Introduction

Human malignant tumors are characterized by abnormal proliferation resulting from alterations in cell-cycle regulatory mechanisms. The regulatory pathways controlling cell-cycle phases include several oncogenes and tumor suppressor genes, which display a range of abnormalities with potential usefulness as markers of evolution or treatment response in cancer. This chapter summarizes the current knowledge about these aberrations in malignant transformation.

1.1. Cell Cycle: The Importance of its Control

Cancer is frequently considered to be a disease of the cell cycle. Alterations in different families of cell-cycle regulators cooperate in tumor development. Molecular analysis of human tumors has shown that cell-cycle regulators are frequently mutated in human neoplasms, which underscores how important the maintenance of cell cycle commitment is in the prevention of human cancer. Mammalian cell division is precisely regulated in a timely manner by a family of protein kinases, the cyclin-depend-ent kinases (CDKs), which is a group of serine/threonine kinases that form active heterodimeric complexes following binding to cyclins, their regulatory subunits. Regulation of CDK activity occurs at multiple levels, including cyclin synthesis and degradation, phosphorylation and dephosphorylation, CDK inhibitor (CKi) protein synthesis, binding and degradation, and subcellular localization. Orderly progression through the cell cycle involves coordinated activation of the CDK protein by binding to the cyclin partner. A succession of kinases (CDK4, CDK6, CDK2, and CDC2) are

From: Current Clinical Oncology: Molecular Pathology of Gynecologic Cancer Edited by: A. Giordano, A. Bovicelli, and R. Kurman © Humana Press Inc., Totowa, NJ

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Fig. 1 (Color Plate 1, following p. 50). A schematic model of the normal mammalian cell cycle. Gj to S transition in normal cells requires phosphorylation of the retinoblastoma proteins by CDKs, which causes the release of E2F transcription factors controlling various genes required for DNA synthesis during S phase. The CKIs p21WAF1 and p27KIP1 act by binding to cyclin-CDK2 complexes to inhibit their catalytic activity and induce cell cycle arrest, whereas p16INK4a inhibits CDK4/6. Wild-type p53 activates the transcription of p21 gene.

Fig. 1 (Color Plate 1, following p. 50). A schematic model of the normal mammalian cell cycle. Gj to S transition in normal cells requires phosphorylation of the retinoblastoma proteins by CDKs, which causes the release of E2F transcription factors controlling various genes required for DNA synthesis during S phase. The CKIs p21WAF1 and p27KIP1 act by binding to cyclin-CDK2 complexes to inhibit their catalytic activity and induce cell cycle arrest, whereas p16INK4a inhibits CDK4/6. Wild-type p53 activates the transcription of p21 gene.

expressed along with a succession of cyclins (D, E, A, and B) as cells go from G1 to S and then to G2 and finally, to M phase (Fig. 1; see Color Plate 1, following p. 50).

Different CDK-cyclin complexes operate during different phases of the cell cycle. Active CDK-cyclin complexes phosphorylate target substrates, including members of the "pocket protein" family (pRb, p107, and pRb2/p130) (1,2). G1/S transition in normal cells requires phosphorylation of the retinoblastoma protein pRb and the related proteins pRb2/p130 and p107 by CDKs, which causes the release of E2F transcription factors controlling various genes required for DNA synthesis and cell-cycle control.

Endogenous inhibition of CDKs is also caused by two families of regulatory proteins induced under mitogenic stimuli: (1) the INK4 family, consisting of p16INK4a, p15INK4b, p18INK4c, and p19INK4d, which specifically inhibit CDK4 and CDK6 (3) and (2) the CIP/KIP family including p21CIP1/WAF1, p27KIP1, and p57KIP2, which cause a broader range of inhibition and act in a concentration-dependent manner (4). All CKIs cause G1 arrest when overexpressed in cells by association and inhibition of the CDKs. INK4 proteins dissociate cyclin D/CDK complexes and redistribute the CIP/KIP proteins to CDK2, producing a double inhibition. At low concentrations, CIP/KIP family proteins enhance CDK4 association with cyclin D, increasing the activity of the complex. Whereas at high concentrations they inhibit kinase activity, presumably by increasing the stechiometry in the CDK complexes (5). The best-studied events of the cell cycle are the G1 phase preceding the DNA synthesis (S) phase and the mechanism that drives the cell across the restriction (R) point in late Gp which is crucial for the cell's destiny toward division, differentiation, senescence, or apoptosis. Several studies suggest that traversion of the R point within the G1 phase is the key event in cell-cycle regulation, and that the rest of cell-cycle progression occurs almost automatically once the R point has been overcome (6). Several proteins can inhibit the cell cycle in G1 phase; if DNA damage occurs, p53 accumulates in the cell and induces the p21-mediated inhibition of cyclin D/CDK. The frequent loss of G1 regulation in human cancer has revealed targets for possible therapeutic intervention. D-type cyclins are transcribed in the G1 phase of the cell cycle. The isoforms D1, D2, and D3 are functionally equivalent, and are expressed in a tissue-specific manner. CDK4 and CDK6 are activated by D cyclins to phosphorylate the retinoblastoma protein pRb, a known cell proliferation regulator. The members of the INK4 family exert their inhibitory activity by binding to the CDK4 and CDK6 kinases and preventing their association with D-type cyclins.

Genetic analysis of human tumors has revealed that some of the molecules most often altered in cancer are those involved in the control of the G1/S transition of the cell cycle, a time when cells become committed to a new round of cell division. During the G1/S transition, the cyclin E/CDK2 and cyclin D/CDK4 complexes promote progression and are each inhibited by the associated CKI p27kip1. The transition to S phase is triggered by the activation of the cyclin D/CDK complex, which phosphorylates pRb.

In contrast to G1 regulators, less is known about the genes, which regulate the S, G2, and M phases of the cell-cycle like cyclin A- and cyclin B-kinase complexes and their inhibitors. The significance of cell cycle regulatory genes in carcinogenesis is underlined by the fact that most of them have been identified as proto-oncogenes or tumor suppressor genes.

In S phase, phosphorylation of components of the DNA replication machinery by cyclin A-CDK is believed to be important for initiation of DNA replication and to restrict the initiation to only once per cell cycle. Transition from G2 to M phase involves destruction of cyclin A and ascendancy of cyclin B. The protein phosphatase CDC25 removes inhibitory phosphates from CDK1/cyclin B complexes. During the normal cell cycle, negative regulation by phosphorylation of cyclin B/CDC2 prevents premature mitotic entry before the completion of S phase.

1.2. Alterations in the Cell Cycle Leading to Malignant Transformation

The knowledge about the molecular mechanisms required for tumor formation has greatly increased during the last 40 years. Key molecular mechanisms required for malignant transformation have been identified. Tumor growth is a dynamic process in which it is difficult to identify a unique event that caused the process. It is well-established that numerous events together contributed to the acquisition of the malignant phenotype. It is commonly accepted that a thorough understanding of the molecular mechanisms that lead to uncontrolled proliferation of cancer cells will allow the identification of targets that can be therapeutically manipulated to arrest or kill tumor cells. For many years, considerable effort has been made to understand the machinery that controls normal cell cycles, thereby aiding the identification of molecules or processes altered in tumor cell cycles. Alterations in the machinery that controls the decision to progress from a resting state into the cell cycle (the so-called G0/G1 transition) or to progress from G1 into S phase are found in virtually all tumor cells.

1.3. Alterations of the Gj to S Regulatory Machinery in Cancer

Cyclin E-CDK2 has long been considered an essential and master regulator of progression through G1 phase of the cell cycle. Cyclin E-CDK2 activity is the highest in Gj/S cells and the lowest in quiescent cells (7-9). This periodicity results from many factors including transcriptional and post-transcriptional control of cyclin E abundance, the binding of CIP/KIP CKIs (10), and modification of CDK2 activity by inhibitory and activating phosphorylations. These multiple layers of control ensure that cyclin E activity is tightly regulated during normal cell cycles. In contrast, cyclin E-CDK2 is often deregulated in cancer cells, and this likely contributes to the development of cancer. Many cancers overexpress cyclin E protein or mRNA including carcinomas (breast, lung, cervix, endometrium, gastrointestinal tract), lymphoma, leukemia, sarcomas, and adrenocortical tumors (11-20). Several mechanisms deregulate cyclin E expression in tumors. A large number of oncogenes function within the mitogenic signal transduction pathways that regulate the pRb pathway, and oncogenic mutations within these pathways may increase cyclin E abundance through increased E2F activity. The most common means of activating cyclin E expression in cancers might thus involve mutations in regulatory pathways, rather than within cyclin E itself.

The improper formation of cyclin D1 complexes with CDK4/6 or other aberrant hyperactivation of these complexes could act equivalently to pRb loss to render a cell insensitive to a need for mitogenic signaling. Such aberrant CDK activation or loss of pRb has obvious implications for cancer cell generation and, indeed, pRb loss or hyper-activation of CDK4 and/or CDK6 is found in most human tumor cells. Hyperactivation of CDK4 and CDK6 can be achieved through deregulated expression of D-type cyclins, loss of p16INK4a or other members of the INK4 family more commonly involved in differentiation or transforming growth factor-signaling (21), or mutation-based insensitiv-ity to the inhibitory effects of p16INK4a. Hence, every element of the core pRb pathway (p16INK4a, D-type cyclins, CDK4/6, and pRb itself) represents a potential oncogene or a tumor suppressor.

Molecular analysis of human cancers strongly support this notion. For instance, amplification or rearrangement of the cyclin D1 gene located on chromosome 11q13 as well as overexpression of cyclin D1 protein has been described in a wide spectrum of human cancers, such as squamous cell carcinomas of head and neck, esophagus, tongue and larynx, carcinomas of uterine cervix, astrocytomas, nonsmall-cell lung cancers, soft-tissue sarcomas, and others (22-27). The best-documented of these alterations is a frequent involvement of cyclin D1 in pathogenesis of human breast cancer. Thus, approx 15-20% of human mammary carcinomas contain amplification of the cyclin D1 gene (28-30), whereas cyclin D1 protein is overexpressed in more than 50% of human breast cancers (31-35). Cyclin D1 overexpression is seen at the earliest stages of breast cancer progression, such as ductal carcinoma in situ, but not in premalignant lesions (such as atypical ductal hyperplasia). Hence, overexpression of cyclin D1 can serve as a marker of malignant transformation of mammary epithelial cells (36). Once cyclin D1 overexpression is acquired by the tumor cells, it is maintained at the same level throughout breast cancer progression from ductal carcinoma in situ to invasive carcinoma and is preserved even in metastatic lesions (31,35).

1.3.3. Cyclin D2 and Cyclin D3

Cyclin D2 and D3 genes are also amplified and the encoded proteins are overex-pressed in many human cancers. Cyclin D2 is involved in B-cell lymphocytic leukemias and lymphoplasmacytic lymphomas (37), chronic lymphocytic leukemias (38) as well as in testicular and ovarian germ cell tumors. Cyclin D3 overexpression has been found in glioblastomas, renal cell carcinomas, pancreatic adenocarcinomas, and several B-cell malignancies, such as diffuse large B-cell lymphomas or multiple myelomas (39-43).

Similarly, overexpression of CDK4 is found (often as consequence of gene amplification) in breast cancers (44), in gliomas, glioblastomas multiforme, sarcomas, and urinary bladder cancers (45-49). Moreover, in several human malignancies, the kinase activity of CDK4 is hyperactivated because of the loss, mutation, or silencing of the gene encoding the CDK4 inhibitor, p16INK4a (50-54). Yet, another set of tumors, including retinoblastoma, osteosarcoma, small-cell lung carcinoma, and bladder carcinoma, is associated with the loss of the pRb protein (55). Cyclin A is a particularly interesting member of the cyclin family because it can activate two different CDKs and functions in both S phase and mitosis. In mitosis, the precise role of cyclin A is still obscure, but it might contribute to the control of cyclin B stability. Consistent with its role as a key cell-cycle regulator, expression of cyclin A is found to be elevated in a variety of tumors.

1.3.5. Alterations of the CKIs

CKIs are negative regulators of the cell cycle. Thus, perturbation in their activity results in severe disregulation of cell proliferation and failure to suppress tumor growth

(56). The INK4 CKIs are lost through mutation, deletion, and/or promoter methylation in a variety of human neoplasms and in this sense are true tumor suppressor genes (21). On the contrary, the CIP/KIP CKI p27kip1 does not fit the classic tumor suppressor paradigm in humans, as mutations in the p27kip1 gene in human tumors are extremely rare

(57). However, p27kip1 has been defined "tumor suppressor protein" because inactiva-tion of its function has been implicated in the development of human tumor (58).

Two different mechanisms have been implicated in p27kip1 inactivation during the process of human carcinogenesis: downregulation of its expression and exclusion from the nuclear compartment. A drastic reduction in the level of p27kip1 protein (or even a complete loss) is observed in approx 50% of all types of human cancer (59). Reduced p27kip1 expression has been associated with the development of human epithelial tumors originating from the majority of human organs, including lung (60), breast (61), colon (62), ovary (63), esophagus (64), thyroid (65), and prostate (66). Loss of p27kip1 expression is detected also in a subset of malignancies originated from the central nervous system (67) and from the lymphoid tissue (68).

In most human tumors the loss of p27kip1 protein results from altered proteasome-mediated degradation (62). Fast, specific, and timely proteolysis of cell-cycle regulators by the ubiquitin-proteasome system represents an important mechanism, which ensures proper progression through the cell division in a unidirectional and irreversible manner (69). A finding that is crucial for its clinical implications is that low or absent p27kip1 expression represents an important marker of disease progression in a number of tumor types (60,63,66).

Cytoplasmic sequestration of p27kip1 in tumors has been identified only recently as a mechanism, whereby cancer cells promote cancerogenesis in humans. Displacement of p27kip1 into the cytoplasm has been shown to contribute to the anchorage-independent growth of human transformed fibroblasts. It is performed by maintaining high cyclin-CDK activity in the nucleus (70), and the increased proliferation associated with the loss of the tuberous sclerosis complex-2 gene product (tuberin), a GTPase-activating protein for Rapla and Rab5 GTPases (71).

1.4. p53 Pathway in Cancer

Cells contain numerous pathways designed to protect them from the genomic instability or toxicity that can result when their DNA is damaged. The p53 tumor suppressor is particularly important for regulating passage through G1 phase of the cell cycle, whereas other checkpoint regulators are important for arrest in S and G2 phase. The phase of the cell cycle in which the cells arrest depends on their p53 status. Cells with wild-type p53 arrest predominantly in the G1 phase, whereas cells with mutant p53 fail to arrest in G1, but rather accumulate in the S and G2 phases. Once repair is complete, cells might recover, proliferate, and divide. Premature progression through the cell cycle can be lethal.

Wild-type p53 can prevent abrogation of arrest by elevating levels of p21waf1 and by decreasing levels of cyclins A and B. p21waf1 regulates cyclin E/CDK2 and cyclin A/CDK2 complexes, both of which phosphorylate pRb. Thus, it contributes to the transition into the S phase and cell-cycle progression, even in the absence of growth signals.

The accumulation of p21waf1 followed by inhibition of cyclin E/CDK2 and cyclin A/CDK2 complexes blocks the progression from G1 to S phase (72). Moreover, p21waf1 is also involved in the apoptotic process by increasing the phosphorylation and inactiva-tion of pRb. During tumorigenesis, tumor cells frequently lose checkpoint controls, which causes the development of the tumor. However, these defects also represent an Achilles heel that can be targeted to improve current therapeutic strategies. Virtually, all human tumors deregulate either pRb or p53 pathways, and often both simultaneously. The importance of these pathways in cellular growth control is underscored by the observation that members of these pathways are found mutated in all human cancers. For example, many studies have pointed out the aberrant expression and prognostic significance of individual proteins in either the pRb (particularly cyclin D1, p16INK4a, and pRb) or the p53 (p53 and p21waf1) pathways in nonsmall-cell lung cancer (73).

1.5. pRb Pathway in Cancer

The protein product of the retinoblastoma gene, pRb, and the related p107 and p130 proteins regulate transitions between cell proliferation and terminal differentiation. A common relevant biological activity shared by the three members of this family is the ability to negatively control the cell cycle (74-77). In fact, they negatively modulate the transition between the G1 and S phases, using mechanisms mostly related to inactivation of transcription factors, such as those of the E2F family, that promote the cell entrance into the S phase. pRb, p107, and p130 all bind to E2F, a transcription factor that regulates the expression of numerous genes needed for cell-cycle entry and DNA synthesis (78). pRb associates with each member of the E2F family, except E2F5 and E2F6, whereas p107 binds E2F4 exclusively, and p130 binds both E2F4 and E2F5 (79), switching from E2F5 in G0 to E2F4 complexes as the cell re-enters the G1 phase (80,81). Complex formation between E2F and pRb families is cell-cycle-dependent: CDKs phos-phorylate the pRb family in late Gj, liberating free E2F (82,83). The three members are active in complexes at different time of the cell cycle: pRb2/p130 is primarily active in arrested G0 or differentiated cells (84), active pRb is found in quiescent and differentiated cells as well as in mid to late G1, and p107 complexes are the most abundant in cycling cells, in G1/S and S phase complexes (81). The full-length pRB protein contains 16 consensus CDK-phosphorylation sites. Phosphorylation at specific sites inhibits the binding of pRB to cellular proteins, thereby disrupting the antiproliferative activity of RB (85,86). Therefore, overexpression of proteins which causes excessive or deregulated phosphorylation of pRB is a common event in human tumors (10,87,88). The pRb-, p107-, and pRb2/p130-E2F complexes can each be disrupted by viral oncoproteins (E1A, SV40, and E7), resulting in the deregulation of E2F transcriptional activity (89,90).

p107 is mostly predominant during the late G1 phase through G2/M and its expression is strictly regulated by its E2F dependent promoter. In contrast to p107, lack of pRb2/p130 expression has been observed in several different tumor types supporting its bonafide tumor suppressor function. During the last 10 years, a large number of studies have examined the diagnostic and prognostic significance of pRb expression in various tumors. Almost all studies report decreased pRb expression in a broad spectrum of tumors. pRb is lower in more aggressive myelogenous leukemia (91-93), as well as in nonsmall-cell lung carcinoma (94,95), in papillary thyroid carcinoma (96), in bladder (97), prostate (98,99) and ovarian (100) carcinomas, malignant astrocytoma (101), non-Hodgkin's lymphoma (102), and other types of cancer. The loss or decreased expression of pRb2/p130 was found in lung carcinomas (95,103), endometrial cancer (104-106), choroidal melanoma (107), non-Hodgkin lymphoma (108), vulvar cancer (109), prostatic (110) and ovarian carcinomas (111).

1.6. Alterations of the G2 to M Regulatory Machinery in Cancer

The G2/M checkpoint prevents cells from initiating mitosis when they experience DNA damage during G2, or when they progress into G2 with some unrepaired damage inflicted during previous S or G1 phases (112,113). The accumulation of cells in G2 might also reflect a contribution of the so-called DNA-replication checkpoint that may detect some of the persistent DNA lesions from the previous S phase as being inappropriately or not fully replicated DNA.

The critical target of the G2/M checkpoint is the mitosis-promoting activity of the cyclin B/CDK1 kinase. Its activation after various stresses is inhibited by ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR), Checkpoint kinase CHK1/ CHK2, and/or p38-kinase-mediated subcellular sequestration, degradation, and/or inhibition of the CDC25 family of phosphatases that normally activate CDK1 at the G2/M boundary (113-115). In addition, other upstream regulators of CDC25C and/or cyclin B/CDK1, such as the Polo-like kinases PLK3 and PLK1 seem to be targeted by DNA-damage-induced mechanisms (113).

The maintenance phase of the G2/M checkpoint probably partly depends on the tran-scriptional programs regulated by BRCA1 and p53, leading to the upregulation of cell-cycle inhibitors such as the CKI p21waf1, GADD45a (growth arrest and DNA-damage-inducible 45-a), and 14-3-3 sigma proteins (113,116). The fact that even tumors defective in other checkpoints, such as those with mutant p53, tend to selectively accumulate in G2 after DNA damage, indicates that p53-independent mechanisms are sufficient to sustain the G2/M arrest. At the same time, this phenomenon has inspired efforts to interfere with the G2/M checkpoint as a potential strategy to sensitize cancer cells, which are deficient in their G/S checkpoint pathways, to radiation- or drug-induced DNA damage (117).

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