH-727965

Group I Group I Group I Group I

NA I (2007) I (2006) I (2008)

47 49, 51, 52 49, 51 49

Nicolas Piromal

P276-00

Group II

I/II (2007)

49, 51

Pzer NMS Bayer Schering Pharma AG NMS GPC Biotech ONO Pharmaceuticals

PD-0332991 PHA-690509 ZK304709 PHA-848125 RGB-286638 ON-01910.Na

Group II Group II Group III Group III Group III Group III

I (2006) I (2008) I (2006) I (2008) I (2008) I (2008)

51 50 49, 51 49, 51, 52 49, 52 49

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