Cell Cycle and Cancer - Unit I IV/I

CELL CYCLE AND CANCER

Cancer is in part a disease of uncontrolled proliferation. Because the proliferation of cells within an organism is normally tightly controlled by redundant regulatory pathways, it is not surprising that cell-cycle and checkpoint genes are often found misregulated or mutated in cancer. Genes in which mutations give rise to a gain of function or an enhanced level of function, leading to malignancy, are referred to as protooncogenes. Protooncogenes usually encode growth- or division-promoting proteins. Genes that give rise to loss of function mutations that lead to malignancy are referred to as tumor suppressor genes. Tumor suppressor genes usually encode negative regulators of growth and proliferation that protect cells from malignancy. Some cell-cycle genes commonly mutated or misregulated in cancer. Whereas mutations that create oncogenes tend to be dominant, mutations in tumor suppressor genes are usually recessive. This has led to the two-hit model of carcinogenesis. Briefly, recessive mutations occur in tumor suppressor genes but are latent due to the persistence of a wild-type allele. The tumor suppressor phenotype, therefore, requires mutation or loss of the second allele, a process known as loss of heterozygosity. A number of genes encoding negative regulators of the cell cycle conform to this two-hit paradigm.

In theory, to achieve uncontrolled cell division, two basic requirements must be met. First, cells need a strong constitutive proliferation signal capable of overriding the environmental and internal restraints on division that normal cells experience. Second, the barrier of senescence needs to be dismantled to render tumor cells immortal. Mutations in a large variety of cell-cycle control and related genes are associated with malignancy, and most of these can be accommodated within this framework. This model of tumorigenesis has been confirmed in rodent tissue culturebased in vitro models. Transfection of primary rodent fibroblasts with individual plasmids programmed to express proteins that promote either growth or immortalization does not result in malignant transformation. However, cotransfection of two plasmids, one in each category, does promote transformation. However, these results need to be interpreted cautiously in the context of human cancer because immortalization of rodent cells in

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culture most likely does not involve telomeres. One idea that has emerged is that strong growth signals and other environmental pressures exerted on premalignant cells produce potent stress responses, leading to cell-cycle blockade or cell death. Therefore, genetic alterations are likely required to neutralize these stress responses to immortalize rodent cells. Transformation of human cells requires these same genetic alterations, but also telomere attrition must be reckoned with, requiring additional mutations.

ALTERATIONS PROLIFERATION

IN

PATHWAYS

AFFECTING

GROWTH

AND

Mutations that regulate cell growth and proliferation can occur at many levels, ranging from cell surface receptormediated signaling pathways that control proliferation to elements of the core cell-cycle machinery itself.

Growth and Proliferation Signaling Pathways Because a large number of receptors and pathways can influence cell proliferation, many mutations in elements of these pathways have been recovered in human malignancies. Only a few examples are cited here. One way to provide a strong constitutive proliferation signal is to overexpress or deregulate growth factor receptors. HER2/neu, a transmembrane tyrosine kinase receptor found on many epithelial cell types, is often overexpressed due to gene amplification in breast and other cancers. Presumably, in such tumors the amplitude of proliferation signaling is abnormally high or completely deregulated. Similarly, signaling elements downstream of mitogen receptors can be mutated to produce constitutive signaling. Perhaps the best-known example is the case of the Ras guanosine triphosphatases, which serve as signal transducers for a number of key proliferation pathways. Dominant mutations in Ras isoforms that stabilize the activated state confer strong constitutive proliferation signaling. One of the pathways stimulated by Ras is the PI3-kinase pathway. A PI3 phosphatase, PTEN, normally reverses this phosphorylation, keeping the signal in check. However, mutational loss of PTEN similarly to oncogenic mutations in Ras can lead to constitutive signaling contributing to carcinogenesis.

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Cell-Cycle Machinery Signaling pathways that stimulate proliferation impinge on the cell-cycle machinery by stimulating the biosynthesis of D-type cyclins and promoting the degradation of cdk inhibitors. Accumulation of D-type cyclins and concomitant activation of cdk4 and cdk6 have been shown to activate the cell-cycle program primarily by phosphorylation and inactivation of the retinoblastoma protein, pRb, and related proteins p107 and p130. These proteins form potent repression complexes with transcription factors that are critical for S-phase entry and progression, notably the E2F family, effectively blocking cell-cycle progression. In addition, INK4 family inhibitors specifically down-regulate cdk4 and cdk6, buffering their capacity to phosphorylate pRb and related proteins. Virtually all components of this pathway have been found to be misregulated or mutated in cancer to provide a constitutive proliferation signal. The genes encoding D-type cyclins are found amplified in a broad spectrum of tumors. On the other hand, the gene encoding the INK4 inhibitor, p16, is mutated and lost in some types of cancer, whereas cdk4 has been found to be mutated so as not to bind p16. In many instances p16, although not genetically altered, is down-regulated at the epigenetic level. The p16 promoter contains a CpG island that is subject to repression via methylation. Finally, pRb is the tumor suppressor on which the so-called two-hit hypothesis was originally formulated. Inherited mutations in the RB gene and subsequent loss of heterozygosity invariably lead to childhood retinoblastoma and eventually other malignancies. However, somatic mutation of RB1 and loss of heterozygosity are found in many sporadic noninherited cancers.

Like D-type cyclins, cyclin E is frequently found up-regulated in cancer. The fact that deregulated expression of cyclin E can drive cells prematurely into S phase suggests that cyclin E provides a growth/division stimulus during carcinogenesis. Furthermore, cells from cyclin E nullizygous mice are resistant to malignant transformation in tissue culture models.12 However, other evidence suggests that deregulation of cyclin E may promote carcinogenesis principally by inducing genomic instability rather than by promoting growth (see Mutations Causing Genetic and Genomic Instability, later in this chapter).

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Likewise, the cdk2 inhibitor p27Kip1 is often found down-regulated in cancer, although never behaving as a classic tumor suppressor inactivated through mutation and allelic loss. However, as with cyclin E deregulation, it is not clear whether low p27 levels have an impact on carcinogenesis by promoting growth or genomic instability.

ALTERATIONS IN PATHWAYS AFFECTING SENESCENCE In addition to a constitutive growth stimulus that overrides natural restraints, tumors need to have the capacity for unlimited proliferation. Normally, the limited life span of somatic cells imposed by the process of clonal senescence constitutes a natural barrier to tumorigenesis. Therefore, genes that mediate senescence are commonly mutated in cancer. However, the issue of senescence is complicated by functional overlap between senescence pathway genes and stress pathway genes that also require inactivation. Because senescence is a result of checkpoint responses to acute telomere attrition, genes that encode DNA checkpoint signaling elements and transducers are targeted. One of the most commonly mutated genes in human cancer encodes the checkpoint effector p53. Inherited mutations in TP53, the gene encoding p53, confer a syndrome known as LiFraumeni characterized by early-onset cancer. However, the majority of sporadic cancers are also mutated at the p53 locus. The role of p53 mutation in cancer as a promoter of immortalization is supported by the finding that cells from p53 nullizygous mice are immortal. However, this conclusion is complicated by the fact that p53 is central to cellular stress responses that also require inactivation during malignant transformation. Nevertheless, an observation supporting the idea that checkpoint genes likely to be triggered by telomere attrition are targeted to immortalize premalignant cells is that chk2, a signaling element of the DNA damage checkpoint response, is mutated in a subset of Li-Fraumeni patients rather than p53. Mutation of the gene encoding Nbs1, required for activation of chk1 and chk2 kinases, is also associated with a hereditary cancer syndrome, Nijmegen disease, as well as sporadic cancers, although the interpretation of this result is complicated by the fact that Nbs1 is also involved in DNA damage repair (see Mutations Causing Genetic and Genomic Instability, later in this chapter). However, the most direct strategy to bypass senescence is to induce directly the expression of telomerase in somatic cells. c-Myc, a transcription factor linked to stimulation of

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proliferation, has also been shown to be a positive regulator of the gene encoding telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. This readily explains the high frequency of human tumors exhibiting c-Myc amplification or overexpression, or both. However, there appear to be a number of different mutational targets that can lead to derepression of the hTERT gene.

MUTATIONS NEUTRALIZING STRESS RESPONSES As stated in Alterations in Pathways Affecting Senescence, cellular stress responses are intimately related to checkpoint responses. Therefore, it is difficult to clearly categorize mutations that affect both. An example is p53, which is required for DNA damage checkpoint responses but also is a key effector of cellular stress responses. Murine double-minute gene-2, which is frequently amplified and overexpressed in human cancer, promotes turnover of p53, consistent with a role in neutralizing stress responses.49 Conversely, p14Arf, a protein that stabilizes p53 by antagonizing murine double-minute gene-2, is frequently found mutated or underexpressed in cancer.49 Indeed, the p53 pathway is so frequently inactivated in human cancer most likely because loss of p53 function simultaneously antagonizes stress pathways and helps override cellular responses to telomere attrition.

MUTATIONS CAUSING GENETIC AND GENOMIC INSTABILITY The pathway to malignancy minimally requires several mutations. In the case of tumor suppressor mutations, secondary genetic events mediating allelic loss are necessary. Therefore, any mutation that itself can confer genetic or genomic instability, or both, is likely to promote carcinogenesis. Mutations in genes required for DNA repair result in a mutator phenotype linked to hyperaccumulation of secondary mutations. In this context, strong association between mutation of the gene encoding Nbs1, which is required for efficient DNA repair as well as checkpoint signaling, and carcinogenesis is easily understood. Similarly, the association between mutation of components of the spindle integrity checkpoint, such as Bub1, and carcinogenesis can be rationalized. Cells defective in this checkpoint experience deregulated mitosis, leading to chromosome instability and ultimately aneuploidy. In principle, aneuploidy potentiates amplification at

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oncogenic loci and allelic losses at tumor suppressor loci. An interesting link between the core cell-cycle machinery and genomic instability is the case of cyclin E. Cyclin E is found overexpressed and deregulated in a broad spectrum of malignancies. Although this correlation might be interpreted in the context of simply promoting proliferation, experiments on cells in culture have revealed that deregulation of cyclin E expression causes chromosome instability and aneuploidy. Interestingly, an essential component of the ubiquitin ligase responsible for cell cycledependent targeting of cyclin E for proteolysis, hcdc4, is often found mutated in cancer. Thus, genetic alterations that interfere with proper regulation of cell-cycle machinery have the potential of affecting not only the cell cycle itself, but also the genetic and genomic integrity of the cell.

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