Cell Cycle and the Induction of Cancer

Classically, neoplastic diseases have been defined as proliferative disorders characterized by unregulated cell growth and proliferation.18 The multitude of regulatory proteins and their specificity of action upon distinct phases of the cell cycle make their genes highly plausible candidates as targets for mutagenic agents that induce neoplastic disease. Each step in the cell cycle is characterized by the activation or inactivation of various proteins and protein complexes. Thus, mutation events that result in loss of function or abrogation of gene expression of CKIs, cyclins, and pRB could result in unregulated cellular proliferation and cancer formation.

Fig. 4.3. Schematic model for the action of p53 and MTS1 (p16) in cell cycle control. (A) the mutational inactivation of p53 would result in loss of p21 transcription and activation resulting in loss of normal control in cell cycle progression checkpoints. (B) The mutational inactivation or loss of homology of MTS1 gene would result in p16 protein inactivation. Conceptually, this event would result in continued cyclin D-Cdk4 activity with maintenance of pRb phosphoryla-tion and E2F activation resulting in loss of G1/S cell cycle checkpoint. These mutational events are thought to play a major role in GI cancer formation with inappropriate entry of cells into the phases of DNA synthesis and cell division. Reproduced with permission, Surgical Clinics of North America 1995; 75:862, ©W.B. Saunders Company.

Fig. 4.3. Schematic model for the action of p53 and MTS1 (p16) in cell cycle control. (A) the mutational inactivation of p53 would result in loss of p21 transcription and activation resulting in loss of normal control in cell cycle progression checkpoints. (B) The mutational inactivation or loss of homology of MTS1 gene would result in p16 protein inactivation. Conceptually, this event would result in continued cyclin D-Cdk4 activity with maintenance of pRb phosphoryla-tion and E2F activation resulting in loss of G1/S cell cycle checkpoint. These mutational events are thought to play a major role in GI cancer formation with inappropriate entry of cells into the phases of DNA synthesis and cell division. Reproduced with permission, Surgical Clinics of North America 1995; 75:862, ©W.B. Saunders Company.

The research on tumorigenesis has led to the discovery of proto-oncogenes, which are homologs of normal growth regulatory genes. Proto-oncogenes are activated to oncogenes resulting in uncontrolled cell proliferation, and are thought to be one factor contributing to tumor progression. The activation events include tumor-specific clonal DNA abnormalities such as gene amplification, gene rearrangement, or point mutation, which result in deregulation of proto-oncogene expression and/or the alteration of mRNA which results in altered protein structure. Certain human cyclin genes have been associated with various clonal genetic aberrations associated with neoplastic disease, which suggests their involvement in tumorigenesis. Cyclins compose a family of proteins responsible for the regulation of Cdk activity and phase-specific cell cycle progression, and have been targets of mutagenic processes. These events lead to abnormal levels of cyclins, which are required for cell cycle progression, and result in uncontrollable proliferation.5,19 Cyclin D is one of the most frequent cyclin abnormalities found in cancers, and is a good model to describe the cyclin role in tumorigenesis.19 The cyclin D1 gene has been localized to the long arm of chromosome 11, at the q13 band. This location has been associated with the t(11;14) translocations which are involved in hemoproliferative malignancies.19-21 Therefore, the 11q13 locus was termed BCL1 because of its proto-oncogene characteristics and involvement in tumor-associated translocation events.22 The cyclin D1 gene is located 110 kb downstream from the BCL1 breakpoint, indicating that cyclin D1 is a target gene for this translocation event.22 This oncogenic event is the result of uncontrollable activation of the cyclin D1 gene and subsequent overexpression of mRNA and normal protein, indicating these mutations are in the regulatory regions of the gene. Cyclin D1 regulates the G! phase of cell cycle control, and therefore, overexpression may prevent cells from exiting the cell cycle for differentiation.23 The most frequent abnormality of cyclin D1 is DNA amplification.19 The amplification factors or driver of the 11q13 amplicon gene are believed to be related to CCND1 genes, and include fibroblast growth factor 3 (FGF3)-int2 and FGF4-hst1 signaling pathways.24 These genes were first identified in gastric cancer, but have subsequently been found in a variety of human malignancies.19 Gene amplification has been shown to be a major factor in cyclin D1 overexpression in head and neck tumors and esophageal cancers.25,26 Although translocation amplification is an attractive mechanism for cyclin D1 overexpression, there is evidence that other factors may be involved according to tissue type. In many tumors the 11q13 translocation amplicon is a rare event or has never been detected despite the aberrant accumulation of cyclin D1 in over 40% of the cases.27 Thus, overexpression may be a consequence of mutations that regulate mRNA turnover. Whether amplification, or post-tran-scriptional/translational modifications occur, these events result in the stable overexpression of cyclin D1 and the loss of cell cycle control with the acquisition of tumor cell characteristics. These findings strongly implicate the level of expression of the cyclin D1 gene and its products as major factors in the induction of some cancers through cell cycle aberrations.

The counterpart to the oncogene is the tumor suppressor gene, which normally confers negative control on cellular proliferation. Negative control of cell cycle progression plays an important role in embryonic tissue development, differentiation, senescence, and cell death. Therefore, the role of CKI proteins on the progression of the cell cycle is an attractive target for the study of regulatory processes in tumorigenesis. In many cases, cell cycle arrest results from DNA damaging agents, many of which are potent carcinogens. Failure to arrest proliferation would result in propagation of highly unstable genomes, which is an early event and primary characteristic of cancer cells. The protein p21Waf1 is one of several negative regulatory proteins involved in the inactivation of Cdks and cyclin/Cdk complexes,2,6,11 which are the regulatory kinase components that target pRB for phosphorylation. Although the incidence of p21 gene mutations appears to be a rare event,5,28 the mechanism for its inactivation in most tumors could be the result of changes in its protein pool levels. This is based on the observation that mutations of p53, which is a transactivating factor that regulates the transcription of p21, are prevalent events in human cancer, and could have a negative effect on p21 expression (Fig. 4.3A).29 Interestingly, the induction of p21 has also been shown to occur in a p53-independent pathway.30 The role of this pathway in human cancer production is still subject to experimental study. The CKI proteins p15, p16, and p18 can complex with both Cdk4 and Cdk6, inhibiting their catalytic regulation of pRB phosphorylation (Fig. 4.3B).6,19,29 Therefore, loss of p15, p16, or p18 function through mutation events would cause an increase in G1 cyclin/Cdk activity that would lead to phos-phorylation and inactivation of pRB with uncontrolled cell cycle progression and cell proliferation. Both p15 and p16 genes are located on the short arm of chromosome 9 (9p21), while the p18 gene is located on chromosome 1p32. These genes are located in regions involved in translocations and mutational deletions in various human cancers,5,29 which suggests a causal role of these inhibitory proteins in cancer induction.

The cell cycle regulatory protein pRB is a prototype tumor suppressor, which functions to regulate DNA binding proteins and transcription factors important for progression through the cell cycle. In the hypophosphorylated state, pRB molecules can interact with viral proteins such as SV40 large T antigen, the adenovirus E1A protein, and the papillomavirus E7 protein inactivating pRB function, which renders the cell immortal.5 The deregulation of cell proliferation by inactivated pRB is compensated by p53 function, another tumor suppressor gene.31 Therefore, the loss of pRB and p53 function would result in a continuous high level of E2F activity resulting in unregulated cell proliferation and tumor formation.31,32 Furthermore, since the tumor virus proteins that inactivate pRB can also inactivate p53, these proteins appear to be components of a common pathway for tumorigenesis in certain tissue neoplasms. Although the mechanisms of these tumor suppressor genes have been elucidated using cells in culture, these findings are supported by experiments utilizing RB and/or p53 knockout murine models, which clearly demonstrate their biological effects. Although homozygous RB mutations result in death in utero,33,34 p53-homozygous and p53- or pRB-heterozygous mice which are viable, develop a high incidence of a variety of tumors. The additive effect of p53 and RB mutations has been investigated, and reveals an increased tumor burden and metastasis.35

Colorectal Cancer

The expression of cyclin D1 protein was examined in normal colonic mucosa and primary and metastatic lesions.36 Normal tissue and 56% of primary tumors revealed weak expression of cyclin D1 gene products, whereas 23% and 21% of primary carcinomas expressed moderate to strong overexpression of cyclin D1, respectively. Moreover, the metastatic lesions examined exhibited immunohistochemical staining characteristics similar to those of the primary lesion. Furthermore, in cell lines overexpressing cyclin D1, the antibody-mediated knockout of activity demonstrated a positive regulatory role of the cyclin D1 protein whose function was required for progression through the G1 phase of the cell cycle.36 These data suggest that cyclin D1 is overexpressed in a subset of colorectal tumors, but it is not clear whether this is a result of abnormalities of gene amplification. Although this study does not directly address the oncogenic role of the cyclin D1 gene product in the induction of colorectal cancers, the knowledge of cyclin D gene amplification and overexpression in other tumors supports a mechanism in colorectal cancer induction.19 Many mechanisms of cyclin D overexpression have been described. There is also documentation that there is an abundance of tumor types that lack any significant frequency of 11q13 amplification but also exhibit cyclin D1 overexpression. These data imply that the expression of this cell-cycle regulatory proto-oncogene which plays a significant role in human oncogenesis has multiple levels of regulation. Since cyclin D is involved in the inactivation of the growth-restraining function of wild type pRB through the cyclin D-kinase associated catalytic unit, this has been proposed as one step in the multistep process of colorectal tumorigenesis.37 Interestingly, some colorectal carcinomas exhibit concurrent increased expression of cyclin E and Cdk2 genes,38 which may act through pRB regulation to induce uncontrolled proliferation in a similar manner to that of cyclin D1.

Tumor suppressor genes exert a negative effect on cell cycle control and proliferation, which includes CKI proteins. Although prevalent in many cancers, mutations in p16 and other INK family Cdk inhibitors have not been found in colon cancer.39 However, loss of p16 transcription by de novo methylation has been found in >92% of colon cancer cell lines.40

The development of null mutation-gene knockout technology has provided rodent transgenic models and a novel approach for the investigation in tumorigenesis. The induction of human colon cancer is achieved through a progression of mutations that encompass the progression of change from normal mucosa to adenomas and then carcinomas. Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary condition linked to the inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene, which results in numerous adenomatous polyps in the colon that progress to colorectal carcinoma at a high rate.41,42 The murine APC knockout model of multiple intestinal neoplasm, (Min)/+, is a good model to study the molecular mechanisms associated with the progression of adenomatous disease to colon cancer, because its progression is similar to that seen in the human FAP disease.43 Recent investigation using this in vivo model system has revealed that cyclin D1 and Cdk4 exhibit concurrent overexpression in the intestinal adenomas which was accompanied by an increase in cell proliferation indicated by 5-bromo-2'-deoxyuridine incorporation.23 These investigators compared their rodent model observations with human tissue and found a similar cyclin D1 and Cdk4 overexpression in the majority of intestinal adenomas from FAP patients. These results indicate that overexpression of G1 cell cycle proteins is an early event in the progression of premalignant disease to cancers of the colon. Further studies using this model have shown an inverse correlation of TGF-PII receptor expression to cyclin D1 and Cdk4 expression at the transcriptional and translational levels.44 Since TGF-P functions to inhibit proliferation through the induction of various CK1 protein expressions, the loss of this signal transduction pathway may be one of the mechanisms involved in the upregulation of these positive G1 cell regulators. This event subsequently leads to the failure of adenomatous cells to exit the cell cycle23 as should normally occur during the process of intestinal cell differentiation.

Recently, other Cdks have been associated with colorectal adenomas and carcinomas. In a study of 50 colonic adenomas, and 15 adenomas with focal carcinoma, overexpression of Cdk2 and cdc2 (Cdk1) were found in 28% and 87%, respectively.45 Although loss or deletion of RB gene has been demonstrated in several human malignancies,46,47 this event is rare in carcinomas of the colon and rectum. However, since pRB is inactivated through phosphorylation,16,48 the mechanism of action in rendering these cells into a state of unregulated proliferation lies in the regulatory role of these overexpressed Cdks in the phosphorylation and inactivation of pRB.49

Cancers of the Esophagus and Stomach

Several genetic abnormalities involving cell cycle regulators have been implicated in the induction and progression of esophageal squamous cell carcinoma, and includes amplification of the chromosome 11q13 region associated with the hst-1, int-2 and cyclin D genes.19 Esophageal cancers exhibit an amplification of cyclin D expression in 34% of combined series investigations.19 Studies have concluded that amplification of cyclin D1 in esophageal cancer closely correlated to tumor staging, depth of tumor invasion, distant metastasis, and indicates a reduced overall survival.19,50 Therefore, overexpression of cyclin D gene is a good biological marker of high malignancy for esophageal carcinoma. As indicated in colorectal carcinomas, the role of cyclin D in regulation of G1 phase progression makes it likely that its overexpression could lead to uncontrollable cell growth and proliferation of these tumor cells. Transforming growth factor-a (TGF-a) and epidermal growth factor (EGF) are autocrine growth factors synthesized and released by esophageal cancer cells.51 Since cyclin D acts as a growth factor sensor to promote cell proliferation, it has been hypothesized that overexpression of EGF/TGF-a by tumor cells could lead to cyclin D amplification in esophageal tumors.

Mutations in the multiple tumor suppressor (MTS1) gene, which encodes the Cdk4 inhibitor p16, are observed in approximately 50% of esophageal squamous cell cancer.52 In a review of the literature, p16 was mutated in about 30% of esophageal cancers.19 In addition to p16, it has been shown that p15 has both point mutations and homozygous deletions in primary esophageal tumors and tumor-derived cell lines.53 The inhibitory role of p15 and p16 in G1 cyclin/Cdk complexes may be another pathway involved in the overexpression of cyclin D, and inactivation of pRB resulting in accelerated cell proliferation (Fig. 4.3B) seen in esophageal cancers.

Finally, loss of heterozygosity of various tumor suppressor genes has been found in esophageal cancers and includes the p53, RB, and MTS1 genes.50,53 Point mutations in p53

have been found in primary squamous cell carcinoma of the esophagus54 as well as Barrett's associated adenocarcinoma and adjacent dysplasia or metaplasia.55 Series studies have shown a high incidence of p53 mutations in both squamous and adenocarcinoma of the esophagus.50,53 Similar p53 mutations have been found in premalignant and early cancers of the esophagus, which lead to the conclusion that p53 mutation is an early event in esophageal cancer induction.50,53,55 There also appears to be a unique mutation profile of p53 in esophageal cancer that consists primarily of nonsense mutations.55 However, p53 mutation was found to confer no prognostic significance on patient survival in esophageal cancer.56

Other mutations include the loss of homology of RB gene on chromosome 13q, and the MTS1 or p16 gene on chromosome 9p.52,55 Aberrant transcripts of RB and DNA amplification of cyclin D have been noted in esophageal cancer.50,53 The role of pRB is to sequester and release transcriptional factors for DNA replication and progression through Gi/S cell cycle transition. Cyclin D regulates its Cdk4 partner to phosphorylate pRB which releases these transcription factors. The CKI protein, p16, blocks the cyclin D/Cdk4 complex to prevent pRB phosphorylation and cell cycle progression (Fig. 4.3B). Therefore, one can see how the mutations of pRB and p16, as well as the amplification of cyclin D, are involved in promoting uncontrolled cellular proliferation in esophageal cancers.

Gastric cancers express a broad spectrum of growth factors that act as autocrine and paracrine growth regulators. Overexpression of EGF/TGF-a and EGF receptors contributes to the biologic malignancy of gastric cancers. TGF-P is often overexpressed in poorly differentiated adenocarcinoma and scirrhous carcinomas.57 Growth factors regulate cell proliferation through their signaling pathway interactions with cell cycle regulators. As in esophageal cancers, increased EGF/TNF-a and EGF receptors are linked to an increase in cyclin D expression. Aberrant CKI expression has been implicated in gastric cancers, and involves p21. Activation of wild type p53 by genetic damage is known to induce p21 expression at the transcriptional level, whereas mutant p53 loses this ability (Fig. 4.3A). It has been observed that gastric cancer cell lines expressing the mutant p53 gene product are associated with low to undetectable levels of p21 mRNA.50,57,58 Since p21 is known to be an inhibitor of G1-Cdk/cyclin complexes, it is not surprising that increased levels of cyclin E is frequently associated with gastric cancers.50 These findings suggest that mutated p53 is not capable of inducing p21 expression, leading to the subsequent overexpression of Cdk2 and G1 cyclins, which are responsible for the deregulated growth characteristics of gastric cancer cells with mutant p53. Gastric cancers with cyclin E amplification and overexpression are associated with a more aggressive characteristic with a higher incidence of lymph node metastasis.59

Allele loss and mutation of the p53 gene is detected in >60% of gastric cancers regardless of histological type.57,58 A good correlation has been found between the nature of p53 gene mutation and histological atypia of gastric adenomas. Nonsense mutations or frame shift mutations that affect the structure of the gene product results in a high-grade atypia, whereas silent mutations have a low-grade atypia.60

Pancreatic Cancer

As with other tumors, the induction of pancreatic cancer is facilitated through the activation of proto-oncogenes and the inactivation of tumor suppressor genes. In pancreatic cancer, there is an increased incidence of mutations in the tumor suppressor genes p53 and MTS1.29,61 As described above, p53 is a nuclear phosphoprotein, which binds to specific DNA sequences to activate gene transcription and induce cell cycle arrest or apoptosis. Mutations resulting in changes of the p53 amino acid sequence can prevent its binding to these DNA regulatory sites.29 Sequence analysis has found that 50 to 70% of pancreatic carcinomas had mutations of the p53 gene. One of the target genes for p53 activated transcription is the CKI protein p21. This negative regulator of cell cycle progression complexes with G1 phase-specific cyclin/Cdk complexes to inhibit phosphorylation of pRB, which is required for initiating DNA transcription and cell cycle progression. Mutated p53 exhibits a loss in p21 transcription ability (Fig. 4.3A), and would therefore result in the maintenance of cyclin/Cdk activity in the G1 phase with subsequent loss of pRB regulatory function of DNA transcription factors.

Pancreatic cancers were the first type of cancer found to have high frequency of p16 inactivation.62 MTS1/p16 tumor suppressor gene was found to be mutated in 38% of pancreatic carcinomas with loss of the wild type allele, and homozygously deleted in another 40% of pancreatic carcinomas.61 Furthermore, methylation associated with the silencing of p16 gene expression can be found in the majority of the remaining cases.63 The loss of inhibition of G! cyclin/Cdk activity, and thus pRB regulation, through mutation or loss of p16 function (Fig. 4.3B) is one of the possible mechanisms involved in the uncontrolled proliferation seen in pancreatic cancers. Recent evidence suggests that loss of p16 protein expression in pancreatic cancer is associated with advanced clinical stage and decreased survival rates.64

Growth factors regulate cell cycle progression by increasing cyclin/Cdk activity. Recent studies have shown that cyclin D1 mRNA and protein levels are overexpressed in pancreatic cancer tissues and cell lines when compared to normal pancreatic tissue.65 Furthermore, cyclin D1 overexpression was found to be associated with decreased survival rates (6.5 vs 15.5). This observation, coupled to the increased susceptibility of pancreatic cancers to growth factor regulation, is a proposed mechanism for loss of cell cycle control and tumorigenesis.3

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