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Cell Cycle Checkpoint Genes and Cancer

  1. Jens Oliver Funk1,2

Published Online: 27 JAN 2006

DOI: 10.1038/npg.els.0006046

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How to Cite

Funk, J. O. 2006. Cell Cycle Checkpoint Genes and Cancer. eLS. .

Author Information

  1. 1

    University of Erlangen-Nuremberg, Erlangen, Germany

  2. 2

    Oncology Research Darmstadt, Global Preclinical R&D, Merck KGaA, Darmstadt, Germany

Publication History

  1. Published Online: 27 JAN 2006

Introduction

  1. Top of page
  2. Introduction
  3. Principles of Cell Cycle Regulation
  4. Cell Cycle Dysregulation and Cancer
  5. Conclusions
  6. See also
  7. References
  8. Further Reading
  9. Web Links

Control of the cell division cycle is central for governing when the cell should commit to deoxyribonucleic acid (DNA) synthesis and proliferation versus growth arrest, DNA repair or apoptosis. Consequently, the pathways regulating the cell cycle incorporate both oncogenes and tumor suppressors and are frequently dysregulated in human cancers. These alterations commonly lead to increased genetic instability. Unraveling the underlying regulatory signaling networks provides insight into the balance of normal and cancerous cell proliferation and is central for the design of novel anticancer strategies.

Principles of Cell Cycle Regulation

  1. Top of page
  2. Introduction
  3. Principles of Cell Cycle Regulation
  4. Cell Cycle Dysregulation and Cancer
  5. Conclusions
  6. See also
  7. References
  8. Further Reading
  9. Web Links

‘Decisions’ to replicate the DNA and proliferate, arrest at distinct phases of the cell cycle or differentiate are based on multiple internal and external stimuli. It is instrumental for cells that these processes are tightly regulated and well balanced. These processes are orchestrated within the basal cell division cycle. The cell cycle consists of distinct phases. When cells leave a state of quiescence (G0), they enter a first gap phase (G1) before they commit to DNA synthesis (S phase). Many signaling pathways feed into the cell cycle machinery in G1. Also, during this phase, all prerequisites for proper S-phase progression are being checked. Subsequently, a second gap phase (G2) follows, before cells enter mitosis (M), the actual cell division phase (Figure 1).

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Figure 1. Simplified model of the mammalian cell cycle. Different cyclin-dependent kinase (CDK)/cyclin complexes are the key regulators of the cell cycle phases and are involved in the checkpoint mechanisms at the transition of one cell cycle phase to the next. Two core signaling pathways of the G1 phase are indicated. The CKI and tumor suppressor p16 inhibits CDK4 and -6 which contribute to retinoblastoma protein (RB) inactivation by phosphorylation, so that E2F transcription factors are released and transactivate S-phase genes. Secondly, the CKI p21, for example, induced by the p53 tumor suppressor, inhibits many CDK/cyclin complexes, thereby inhibiting the G1–S transition.

On the molecular level, the cell cycle is governed by the temporally and spatially fluctuating activities of protein complexes. The core of each complex comprises a cyclin, the essential regulatory subunit, and a cyclin-dependent kinase (CDK), the catalytic subunit (Figure 1). The activity of CDK/cyclin complexes is regulated by a complex network of ordered events including induction and degradation of the cyclin subunit, activating and inhibitory phosphorylation of the CDK subunit, spatial distribution within different cellular compartments and binding of CDK inhibitors (CKIs) to the CDK/cyclin complex (Morgan, 1995). The phosphorylation events of, as yet largely unidentified, substrates regulate the length of each phase of the cell cycle and the transition into the next phase. These events are end points of the signaling pathways involved in the cell cycle checkpoints and tumor suppression.

The concept of cell cycle checkpoints

Cell cycle checkpoints exist at the G1–S and the G2–M transitions as well as in S phase and mitosis. Checkpoints are complex signal transduction pathways that serve as control positions to regulate the order of events in the cell cycle and integrate cell cycle progression with DNA repair (Hartwell and Weinert, 1989). By arresting the cell cycle, checkpoints presumably allow cells to repair DNA. Conceptually, checkpoints can be seen as a network of surveillance systems that interrupt cell cycle progression when damage to the genome or failure of a previous activity in the cell cycle is detected. Some cell types may preferentially undergo programmed cell death (apoptosis), avoiding the risk of generating genetically altered progeny.

In order to signal cell cycle arrest, for example after DNA damage, checkpoint control pathways must sense the damage and then transduce the signal. To delay cell cycle progression after DNA damage, these mechanisms affect the activity of critical cell cycle regulators. The integrity of these checkpoints is therefore considered pivotal in maintaining genetic stability (see below). Mutations in the checkpoint components may lead to aberrant cell cycle progression in the presence of perturbing stimuli, including DNA damage, and subsequently to genetic instability (Hartwell, 1992).

Mammalian cells commit to cell division during mid-G1, termed the restriction point, following phosphorylation of the retinoblastoma protein (RB) (Weinberg, 1995). RB becomes inactivated by phosphorylation and releases the transcription factor E2F (Figure 1). Free E2F transactivates numerous S-phase genes, including those for cyclins D, E and A, prior to the progression of cells into S phase. At least two CDK/cyclin complexes phosphorylate RB, CDK4 or -6/cyclin D and CDK2/cyclin E. While RB appears to be an exclusive target of cyclin D-associated kinases, cyclin E probably targets additional factors necessary for cell cycle progression (Resnitzky and Reed, 1995).

Two pathways contribute to entry into S phase in an independent though partially linked fashion. The release of E2F after phosphorylation of RB by CDK/cyclin complexes leads to entry into S phase. Consistent with the notion that one prime task of G1/S CDKs is to provide active E2F, the overexpression of E2F may be sufficient to induce S phase under certain conditions. However, cyclin E-associated kinase activity constitutes a second prerequisite signal for entry into S phase. Overexpression of cyclin E may lead to progression through at least one cell cycle in the absence of E2F activity and overcome a G1 arrest (Lukas et al., 1997).

CDK inhibitors and checkpoint components

The inhibition of CDK activities by CKIs constitutes a powerful mechanism for cell cycle control and provides an important link to other signaling pathways during proliferation, differentiation and senescence (Harper and Elledge, 1996). Two main families of CKIs have been identified (Figure 1), whose members share structural and functional homologies (Table 1). The CIP/KIP family members p21CIP1, p27KIP1 and p57KIP2 share broad specificity for binding to and inhibition of most CDK/cyclin complexes through conserved motifs for CDK and cyclin binding. The CIP/KIP CKIs can also function as adaptors to promote CDK/cyclin complex assembly, thereby acting as positive cell cycle regulators.

Table 1. Cyclin-dependent kinase (CDK) inhibitors and cancer
ProteinChromosomeFunctionRole in human cancerMouse knockout models
p21CIP16p21Binds to and inhibits multiple CDK/cyclin complexes and proliferating cell nuclear antigen (PCNA) to block G1 and S phase; induced by p53Rare mutations in prostate, bladder and breast carcinomasNo spontaneous tumors, defective DNA damage G1–S checkpoint; no tumor suppressor
p27KIP112p13Binds to and inhibits multiple CDK/cyclin complexes to induce G1 arrestLoss of heterozygosity not uncommon; variable loss of protein expression in many malignanciesGigantism, organomegaly, pituitary hyperplasia/adenoma; haploinsufficient tumor suppressor
p57KIP211p15.5Binds to and inhibits multiple CDK/cyclin complexes to induce G1 arrest; imprintedFew inactivations identified; mutations found in patients with Beckwith–Wiedemann syndromeNeonatal lethality, developmental defects, adrenal hyperplasia; no spontaneous tumors
p16INK4a9p21Binds to and inhibits CDK4/6 to induce G1 arrestFrequently inactivated in cancers, especially melanoma, pancreatic adenocarcinomas, lung and bladder carcinomasLow incidence of spontaneous tumors, carcinogen-induced increase in melanomas; cooperative effects with haploinsufficient p14ARF status
p14ARF9p21Blocks MDM2 inhibition of p53, thereby inducing G1 and G2 arrestFew exclusive deletions identified in melanoma cell lines, gliomas; targeted in acute T-cell leukemiaHigh incidence of spontaneous and induced tumors; p16INK4a−/− p19ARF−/− show very similar phenotype

P21CIP1 is predominantly regulated by transcriptional activation of p53 and functions as a key regulator in the DNA damage-induced G1 arrest. Interestingly, evidence from various p21CIP1−/− cell models not only confirmed the requirement of p21CIP1 for an intact G1–S checkpoint but also pointed to additional functions of p21CIP1 in the positive regulation of G2–M, thereby preventing uncoordinated timing of cell division (Waldman et al., 1996). p27KIP1 is mostly regulated in various DNA damage-independent pathways, for example those for growth factor signaling. Thus, p27KIP1 functions rather as an intrinsic G1 regulator, which is underscored by the gross phenotype of the p27KIP1−/− mice (Table 1). Importantly, p27KIP1 is a haploinsufficient tumor suppressor (see below) (Fero et al., 1998).

The second family (INK4) of CKIs comprises four members, p16INK4a, p15INK4b, p18INK4c and p19INK4d, which are all specific for CDK4 and -6 kinases and thus have a role in early G1. In the context of tumorigenesis, the focus here shall be on p16INK4a and the CDKN2A tumor suppressor locus. The p16INK4a gene cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) (CDKN2A) is biologically unique as it encodes two structurally and functionally different proteins from two independent promoters in overlapping reading frames, p16INK4a and p14ARF (Chin et al., 1998). The expression pattern of p16INK4a and p14ARF in tissues is different. Both are bona fide tumor suppressors, but the detailed analysis of the individual contribution of each of these cell cycle inhibitors remains a matter of debate (Table 1).

P16INK4a is the key representative of the INK4 family of CKIs. In accordance with its function upstream of the RB pathway it is one of the most commonly inactivated targets in human cancers (see below) (Serrano et al., 1996). In contrast, p14ARF leads to cell cycle arrest in both the G1 and G2 phases, which is not based on direct inhibition of CDKs (Pomerantz et al., 1998). Instead, p14ARF complexes with MDM2 and thereby neutralizes the MDM2 inhibition of p53, which subsequently leads via p53 induction either to transcriptional activation of cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and G1 arrest or to apoptosis, depending on the cellular context (Weber et al., 2002). These p14ARF effects appear to depend on intact p53. Furthermore, levels of p14ARF and p53 proteins appear to be inversely correlated.

Cell cycle pathways of tumor suppression

Collectively, available data provide conclusive evidence for the ordering of p16INK4a, CDK4/cyclin D and RB in a G1 regulatory pathway (Figure 1), with one function of providing active E2F to stimulate S phase. The other prominent, only partially overlapping G1 pathway is governed by p53. The p53 tumor suppressor protein has been shown to be a key regulator in interpreting the extrinsic signals that induce a cell cycle response. Following exposure to a variety of genotoxic stressors p53 is involved in signal transduction pathways resulting in either cell cycle arrest at several points in the cell cycle or apoptosis (Levine, 1997). Biochemically, p53 is a sequence-specific transcriptional activator and a nonspecific transcriptional repressor. Among the transcriptional targets of p53, the CKI p21CIP1 plays a key role in mediating G1 arrest. p53 also regulates the G2–M checkpoint through induction of 14-3-3σ, a protein that sequesters CDK1 in the cytoplasm. 14-3-3σ is a component of the G2–M checkpoint since its overexpression leads to G2 arrest, and cells lacking 14-3-3σ are defective in a stable G2 arrest (Chan et al., 1999). However, additional p53-independent mechanisms exist to initiate the G2–M checkpoint, for example, through posttranslational modifications of CDK1. Finally, another p53-responsive gene product, MDM2, functions in a feedback loop to promote the degradation of p53. This interaction appears to be blocked by the p14ARF protein.

A hallmark of cancer cells is that the normal balance of these processes is perturbed and that the cells are prone to genetic instability, that is, a state of instability seen at the chromosomal level, at the nucleotide level or reflected by chromosomal translocations and gene amplification. Multiple pathways are involved in the maintenance of genetic integrity, most of which feed into the central control mechanisms of the cell cycle. The inactivation of these pathways as part of a multistep process contributes significantly to the origin of tumors.

Cell Cycle Dysregulation and Cancer

  1. Top of page
  2. Introduction
  3. Principles of Cell Cycle Regulation
  4. Cell Cycle Dysregulation and Cancer
  5. Conclusions
  6. See also
  7. References
  8. Further Reading
  9. Web Links

Dysregulation of cell cycle checkpoint control may lead to independence of growth regulating signals. Common causes in cancer include either the aberrant expression of positive regulators, such as cyclins, or the loss of function of negative regulators, such as CKIs. Note that not all cell cycle inhibitors are tumor suppressors. The precise definition of a tumor suppressor needs to include evidence of loss-of-function mutations, of somatic mutations in sporadic tumors and of inactivations in hereditary tumor syndromes. More recently, the evidence of a tumor-prone phenotype of the mouse knockout model has also been named as a criterion. For both mechanisms, multiple examples have been identified in human cancers (Table 2 and Table 1). In the following, examples of these mechanisms will be discussed.

Table 2. Cell cycle regulators and cancer
ProteinChromosomeFunctionRelevance in human cancer
Cyclin A4(q25–q31)Complexed with CDK2 and 1 to regulate S phase and G2–MOverexpressed in some breast carcinoma, hepatocellular carcinoma
Cyclin B15(q13–qter)Complexed with CDK1 to regulate G2–MOverexpressed in some breast carcinoma
Cyclin D111q13Complexed with CDK4/6 to regulate early G1Overexpressed in multiple tumors, for example, breast cancer, lymphoma, parathyroid adenoma
Cyclin D212p13Complexed with CDK4/6 (in some cell types) to regulate early G1Overexpressed in some colorectal cancers
Cyclin E19q12Complexed with CDK2 to regulate late G1 and the G1–S transitionOverexpressed in multiple tumors including leukemias, carcinomas of the breast, colon, prostate
CDK110Complexed with cyclin B1 to regulate G2–MOverexpressed in some breast cancers
CDK412q13Complexed with D-type cyclins to regulate early G1Amplified in brain tumors, infrequently mutated in melanomas

Cyclins and cancer

One of the best explored examples of a cyclin contributing to cancer is cyclin D1. Rearrangements and overexpression of the cyclin D1 gene (cyclin D1 (PRAD1:parathyroid adenomatosis 1) (CCND1)) have been identified in parathyroid adenoma, mantel cell lymphoma and some squamous cell carcinomas. In up to 50% of investigated breast carcinomas, an amplification of the chromosomal region encoding cyclin D1 has been found. Cyclin D1 overexpression has also been detected in multiple other tumors and is sometimes correlated with prognosis. The oncogenic properties of cyclin D1 appear to be cell type-specific, since overexpression of cyclin D1 in a mouse model led to tumors in some tissues while it depended on the coexpression of an oncogene in other tissues.

Other cyclins also share oncogenic properties. Cyclin E overexpression occurs in some breast carcinomas and correlates well with tumor aggressiveness (Porter et al., 1997). In studies with lower numbers of other tumors, including carcinomas of the prostate, colon, pancreas and stomach, further evidence of cyclin E overexpression was found. Overexpression of other cyclins has only been found sporadically. A mechanistically interesting finding is the integration of the hepatitis B virus (HBV) genome in the cyclin A gene in HBV-induced hepatocellular carcinoma. This leads to increased expression of the fusion transcript and decreased degradation of the cyclin A protein.

Examples of a direct involvement of CDKs, through mutation or amplification, in cancer are rare. In some cell lines, overexpression of the cyclin-dependent kinase 4 (CDK4) gene was detected, which could desensitize the cells against the effects of certain growth inhibitory factors. Some melanomas carry a mutant CDK4 that cannot be bound and inhibited by p16INK4a, a scenario that likely disturbs the normal balance in G1/S and leads to loss of proper cell cycle control. Therefore, this represents a different, but functionally equivalent, mechanism of inactivating the RB pathway.

CDK inhibitors and cancer

Given the potentially critical role that CIP/KIP CKIs play in controlling cell cycle checkpoints, it is surprising that their elimination does not result in a tumorigenic phenotype in mice, nor are mutations in these CKIs frequently found in human tumors (Table 1) (Elledge et al., 1996). Perhaps the positive requirement for these CKIs under some conditions in CDK/cyclin assembly or other cell cycle-related functions in G2–M protect against inactivation. Also, other proteins that modulate CIP/KIP activity, especially proteins involved in degradation pathways, may be functionally altered in neoplasias. Alternatively, genetic alterations of the CDKN2A locus that lead to the dysregulation of both the p53 and RB pathways (see above) and their downstream targets may be a more frequent event.

Interestingly, many human tumors, especially breast cancers as well as colon, gastric and prostate tumors, show decreased levels of p27KIP1 protein, while normal tissues express high nuclear p27KIP1 protein levels. Reduced p27KIP1 expression, probably reflecting altered protein degradation, is of independent prognostic significance in many cancers. Also, loss of heterozygosity was observed in some tumors at the cyclin-dependent kinase inhibitor 1B (p27, Kip1) (CDKN1B) locus. However, to this end, the potential that this information may influence treatment decisions has not fully been obtained.

A detailed analysis of p27KIP1−/− mice treated with carcinogens showed that the latency of tumors was decreased and the mean number of several tumor types was increased. The heterozygous mice exhibited an intermediate tumor susceptibility, and the remaining Cdkn1b allele appeared unaffected in these tumors. This is in contrast to the paradigm of two-hit inactivation in tumors. Thus, CDKN1B acts as a haploinsufficient tumor suppressor gene in multiple tissues, that is, inheritance of just one CDKN1B allele resulted in a tumor-prone phenotype (Fero et al., 1998).

Selective downregulation of 14-3-3σ by promoter methylation has been shown to frequently occur in breast and hepatocellular carcinomas. Downregulation of 14-3-3σ in tumor cells may contribute to malignant transformation, while at the same time it may sensitize the cells to the effects of chemotherapeutic DNA damage, as evident from recent data showing that loss of 14-3-3σ also has a proapoptotic effect (Samuel et al., 2001).

The tumor-suppressive activity of p16INK4a is evidenced for example, by the correlation between CDKN2A deletion/mutation and transformation of cells, the functional antagonism of the CCND1 oncogene as well as an increased tumor susceptibility in cases of inherited mutated CDKN2A alleles. Current evidence suggests that CDKN2A is inactivated in about 50–60% of all malignancies, and about equally as many show tumor protein p53 (Li–Fraumeni syndrome) (TP53) inactivations. p16INK4a−/− mice, which have both p16INK4a and p14ARF transcript variants inactivated, develop tumors, primarily fibrosarcomas and lymphomas, at an early age and are very sensitive toward exposure to carcinogens (Table 1). Upon melanocyte-specific expression of activated H-ras in these mice, melanomas arise spontaneously at a young age. p16INK4a−/− mice preferentially develop melanomas.

Most mutations in the p16INK4a/p14ARF locus identified to date inactivate only p16INK4a but not p14ARF, while the common deletions appear to affect both p16INK4a and p14ARF. The p14ARF−/− mice have a very similar phenotype as the p16INK4a−/− mice, which underscores the tumor-suppressive potency of p14ARF. However, the frequency and mode of inactivation of p14ARF alone in human tumors remains controversial.

Recent evidence suggests that p14ARF is activated by aberrant oncogene signaling and is thereby part of an oncogene checkpoint (Sherr, 1998). Present models suggest that the functional connection between p14ARF and p53 explains the very infrequent finding of TP53 mutations in ras-induced melanomas of p16INK4a−/− mice or in p14ARF−/− mouse cells, which appears to mirror the findings in human melanomas. In fact, deletions of p53 and p14ARF are mutually exclusive events during the process of immortalization in mouse fibroblasts. An impairment of the p14ARF-mediated signaling to p53 would also compromise the p53-mediated effects leading to cell cycle arrest or apoptosis. Consequently, there would be no selective pressure to inactivate p53.

Conclusions

  1. Top of page
  2. Introduction
  3. Principles of Cell Cycle Regulation
  4. Cell Cycle Dysregulation and Cancer
  5. Conclusions
  6. See also
  7. References
  8. Further Reading
  9. Web Links

Loss of cell cycle checkpoint control has emerged as a frequent and pivotal cause of genetic instability. Consequently, the chance that these unstable cells will progress to cancer is increased. This notion has several important implications:

  • Since checkpoints are involved in determining the ultimate response of cells (cell cycle arrest versus apoptosis) to various perturbing stimuli, the integrity of checkpoints influences the susceptibility of cells to DNA damage. This is of relevance both to the fate of cells after accumulation of undesired DNA damage and the sensitivity of cells to desired damage during chemo- or radiotherapy.

  • Exploring the early checkpoint defects in cancerous or precancerous lesions may serve as a prognostic or, in certain tissues, as an additional diagnostic marker.

  • Known defects of pivotal checkpoint genes may help to predict treatment outcome or to design more specific therapeutic strategies. In addition, checkpoint components that are defective in certain cancer cells may be targeted during therapy to enhance the antitumor effect, for example, by preventing arrest and/or by forcing cells into apoptosis.

  • Moreover, therapeutic strategies could be considered to restore missing or dysfunctional checkpoints in order to provide additional time for DNA repair and delay the onset of cancer.

  • Finally, since some of the components that are involved in the DNA damage checkpoint are also involved in other cellular regulatory activities, for example, during senescence, differentiation or certain immunological responses, this could lead to cross-signaling into other pathways and might permit new strategies to influence related cellular functions.

References

  1. Top of page
  2. Introduction
  3. Principles of Cell Cycle Regulation
  4. Cell Cycle Dysregulation and Cancer
  5. Conclusions
  6. See also
  7. References
  8. Further Reading
  9. Web Links

Further Reading

  1. Top of page
  2. Introduction
  3. Principles of Cell Cycle Regulation
  4. Cell Cycle Dysregulation and Cancer
  5. Conclusions
  6. See also
  7. References
  8. Further Reading
  9. Web Links

Web Links

  1. Top of page
  2. Introduction
  3. Principles of Cell Cycle Regulation
  4. Cell Cycle Dysregulation and Cancer
  5. Conclusions
  6. See also
  7. References
  8. Further Reading
  9. Web Links