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Modulation of Cell Cycle Components by Epigenetic and Genetic Events Marcella Macaluso,a,b,c Micaela Montanari,a,d Caterina Cinti,a,e and Antonio Giordanoa,c

Cell cycle progression is monitored by surveillance mechanisms, or cell cycle checkpoints, that ensure that initiation of a later event is coupled with the completion of an early cell cycle event. Deregulated proliferation is a characteristic feature of tumor cells. Moreover, defects in many of the molecules that regulate the cell cycle have been implicated in cancer formation and progression. Key among these are p53, the retinoblastoma protein (pRb) and its related proteins, p107 and pRb2/p130, and cdk inhibitors (p15, p16, p18, p19, p21, p27), all of which act to keep the cell cycle from progressing until all repairs to damaged DNA have been completed. The pRb (pRb/p16INK4a/cyclin D1) and p53 (p14ARF/mdm2/p53) pathways are the two main cell-cycle control pathways frequently targeted in tumorigen- esis, and the alterations occurring in each pathway depend on the tumor type. Virtually all human tumors deregulate either the pRb or p53 pathway, and oftentimes both pathways simultaneously. This review focuses on the genetic and epigenetic alterations affecting the components of mechanisms regulating the progression of the cell cycle and leading to cancer formation and progression. Semin Oncol 32:452-457 © 2005 Elsevier Inc. All rights reserved.

Cell cycle progression is monitored by surveillance mech-anisms, or cell cycle checkpoints, which ensure the in- tegrity of the genome and the fidelity of chromosome sepa- ration through ordered execution of cell cycle events. A breakdown in regulation of the cell cycle can lead to uncon- trolled growth and contribute to tumor formation. In fact, deregulated proliferation is a characteristic feature of tumor cells and mutations in the genes involved in controlling the cell cycle are extremely common in human cancer.1,2 For example, several studies have been reported defects in mol- ecules regulating the cell cycle, such as p53, cdk (cyclin-

dependent kinase) inhibitors (p15, p16, p18, p19, p21, p27) and the retinoblastoma protein (pRb) family, in cancer cells.3

Moreover, silencing of many tumor-suppressor genes (among these p15, p16, retinoblastoma [Rb], p73, von Hip- pel Landau [VHL], hMLH1, and E-cadherin) by DNA meth- ylation has been shown to occur during cancer formation and progression.4–6

The pRb (pRb/p16INK4a/cyclin D1) and p53 (p14ARF/ mdm2/p53) pathways are the two main cell-cycle control pathways (Fig 1) frequently targeted in tumorigenesis, and the alterations occurring in each pathway depend on the tumor type.7–10 The important role of these pathways in con- trolling cellular growth is underscored by the observation that components of these pathways are found mutated in all human cancers. Virtually all human tumors deregulate either the pRB or p53 pathway, and oftentimes both pathways si- multaneously.11,12 This review focuses on the genetic and epigenetic alterations affecting various components of the mechanisms regulating the progression of the cell cycle and leading to cancer formation and progression.

The pRb/p16INK4/Cyclin D1 Pathway and Cancer The pRb pathway is inactivated in most human cancers.9

Data generated in the last decade have documented the role

aSbarro Institute for Cancer Research and Molecular Medicine, Center of Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA.

bSection of Oncology, Department of Oncology, University of Palermo, Pal- ermo, Italy.

cDepartment of Human Pathology and Oncology, University of Siena, Siena, Italy.

dInstitute of General Pathology, Giovanni XXIII Cancer Research Center, Catholic University of Sacred Heart, Rome, Italy.

eInstitute of Clinical Physiology, CNR, Siena Unity, Italy. Supported by NIH grants, Sbarro Health Research Organization (www.

shro.org), and AIRC to A.G. M.M. is in part supported by a Fondazione Italiana per la Ricerca sul Cancro (FIRC) fellowship.

Address correspondence to Antonio Giordano, MD, PhD, Sbarro Institute for Cancer Research and Molecular Medicine, Center of Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA 19122. E-mail: giordano@temple.edu

452 0093-7754/05/$-see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1053/j.seminoncol.2005.07.009

of pRB pathway, and its family members p107 and pRb/ p130, in regulating the progression through the G1 phase of the mammalian cell cycle.13,14 In addition to pRb family pro- teins, key components of this pathway include the G1 cyc- lins, the cyclin-dependent kinases (CDKs), and the cyclin- dependent kinase inhibitors (p15INK4B, p16INK4A, p18INK4C, and p19INK4D).13 Mutations and deletions of the Rb gene have been reported in several human tumors, and inherited allelic loss of Rb confers increased susceptibility to cancer forma- tion.11 Nevertheless, the Rb-related Rb2/p130 gene plays a pivotal role in the negative control of the cell cycle and in tumor progression as well.15

Precisely how pRb family members control cell prolifera- tion is not completely understood. However, various data have indicated that pRb family proteins interact with a wide variety of transcription factors and chromatin-modifying en- zymes.16–20 Nevertheless, the binding of pRb family proteins with the E2F family of transcription factors appears to be central in governing cell cycle progression and DNA replica- tion by controlling the expression of cell cycle E2F-depen- dent genes.14 These genes include CCNE1 (cyclin E1), CCNA2 (cyclin A2), and CDC25A, which are all essential for the entry into the S phase of the cell cycle, and genes that are involved in the regulation of DNA replication, such as CDC6,

DHFR, and TK1 (thymidine kinase).21,22 In addition, recent data have suggested an interesting role of the pRb pathway in regulating hematopoietic homeostasis.23 Moreover, alter- ation of the pRb pathway has been reported in transitional cell carcinomas of the urinary bladder and in human endo- metrial cancer.24,25

The INK4a/ARF locus (9p21) encodes two unique and unre- lated proteins, p16INK4a and p14ARF, which function as tumor suppressors by modulating the responses to hyperprolifera- tive signals.26 p16INK4a is one of the cell cycle–regulatory pro- teins involved in tumor suppression in the pRb pathway, and loss-of-function alterations in p16INK4A occur frequently in human cancers.27,28 Several studies have indicated that p16INK4A can control pRb activity and it also seems to be under pRb regulatory control itself.29–31 p16INK4a blocks cell cycle progression by binding Cdk4/6 and inhibiting the ac- tion of D-type cyclins. Moreover, p16INK4a blocks the G1- to S-phase progression of the cell cycle by promoting the inhi- bition of pRb phosphorylation and the formation of a pRb/ E2F-repressive complex.32,33 It has been reported that both cyclin D1 overexpression and p16INK4a protein alteration pro- duce persistent hyperphosphorylation of pRb, resulting in evasion of cell cycle arrest.34

Small homozygous deletions are the major mechanism of

Figure 1 Combinatorial signaling network between pRb and p53 pathways in controlling cell cycle progression. Cell cycle progression is tightly monitored by surveillance mechanisms that ensure that initiation of a later event is coupled with the completion of an early cell cycle event. The pRb (pRb/p16INK4a/cyclin D1) and p53 (p14ARF/mdm/p53) pathways are the two main cell-cycle control pathways. The disruption of pRb/p16INK4/cyclin D1 and the p14ARF/ mdm2/p53 pathways appear to be a common part of the life history of human cancers, independent of age or tumor type.

Modulation of cell cycle components 453

p16INK4a inactivation in various primary tumors such as glial tumors and mesotheliomas, while mutations are not com- monly reported.35–38 Chromosome 9p21 is frequently de- leted in malignant melanoma, and the presence of p16INK4a

point mutations has been demonstrated in familial melanoma and melanoma cell lines in vitro.39,40 Moreover, aberrant methylation of p16INK4a has been shown in a wide variety of human tumors such as tumors of the lung, breast, bladder, head and neck, colon, and esophagus.41–43 Inactivation of the p16INK4a gene through promoter hypermethylation has been frequently observed in non-small cell lung cancer and may constitute a new biomarker for early diagnosis of this dis- ease.44 For example, it has been reported that in patients with early stage non-small cell lung cancer, p16INK4a promoter methylation is a predictor of the patient’s clinical outcome and is significantly related to an unfavorable prognosis.45–48

Moreover, p53 and Rb genes and their pathways involving the G1- to S-phase transition are commonly affected genes in lung cancer.49,50 Although the inactivation of p16INK4a seems to be a crucial event in the development of several human tumors, the relevance of this alteration in mammary carcino- genesis remains unclear.28,51,52 For example, homozygous deletions of p16INK4a are seen in 40% to 60% of breast cancer cell lines, while both homozygous deletions and point muta- tions are not frequently observed in primary breast carci- noma, suggesting that these alterations might have been ac- quired in culture.53–55 Moreover, promoter hyper- methylation of p16INK4a has been reported in breast carci- noma, although the relevance of this p16INK4a alteration is discordant among different studies.56,57 In fact, although dif- ferent studies have indicated that a strong p16INK4a expres- sion is associated with negative prognostic parameters, one study did not show any evident correlation between the ab- errant expression of p16INK4a and histopathologic parame- ters.58–61 Moreover, although methylation of p16INK4a pro- moter is common in cancer cells, it recently has been reported that epithelial cells from histologically normal mammary tissue of a significant fraction of healthy women, present p16 promoter methylation as well.62

Deregulated tumor expression of p16INK4a has been de- scribed in association with clinical progression in sporadic colorectal cancer (CRC) patients. Furthermore, hypermeth- ylation of p16INK4a promoter has been shown to occur in advanced colorectal tumors and has been associated with patient survival.63–65 Increasing evidence indicates that per- turbation of cyclins is one of the major factors leading to cancer initiation and progression. Convincing results indi- cate that a combination of cyclin/cdks, and not a single ki- nase, executes pRb phosphorylation and that each one of these complexes phosphorylates specific pRb-phosphoryla- tion sites.66

Recently, it has been reported that the activation of the mitogen-activated protein kinase (MAPK) leads to pRb inac- tivation by sustaining cyclin levels and consequently activat- ing CDKs.67 Constitutive cell surface kinase receptors and persistent phosphorylation/inactivation of the pRb family proteins (pRb, p107, and pRb2/p130) have been implicated in conferring uncontrolled growth to melanoma cells.68

Moreover, overexpression of cyclin D1 has been found in several neoplasms, including breast carcinoma, endocrine pancreatic tumors, multiple myeloma, mantle cell lym- phoma, colon cancer, various sarcoma.69–73 In addition, it has been shown that there is an absolute requirement for cyclin D1 overexpression in malignant transformation of breast cells that cannot be complemented by the other related cyclins D2 and D3, suggesting a putative anti–cyclin D1 ther- apy highly specific for breast cancer.74,75

The mechanisms disturbing the pRb pathway converge to reach a common goal: uncontrolled expression of regulators that trigger an irreversible transition into the S phase and cell cycle progression, even in the absence of growth signals. Moreover, the pRb pathway is not strictly linear, so that over- expression of cyclin D1 not only accelerates the pRb-E2F program but also leads to p27kip1 sequestration.76,77

It is important to underscore that alterations affecting the components of pRb pathway occur in a mutually exclusive fashion, in that one alteration is unaccompanied by others. Moreover, the frequency of particular genetic and epigenetic events varies among tumor types.

The p14ARF/mdm2/p53 Pathway and Cancer p53 is a key regulator of cell cycle checkpoints and mutations in this gene occur in more than 50% of human cancers.78 p53 can be defined as either a gatekeeper or caretaker tumor suppressor. In fact, as an inducer of cell cycle arrest and apoptosis, it may be considered as a gatekeeper, and as a “guardian of the genome” that preserves the genomic integ- rity it appears to act as a caretaker.79 Moreover, due to the fact that p53 is the most frequently mutated gene in human can- cer, it appears to be a crucial target for therapy with respect to tumor formation and elimination of the tumor cells.80

The p14ARF/mdm2/p53 pathway appears to play a major role in mediating oncogene-induced apoptosis.81 Conse- quently, the suppression of apoptosis by inactivation of the p14ARF/mdm2/p53 pathway appears to play an important role in tumor development.82 The check and balance that exists between the pRb and p53 pathways involves the regu- lation of the G1 to S transition and its checkpoints. Part of this network consists of an array of autoregulatory feedback loops, where pRb and p53 signals exhibit very intricate in- teractions with other proteins known to play important roles in the determination of cell fate.9 p53 is activated in response to ultraviolet irradiation, DNA damage, cellular stress, and the turnover of this short-lived protein is regulated by ubiq- uitination through mdm2 binding, leading to degradation by proteosomes and thereby limiting p53 accumulation.83

Moreover, p53 activates mdm2 transcription, ensuring a neg- ative feedback regulation.83,84 The human p14ARF protein is known to arrest the cell cycle in G1 and G2 phases and acts in the same p53-pathway. p14ARF interferes with all the known functions of mdm2 and it has been shown that p14ARF binds the mdm2-p53 complex, resulting in a stabilization of both proteins.85,86 Significantly, p14ARF expression is positively

454 M. Macaluso et al

regulated by members of the E2F family of transcription factors.87This provides a link between the pRb family mem- bers and p53 pathways, suggesting a mechanism whereby the inactivation of pRb proteins leads to E2Fs release, p53 stabi- lization, and functional activation.88 Furthermore, p53 acti- vates the transcription of p21Cip/Kip, which is largely respon- sible for the p53-dependent G1 arrest in response to cellular stress and DNA damage.84 p21Cip/Kip regulates cyclin E/Cdk2 and cyclin A/Cdk2 complexes, both of which phosphorylate pRb, thus contributing to an irreversible transition into the S phase and cell cycle progression, even in the absence of growth signals. The accumulation of p21Cip/Kip followed by inhibition of cyclin E/Cdk 2 and cyclin A/Cdk2 complexes blocks the progression from G1 into S phase.11,89

In addition, it has been recently shown that p53 modulates radiation sensitivity in the G1 phase of the cell cycle through mechanisms independent of p53-mediated transcriptional activation of p21 and cell cycle arrest.90 Moreover, it has been suggested that p21(WAF1) is also involved in the execution of apoptosis by increasing the phosphorylation and inactivation of pRb.91 The p14ARF protein induces both G1 and G2 phase arrest in a p53-dependent manner.55 Deletion inactivation of p14ARF has been reported in human cancers, but in those studies p16INK4a was always codeleted (p14ARF and p16INK4a

genes are both encoded by the INK4a/ARF locus at chromo- somal region 9p21).92–94 Only germline deletion of p14ARF- specific exon 1b in a family characterized by multiple mela- noma and neural cell tumors has been reported.95

Moreover, recent studies have reported that epigenetic al- terations such as CpG hypermethylation may be the first cause of the genetic silencing of p14ARF, followed by p14ARF

loss of heterozygosity (LOH) and homozygous deletions. Hy- permethylation of p14ARF has been detected in primary colo- rectal, gastric, breast, and lung cancers.27,96,97

Conclusion Virtually, all human tumors deregulate either pRb or p53 pathways, and often both pathways simultaneously. The im- portance of these pathways in cellular growth control is un- derscored by the observation that members of these path- ways are found mutated in all human cancers. In addition, the importance of pRb and p53 in preventing tumor forma- tion was confirmed by mouse knockout studies, which showed that mouse embryo fibroblasts derived from p53!/!, p19!/!, or pRb/p107/p130!/! animals could be transformed by the activated Ras oncogene alone.98 The disruption of pRb/p16INK4/cyclin D1 and the p14ARF/mdm2/ p53 pathways appears to be a common part of the life history of human cancers, independent of age or tumor type. For example, many studies have highlighted the aberrant expres- sion and prognostic significance of individual proteins in either the Rb (particularly cyclin D1, p16INK4A, and pRb) or the p53 (p53 and p21Waf1) pathways in non-small cell lung cancer.99

Moreover, it has been suggested that in human fibroblasts, suppression of both the p53 and pRb pathways is necessary to bypass replicative senescence as well as senescence in-

duced by ectopic expression of a dominant-negative form of the telomere repeat binding factor 2 (TRF2).100 Furthermore, it has been reported that among the cell cycle proteins, the p16INK4a/pRb and ARF/mdm2/p53 cell cycle arrest pathways play a prominent role in glial transformation.101 Many other studies have revealed the molecular and genetic interaction between the pRb and p53 pathways.102–104

Understanding the complex molecular mechanisms that regulate cell cycle progression and are involved in tumor formation and progression still remains the most important goal in cancer research. Indeed, an increased knowledge of the alterations in pRb and p53 pathways will be useful to design effective anticancer treatments. Moreover, a better knowledge of the epigenetic mechanisms affecting key regu- lators of cell cycle could add a new point of view to our understanding of tumorigenesis in a variety of human can- cers.

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