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Chapter 2: Mechanisms of Genomic Instability

Mechanisms of Genomic Instability: Introduction
Cancer arises from a series of genetic alterations that promote resistance to apoptosis, selfsufficiency in growth, cellular immortalization and escape from cell-cycle exit. The acquisition of these properties ultimately facilitates angiogenesis, invasion, and metastasis.1 It has been recognized for more than a century that genetic instability might represent an important pathway for the development of these disease characteristics. Von Hansemann2 identified abnormal mitotic figures in cancers, leading Boveri3to propose that genetic instability, manifest in his experiments as whole-chromosome aneuploidy, could have a causal role in tumor development. The recognition that mutation of genes involved in monitoring genomic integrity underlies inherited cancer syndromes such as hereditary nonpolyposis colon cancer (HNPCC) and familial BRCA-mutant breast cancer provides clear evidence that genomic instability due to a so-called mutator phenotype can be the starting point for tumor development.4,5 However, many important questions remain. Does genomic instability play a central role in oncogenesis in common sporadic tumors? When during tumorigenesis does genetic instability develop, and what are the dominant mechanisms in specific cancer types? Why do inherited mutations in caretaker genes such as BRCA1 and BRCA2 lead to breast and ovarian cancer when their repair function is presumed to be ubiquitous? What is the relative contribution of telomere shortening to the development of genomic instability? Finally, what is the specific role of aneuploidy in cancer development, and what are the defects that promote chromosomal instability? This chapter will outline the basic mechanisms involved in the maintenance of genomic integrity and will address these questions. One theme that has emerged from recent work in this area is that the development of genomic instability during cancer progression involves evolutionary tradeoffs.6–8 Loss of genetic stability is expected to increase the rate of growth-promoting or survival-promoting mutations that could drive tumor growth. However, genomic instability will also increase the rate of deleterious mutations that could kill cells before they develop into tumors. Understanding how these factors balance out will ultimately be the key to understanding tumor development via genome destabilization. Perhaps most importantly, understanding this balance may also have implications for cancer therapeutics. If deleterious, genome-destabilizing mutations are found in the population of developing cancer cells, these defects may provide an ―Achilles heel‖ for therapeutic attack.

Basic Defenses Against Genomic Instability
The roughly 1014 cells in the human body are continually exposed to sources of genomic injury, both spontaneous injury accompanying normal cell division and metabolism and external sources of damage. In addition to the oxidative stresses that are a by-product of cellular metabolism, cell populations that undergo constant turnover are subject to errors that may arise during the processes of DNA replication, mitosis, and telomere maintenance. Cells are also exposed to a variety of exogenous genotoxic insults. Examples include

ultraviolet and gamma irradiation and certain chemicals (as detailed in other chapters). As a result, mechanisms have evolved at a number of different levels to guard against genomic instability and prevent the propagation of cancer-promoting and/or deleterious mutations. At an organismal level, tissues are designed to prevent the accumulation of cells with sustained disruption of genomic integrity.4 For example, those cell types in constant contact with the outside world, including the skin, gastrointestinal tract, and bronchial epithelium, undergo continuous self-renewal, with shedding of those differentiated cells that are exposed most directly to a potentially deleterious environment. In addition to being shielded from this stress, the stem cell compartment undergoes cell division fairly infrequently, with the bulk of exponential growth occurring in transit-amplifying cells that are ultimately discarded at the surface. Thus, in tissues such as the colon, stem cells are normally protected within the crypts, and those cells that proliferate and migrate toward the lumen are ultimately eliminated. Nevertheless, this process is imperfect and subject to persistence of dysplastic clones if a cell sustains a mutation that affords a proliferative advantage. Many cells also possess physiologic characteristics that can shield them from genotoxic injury.4,9 For example, melanin in the skin absorbs ultraviolet radiation, while antioxidants and enzymes such as catalase and superoxide dismutase reduce concentrations of reactive oxygen species generated as a result of cell metabolism. In addition, cytochrome P-450 enzymes detoxify a variety of chemicals, and glutathione-S-transferases (GSTs) conjugate glutathione with electrophilic compounds, neutralizing their mutagenic potential. Defective GST function has been observed in lung, breast, and prostate cancer and has been shown to predispose patients to myelodysplastic syndrome. Conversely, drugs that inhibit GST function are being tested in combination with chemotherapy in an attempt to enhance toxicity to cancer cells.

Barriers to Genomic Instability
Cell Cycle Checkpoints
Coordinated progression through the cell cycle is crucial for the maintenance of genome stability.4,10,11This is particularly the case for the main tasks of the cell cycle—DNA replication and mitosis. Either incomplete DNA replication or overreplication of DNA would generate lesions that could lead to chromosome breaks and rearrangements. Mitotic errors produce chromosome mis-segregation and whole-chromosome aneuploidy. These types of errors do not occur in isolation; a defect in one process can lead to a cascade of downstream events. Chromosome breaks can lead to translocations, chromosomes with two centromeres (dicentric chromosomes), anaphase bridges, and chromosome mis-segregation. Likewise, mitotic errors leading to aneuploidy will generate gene expression imbalances that could, in principle, compromise DNA replication, telomere maintenance, or DNA repair. Both DNA replication/repair and mitotic errors can cause cytokinesis to fail, resulting in tetraploid cells that contain extra centrosomes and are themselves genetically unstable. Although an extensive review of the cell cycle is beyond the scope of this chapter, selected features of the normal cell cycle that are crucial for preventing genome instability and cancer are described here. In particular, the following sections will focus on the restriction point, the DNA damage

checkpoint, and the spindle assembly checkpoint. More extensive summaries of the eucaryotic cell cycle can be found in other chapters and in recent reviews.

Restriction Point
The decision to commit to cell division is controlled by a complex signaling system, the retinoblastoma protein (RB) pathway, that is the major target of human cancer-causing mutations.11,12 RB represses the transcription of genes involved in cell cycle progression by binding to the E2F family of transcription factors and altering the expression of E2F target genes, blocking E2F-mediated transactivation and recruiting active repressor complexes to promoters.13,14 E2F target genes include components of the nucleotide synthesis and DNA replication machinery that are essential for S phase entry and transit. In response to mitogenic signals during G1 phase of the cell cycle, RB is phosphorylated and inactivated by cyclin D/CDK (cyclin-dependent kinase) 4/6 complexes, followed by cyclin E/CDK2 and cyclinA/CDK2 complexes, resulting in E2F-target gene expression and S phase transit. Cyclin E/CDK2 regulates a number of other processes involved in the duplication of chromosomes, including the activation of histone gene transcription, as well as promoting the initiation of DNA replication and centrosome duplication.15,16 Deletion of cyclin E in mice results in defective endoreduplication, while constitutive overexpression of cyclin E has been linked to the generation of polyploidy and chromosomal instability. Thus, the RB pathway integrates intrinsic and external growth signals and is a key mediator of cyclin/CDK complexes that drive cell cycle progression. CDK inhibitors and phosphatases provide other important mechanisms for counteracting the activity of CDKs and restricting cell cycle progression.4,11,16 CDK inhibitors fall into two general categories, including specific inhibitors of CDK4 such as p16INK4A, and those that target CDK activity more broadly such as p21 or p27. CDK2 is also targeted by inhibitory phosphorylation at its active site by the Wee1 family of protein kinases. Activation of cyclin E(A)/CDK2 during the normal G1/S phase transition thus requires the activity of the CDC25A phosphatase. Phosphorylation of CDC25A itself leads to its subsequent ubiquitin-mediated proteasomal degradation and inhibition of S phase progression. Thus, expression of CDK inhibitors and down-regulation of CDC25A phosphatase activity are means by which checkpoint signals are able to mediate downstream cell cycle arrest. Furthermore, PP2A, a protein phosphatase that is also critical to the process of oncogenic transformation, has been shown to regulate an S phase checkpoint by dephosphorylating pRB and licensing recruitment of pRB to chromatin to suppress DNA replication.17 Heralded as the guardian of the genome, p53 integrates the response to DNA damage, replication stress, hypoxia, telomere dysfunction, and activated oncogenes and mediates downstream checkpoint activation.4,18–20 Inherited mutations in p53 or its direct upstream activator CHK2 result in the Li Fraumeni cancer predisposition syndrome, and sporadic inactivation of p53 is one of the most frequent events observed in tumor development. Tumors lacking p53 exhibit widespread genomic instability resulting from an inability to arrest the cell cycle or trigger apoptosis in the setting of DNA damage and the cellular stresses previously described. In normal cells, p53 is maintained at low levels in the cytoplasm because of ubiquitination by MDM2 and proteasomal degradation. In response to

checkpoint activation and phosphorylation, p53 increases in abundance and translocates to the nucleus, where it activates a transcriptional program that promotes cell cycle arrest, senescence, or apoptosis, depending on the cell type and conditions. The CDK inhibitor p21 is a key transcriptional target of p53 that mediates checkpoint arrest while repair is attempted. In response to a variety of signals p53 can trigger an apoptotic program, in part via transcriptional activation of proapoptotic targets such as NOXA and BAX.21

DNA Damage Checkpoints
Activation of cell cycle checkpoints occurs as part of a larger DNA damage response pathway.22,23There are three major DNA damage checkpoints, with two of the checkpoints occurring at the boundaries between G1/S and G2/M. The third checkpoint, however, occurs intra-S phase. In addition to promoting cell cycle arrest through the mechanisms described earlier, these pathways coordinate recruitment of repair proteins to the sites of DNA damage, modulation of transcription, activation of subsequent repair, and apoptosis. The signaling network that controls this response is initiated by the key DNA damage sensors, the ataxia-telangiectasia mutated (ATM) and AT and Rad3-related (ATR) protein kinases.10,11,24,25 ATM is principally activated in response to double-strand breaks, while ATR is activated by replication fork collapse and by bulky DNA lesions. As will be described in more detail later, both proteins phosphorylate multiple targets in coordinating the subsequent DNA damage response. Key signal transducers in this process include CHK2 (activated by ATM) and CHK1 (activated by ATR). p53 is a major substrate for ATM/CHK2 and ATR/CHK1 phosphorylation, and subsequent activation of p53 represents a principal mechanism by which cell cycle checkpoints are activated in response to DNA damage. Replication stress and hypoxia also appear to activate p53 through ATR signaling, while telomere dysfunction contributes to p53 activation through ATM (Fig. 2.1).18 In addition, a variety of stress responses have been shown to activate p38MAPK, which can promote checkpoint activation through both p53-dependent and -independent pathways.26,27 CHK2 activation also leads to phosphorylation and degradation of the phosphatase CDC25A, resulting in activation of an S phase checkpoint.28

Figure 2.1. G1 pathways that can trigger cell cycle arrest, senescence, or apoptosis. A variety of threats to genomic integrity lead to activation of pathways that result in cell cycle arrest. Signaling via ATM/CHK2 and ATR/CHK1 leads to p53 activation, among other effects. One of the principle downstream effects of p53 is activation of p21 expression, with resultant cyclin E(A)/CDK2 inhibition and cell cycle arrest. Senescence, which results in a more sustained cell cycle exit, also involves the up-regulation of p14ARF and p16INK4A. Both proteins ultimately lead to retinoblastoma protein (RB) activation and G1 arrest via cyclin/CDK inhibition. In response to DNA damage during S phase, activation of PP2A can lead to dephosphorylation of RB and inhibition of DNA synthesis.

Oncogene-induced senescence is also triggered by DNA replication stress. the formation of stable heterochromatic foci that envelop and silence E2F target genes. which assemble onto unattached kinetochores and generate a ―wait anaphase‖ signal that prevents activation of anaphase effector proteins. suboptimal culture conditions. inappropriate activation of RAS signaling in other tissues results in p16INK4A-mediated senescence. including prematurely terminated DNA replication forks.34 p14ARF inhibits MDM2 function. Nonetheless. although it can be bypassed by inactivation of p16INK4A and HMGA proteins. In humans. which exists in an alternative reading frame within the p16INK4A locus. also known as oncogene-induced senescence. In the setting of RB and p53 pathway inactivation. in part by sequestering it in the nucleolus. in conjunction with HMGA chromatin proteins. p16INK4Aexpression is associated with both oncogene activation and genotoxic stress.4. The RB and p53 pathways have been shown to mediate the arrest by replicative senescence. and DNA hyperreplication. inhibiting the DNA double-strand break response kinase ATM suppressed the induction of senescence and led to increased tumor size and invasiveness. whereby progressive telomere attrition elicits a DNA damage response similar to that induced by other genotoxic stresses (Fig. but rare malignant clones can emerge. cellular senescence also occurs as a response to oncogene activation.‖ Most cells in crisis will die.35 Cellular senescence induced by this latter program is refractory to RB and p53 inactivation. but progressive telomere shortening results in the accumulation of massive genetic instability and a state of ―crisis. DNA doublestrand breaks. a similar but less well understood crisis event occurs that is. in which telomeres start out long and seldom shorten to a critical length.1). in mice. oxidative stress.Spindle Assembly Checkpoint Errors that occur during mitosis are similarly monitored by a spindle checkpoint. resulting in the accumulation and activation of p53. melanoma. targeted . and lymphoma. which prevents progression into anaphase when chromosomes are improperly attached to the mitotic spindle.37 In a mouse model.29 Key sensors of this response include the spindle checkpoint proteins.36. related to differential sensitivity of mouse cells to oxidative damage in culture. resulting in cellular immortalization. Oncogene-induced senescence has been shown to occur in vivo. 2. Cellular Senescence and Crisis Cellular senescence is another mechanism that limits the progressive accumulation of cells with impaired genomic integrity and oncogenic potential. cells can bypass replicative senescence. Senescence induced by oncogene activation. and chemotherapy. limiting tumor progression in models of lung and prostate cancer. This pathway is outlined in further detail later.30–32 Originally described as an irreversible state of cell cycle exit in response to exhausted replicative potential of cultured cells. results from expression of p16INK4A and p14ARF (note that p14ARF in humans is p19ARF in the mouse).33. promoting activation of RB and. activation of the enzyme telomerase and subsequent maintenance of telomere length allows such cells to bypass crisis.33 Whereas inactivation of the PTEN tumor suppressor and resultant activation of the AKT signaling pathway in prostate epithelial cells appears to promote senescence through p14ARF. By contrast. at least in part.

including early-onset lung cancer. and the mechanisms by which genome destabilization can occur. accelerated mutation rates and chromosomal instability destabilize the genome and facilitate progression through the steps of oncogenic transformation. Cancer predisposition syndromes that result from inherited defects in genome maintenance are highlighted in Table 2. with age. contributing to lymphomagenesis. In this setting.4 One path to genetic instability is inactivation of checkpoint proteins such as those previously described.38. expression of endogenous levels of oncogenic K-RAS can promote proliferation. a potential caveat to their use. . and it has been demonstrated that oncogene-induced senescence due to RAS activation can be dose-dependent. the disruption of which facilitates lymphoma development in response to RAS activation.43 This may occur at least in part through cell division failure and the generation of unstable tetraploid cells (see later discussion). suggesting that this barrier may be readily overcome or that the consequence of RAS expression may vary depending on the context.1.activation of oncogenic K-RAS alleles in somatic tissues in mice predisposes to a wide variety of tumor types. The subsequent deregulation of the cell cycle and impairment of the response to genomic injury allows the progressive accumulation of lesions that can drive oncogenesis. cells can develop genetic alterations that escape detection. mutations can also occur in genes encoding the proteins that repair DNA damage and protect against the development of chromosome abnormalities. Additionally. are outlined in the next sections.39 Moreover. In addition.33 Furthermore. The basic types of genetic alterations observed in tumors. they suggest that emerging epigenetic therapies such as histone deacetylase inhibitors and DNA methyltransferase inhibitors that interfere with chromatin silencing may disrupt this senescence barrier. These findings confirm the in vitro observations that changes in chromatin structure contribute to cell cycle exit by senescence.40–42 RAS-induced senescence in lymphocytes depends on heterochromatin formation via the Suv39h1 histone methyltransferase. Mutations in Cancer Despite multiple levels of protection against the development of genomic instability. disruption of Suv39h1 by itself has been shown to disrupt heterochromatin formation and to promote genetic instability.

As a result.46. a variety of endogenous and exogenous chemical and radiation exposures can introduce additional DNA lesions. revealed that these cancers harbor approximately 100 mutant genes.1.Table 2. pancreatic. B-RAF in melanoma. and genomic rearrangements on a genomewide basis. Notable examples include activating mutations in oncogenic kinases such as K-RAS in colorectal. with oncogenic K-RAS mutations in non–small cell lung cancer (NSCLC) occurring more frequently in smokers and epidermal growth factor receptor (EGFR) mutations in nonsmokers. and JAK2 in myeloproliferative disorders. In some instances. copy number changes. and lung cancer. specific mutations have been linked to epidemiologic features such as tobacco exposure. Mutations arise when such errors are not detected and repaired by this machinery. for example. point mutations are frequently detected via sequencing of both oncogenes and tumor suppressor genes in multiple cancers.4. insertions and deletions.47 Massively parallel DNA sequencing platforms now allow for the routine sequencing of billions of bases of DNA per week and the identification of point mutations. exposure to endogenous or exogenous mutagens. Large-scale sequencing of coding sequences from a panel of colorectal and breast tumors. which can occur when repair pathways are overwhelmed or defective.21 The spontaneous mutation rate per nucleotide per cell division has been estimated to be on the order of 10−9 in somatic cells and 10−11 in stem cells. with computational methods predicting that 14 to 20 of these mutations will be bona fide . requiring the presence of multiple repair pathways for further protection of genomic integrity.45 Next-generation sequencing technology has ushered in a new era in cancer genomics.44 Despite the remarkable fidelity of DNA polymerase and its inherent proofreading capacity. or defects in the ability to detect and/or repair simple sequence errors. Inherited Genome Maintenance Defects with Cancer Predisposition Point Mutations Changes in the nucleotide sequence can arise from spontaneous mutation.

The genomes of a small cell lung cancer. including the enzyme isocitrate dehydrogenase (IDH1). which remains low in most mature tumors.53 Subsequently. including MAPK signaling. there has been a rapid progression from targeted gene sequencing to targeted whole-genome and whole-transcriptome sequencing. In another study. sequencing of coding regions of protein kinases in a large number of cancers identified ―driver‖ mutations in approximately 120 genes across all samples. CDH24. GPR123. and breast tumor have also been described. and the mTOR pathway. with each tumor possessing a relatively unique cancer gene mutational signature. Intriguingly.48 In this unbiased effort. that are targeted by a combination of point mutations.tumor suppressor genes or oncogenes. In the lung adenocarcinoma samples.57–59Interestingly. PCLKC.000 protein coding genes led to the discovery of a variety of genes that were not known to be altered in GBM. are in gene families that are strongly implicated in cancer pathogenesis. Four of the affected genes (PTPRT. the mutations in both the lung cancer and melanoma genomes were not distributed evenly throughout the genome—many were present outside the genecoding regions. In a different study of GBM tumor samples. The first sequencing of a whole cancer genome was reported for AML. including the preferential targeting of repair to transcribed regions compared with nontranscribed regions. were in genes that were not previously implicated in the pathogenesis of AML. EBI2. melanoma. to exons compared with introns.55 More recently. revealed multiple levels of selective DNA repair. The other eight mutations. suggesting that cells had repaired damaged DNA in those key regions. and SLC15A1).51 Both studies integrated the somatic mutation data with other genome-wide characterizations and clinical data. both known and unknown mutations were identified. FLT3 and NPM1. to transcribed DNA strands compared with nontranscribed strands. Sequencing of the melanoma genome. and to the 5′ end of genes compared with the 3′ end. however. which has been shown to lead to an elevated risk of malignant brain tumors. the sequencing data were used to identify multiple pathways.49 Although these studies identified a greater number of mutational events associated with oncogenesis than previously thought (the ―state‖ of genome integrity). p53 signaling. targeted gene resequencing was also applied to lung adenocarcinoma and glioblastoma multiforme (GBM) tumor samples. copy number amplifications and deletions.56 Acquired mutations in coding sequences of annotated genes were identified in ten genes in the AML genome by comparing the genomic DNA of leukemia cells with normal skin cells obtained from a patient with FAB M1 AML. and GRINL1B) are involved in metabolic pathways. Two of the identified mutations were in genes previously described to have a role in leukemogenesis. for example. the sequencing of approximately 20. for example. though. mutations in IDH1 have also been identified in acute myeloid leukemia (AML) genomes. .54. the remaining genes (KNDC1.52 The cancer-associated IDH1 mutations result in the novel ability of the enzyme to catalyze the NADPH-dependent reduction of alpha-ketoglutarate to R(-)-2-hydroxyglutarate (2HG).21 In two early studies. and loss of heterozygosity (LOH).50. they do not necessarily imply a high ―rate‖ of mutation.

structural and copy-number alterations. Although major clinical impact of large-scale sequencing projects is yet to be realized. and therapeutic response. resulting in fusion of two different genes or placement of a gene next to an inappropriate regulatory element.18) in follicular lymphoma.21 (Fig. Translocations Unlike point mutations. The number of sequenced cancer genomes is likely to expand substantially in coming years. though. GBM. The clinical and translational implications of routine cancer genome sequencing are profound and include the identification of new drug targets. resulting in expression of the growth promoting BCR-ABL gene product.22) in chronic myelogenous leukemia.2.61 This approach is more sensitive than microarrays and also provides data that can be used to evaluate for allele-specific expression. and lung squamous carcinoma. The scope of the Cancer Genome Atlas Research Network. and single nucleotide mutations. Examples include t(9. for example.Next-generation sequencing has also been applied to RNA (―RNA-Seq‖) extracted from tumor cells for complete transcriptome characterization. as well as the generation of new insights into the genetic patterns of disease phenotype. small insertions. which may alter gene expression indirectly. the identification of this unexpected class of mutations validates the high-throughput cancer genome sequencing approach. Recent work has also identified a translocation between the immunoglobulin heavy chain and the cytokine receptor CRLF2 in a subset of precursor B-cell acute lymphoblastic leukemia associated with a poor outcome and activating JAK mutations. and deletions may be observed using cytogenetic analysis4. ovarian serous cystadenocarcinoma. The Cancer Genome Atlas Program of the U. Chromosome translocation involves juxtaposition of two different chromosome segments. initially focused its large-scale genomic analysis on only three tumor types. alternative splice isoforms. RNA-Seq was used to identify both known and novel fusion transcripts in prostate cancer samples. Although the role of 2HG in cancer development remains unclear. was applied to four granulosa-cell tumors and identified missense point mutations in the FOXL2 gene. resulting in overexpression of the antiapoptotic protein BCL2 as a result of its fusion with the immunoglobulin heavy chain promoter.S. because the mutations in IDH1 result in a gain of function. One shortcoming of direct sequencing. has now expanded to include more than 20 tumor types and thousands of samples. or deletions of nucleotides.64. is its failure to detect epigenetic changes. there is much excitement about developing small molecule inhibitors of mutant IDH1.2).62 In a different study. which encodes a transcription factor known to be crucial in granulosa cell development. the discovery of IDH1 illustrates the potential of this approach.65 .60RNA-Seq.60. fusion transcripts. amplifications. and t(14. National Cancer Institute. larger chromosomal changes such as translocations. such as DNA methylation. prognosis. however. Also. for example.63 RNA-Seq has also been used to study the role of microRNAs in the regulation of gene expression in both normal and cancerous cells.

Genetic alteration of tumor suppressor genes frequently involves mutation in one allele and deletion of the second allele as part of a larger chromosomal segment. such as TMPRSS2 to ERG and ETV1 in prostate cancer.2. Conversely. has begun to elucidate the mechanisms of some tissue-specific translocations. which results in the generation of DNA double-stranded breaks (DSB). resulting in multiple copies of both oncogenes and their neighboring sequences.Figure 2.69 First. and changes in microRNA expression have been linked to prognostic factors and progression in diseases such as chronic lymphocytic leukemia.71 This approach was used to successfully identify recurrent gene fusions of TMPRSS2 to ERG or ETV1 in prostate cancer. termed cancer outlier profile analysis.70 A clever bioinformatics approach. Normal mouse cells contain a diploid complement of 40 chromosomes. . the intron-bound androgen receptor alters local chromatin architecture and recruits the ligand and genotoxic stress-induced enzymes. was used to first identify this recurrent translocation in prostate cancer.5 to 10 megabases of DNA. Second.66. The DSB are subsequently ligated by the nonhomologous end-joining (NHEJ) machinery to create translocations. including the activation-induced cytidine deaminase and LINE-1 repeat-encoded ORF2 endonuclease. Common cytogenetic abnormalities.and interchromosomal interactions that result in the spatial proximity of tumor translocation partners. with multiple dicentric chromosomes and nonreciprocal translocations.69. Individual chromosomes are highlighted by spectral karyotyping (SKY) using fluorescent chromosome probes (right panel). resulting in regions of uniform sequence with LOH. In this sample. but can also affect the expression of microRNAs. Recent work. however. and can involve small interstitial segments or entire chromosome arms. ligand-dependent binding of the androgen receptor to intronic binding sites near the tumor translocation sites creates specific intra. as well as possibly facilitating the development of new therapeutic strategies.67 MicroRNAs are short regulatory RNAs that control mRNA stability and/or translation. Such ―amplicons‖ may range in size from 0. to these regions. deletions result in loss of chromosomal regions. the mechanisms underlying their genesis are unclear. Further elucidation of the mechanisms leading to geneand tissue-specific translocations will advance the understanding of basic mechanisms of cancer. Metaphase spread derived from a mouse tumor model (combined telomerase and p53 deficiency). Further mechanistic work has shown that this translocation requires two roles of the androgen receptor. One exciting recent development is that translocations not only create chimeric genes or alter promoter sequences.68 Although translocations and gene fusions are a hallmark of cancer. more than 260 chromosomes are observed. Amplifications and Deletions Amplifications can be detected cytogenetically as double-minute chromosomes or regions of excess signal intensity using fluorescence in situ hybridization.

Whole-chromosome loss may be underestimated by karyotypic analysis. while melanomas often show gain of chromosome 7. The focal SCNAs occurred at a frequency inversely related to their lengths. including the BCL2 family of apoptosis regulators and the NF-kB pathway. childhood acute lymphoblastic leukemia. Bub1 insufficiency predisposed p53+/−mice to thymic lymphomas and ApcMin/+ mice to colonic tumors. or acquisition of improper epigenetic patterns. proliferation. Whole-Chromosome Loss/Gain Nearly all solid tumor types exhibit whole-chromosome loss or gain. Epigenetics Significant evidence now indicates that epigenetic modifications. resulting in an abnormal ―allelotype‖ with accompanying LOH. Glioblastomas. breast cancer. including primary and secondary AML. frequently exhibit loss of chromosome 10.A recent study reported the high-resolution analysis of somatic copy-number alterations (SCNAs) from 3. or heritable changes in gene expression that are not caused by changes in DNA sequence. This observation was seen across all cancer types and applied to both copy gains and losses. Arm-level SCNAs occurred approximately 30 times more frequently than expected by the inverse-length distribution associated with focal SCNAs. are critical factors in the pathogenesis of cancer. for example.21. multiple myeloma.8 megabases. epigenetic mechanisms controlling the transcription of genes involved in cell differentiation. and glioblastoma. or copy neutral loss of heterozygosity (CN-LOH).131 cancer specimens belonging to 26 histologic types. such defects are generally the result of chromosomal mis-segregation during mitosis. the finding that most of the SCNAs were found in multiple cancer types suggests that the diversity across cancer genomes may reflect the combinations of a limited number of functionally relevant events. Several gene families were enriched among the regions of focal SCNA.79 Although beyond the scope of this chapter.77 In mice. chronic lymphocytic lymphoma. it has been demonstrated that Bub1 insufficiency can drive tumor formation through tumor suppressor gene LOH.74 CN-LOH. of which 122 could not be explained by the presence of an oncogene located within the region.75 Furthermore. basal cell carcinoma. Interestingly.73 As will be described later. as loss of one parental chromosome may be accompanied by duplication of the other parental chromosome. from which B-RAF is expressed. myelodysplastic syndrome. These tumors demonstrated CN-LOH and lacked the nonmutated tumor suppressor allele. Monosomy 7 and trisomy 8 are associated with myelodysplasia and AML. with a median length of 1. but had gained a copy of the mutant allele. is common in cancer and has been described in AML. .78 Specifically.72 The most prevalent SCNAs were either very short (focal) or almost the length of a chromosome arm or whole chromosome (arm level). also referred to as uniparental disomy. but possible mechanisms include the duplication of oncogenes. and survival are often targets for deregulation in the development of cancer. The study also identified 158 regions of focal SCNAs that were altered at significant frequency across several cancer types. inactivating the tumor suppressor PTEN. CN-LOH has been shown to have prognostic significance in a number of these cancer types.76 The specific role of CN-LOH in tumorigenesis remains undefined. resulting in alterations in chromosome number or aneuploidy. loss of tumor suppressors.

The drug-tolerant subpopulation can be selectively ablated by treatment with IGF-1 receptor inhibitors or chromatin-modifying agents. particularly in regions of simple repeat sequences known as microsatellites. covalent modifications of histones. Larger insertion/deletion mispairs due to slippage of the replication machinery in repetitive sequences or recombination errors form a loop structure that is alternatively recognized by a complex of MSH2 and MSH3. this research suggests that the potentially reversible nature of epigenetic changes. with the drug erlotinib results in the death of nearly all of the parental cells. with recruitment of an MLH1/MLH3 complex promoting . and ligation (Fig. DNA synthesis. Linkage analysis in kindreds with HNPCC revealed germ line mutations in hMSH2 and hMLH1. Most human SNF5-deficient cancers are diploid. had been described in bacteria and yeast mutants defective in mismatch repair.83 It had been recognized that a subset of sporadic colon cancers and a majority of cancers derived from patients with HNPCC exhibited frequent mutations. Mismatch repair corrects mispaired bases that can result from errors during DNA replication.11. the epigenetically based changes in transcription that occur following loss of SNF5 correlate with the tumor phenotype. demonstrate significantly reduced drug sensitivity and remain viable through activation of IGF-1 receptor signaling and an altered chromatin state that requires the histone demethylase RBP2. There has been much discussion and controversy about the existence of drug-resistant cancer stem cells. hPMS2.84 Mismatched bases are recognized by a complex of MSH2 and MSH6. as well as mismatched bases occurring in recombination intermediates or occurring as a result of some types of chemical damage to DNA. This drug-tolerant phenotype is transiently acquired at low frequency by individual cells within the population.4. unlike genetic mutations. Mechanisms of Genome Destabilization in Human Tumors Microsatellite Instability One of the earliest insights into the contribution of genome destabilization to carcinogenesis came from the study of the familial cancer syndrome HNPCC. recruiting MLH1 and PMS2 to the site to initiate the subsequent steps of repair.3). termed microsatellite instability (MIN). including excision. The role of epigenetic modifications in cancer pathogenesis is illustrated by the tumor suppressor SNF5. and are genomically stable. however.80 Biallelic inactivation of SNF5 is found in the majority of malignant rhabdoid tumors. has also identified a novel role for epigenetics in acquired drug resistance. the epigenetic effects described in this work provide a potential mechanism for the generation of some ―cancer stem cells.These epigenetic alterations include DNA methylation. 2. which have an activating mutation in the EGFR. such as HDAC inhibitors. are also observed in families with HNPCC. and hMSH6. may provide a unique therapeutic avenue in the treatment of cancer. PC9. It is now known that mutations in other components of the human mismatch repair process. which are human homologues of the mutL and mutS mismatch repair genes in Escherichia coli. A small percentage of the PC9 cells. lack genomic amplifications/deletions. Recent work with an NSCLC cell line.81 Furthermore.82 Treatment of PC9 cells.‖ Furthermore. which regulates the epigenome as a member of the SWI/SNF chromatin remodeling complex. and noncovalent changes in nucleosome position. This type of genetic instability.

and a potential resistance to 5-fluorouracil chemotherapy. the nucleotide excision repair (NER) and base excision repair (BER) pathways respond principally to lesions created by exogenous or endogenous DNA damaging agents. DNA synthesis.9 Figure 2. MLH1 and MSH2 have also been shown to have functions outside mismatch repair. in contrast to the remaining 85% of cases. or MLH1 and MLH3 to insertion/deletion loop sites. as defects in these proteins have been associated with an impaired G2/M cell cycle checkpoint in response to alkylating agents as well as abnormalities in meiotic recombination in mouse knockout models. helix-distorting lesions that are recognized by the NER machinery. In both sporadic cases and HNPCC. prior to anaphase-promoting complex (APC) inactivation.9.3.84. which are associated with chromosomal instability (CIN).86 Experimental evidence supports the idea that MIN occurs very early in sporadic colorectal cancer formation. Mismatch repair pathways. Components of the NER pathway were in . lack of p53 mutation.subsequent repair. HNPCC is associated with a 60% to 80% lifetime risk of developing colorectal cancer and is responsible for 2% to 5% of all cases of colorectal cancer. with median age at colon cancer diagnosis being significantly lower in MLH1 mutation carriers and in males. often due to epigenetic silencing of mismatch repair genes such as MLH1. but at the same time compromises fitness of cells. presumably by the accumulation of deleterious mutations. CIN. and ligation to complete the repair. This is followed by excision of the respective lesions. or recognition of insertion/deletion loops by MSH2 and MSH3 (lower panel). Cancer cells that exhibit MIN from defects in these components have a nucleotide mutation rate that has been estimated at two to three orders greater than that of normal cells. increasing genomic instability and thus obviating the selection pressure to develop another mechanism of genomic instability. Subsequent steps involve recruitment of MLH1 and PMS2 to mismatch sites. Nucleotide Excision Repair/Base Excision Repair Defects Whereas the mismatch repair pathway functions primarily in the recognition of and repair of replication errors. MIN has been associated with more favorable prognosis. The initial step involves recognition of simple mismatches by MSH2 and MSH6 (upper panel).4. and with more frequent extracolonic tumors observed in MSH2 carriers.85 Nearly 85% of HNPCC patients have mutations in MLH1or MSH2. Colorectal cancers exhibiting MIN are typically diploid.88 Ultraviolet radiation or exogenous chemicals such as polycyclic aromatic hydrocarbons and platinum chemotherapeutic drugs can result in bulky.87 It is becoming apparent that defects in components of these repair pathways have impacts on both cancer pathogenesis and the efficacy of cancer therapy. These observations are in accordance with the hypothesis that the accelerated mutation rate facilitates cancer evolution. The MIN phenotype is also observed in 15% of sporadic colon cancers. Mispaired bases due to errors in DNA replication or other causes are recognized by the mismatch repair machinery.

Notably. with a more pronounced cancer phenotype and evidence of premature aging.90 Deletion of CSB has been shown to impair tumor formation in cancer-prone mice. by UV irradiation (upper panels). Subsequently. an endonuclease involved in excision of the lesion. Figure 2. Furthermore. the resulting cancers are ―stuck‖ with the deleterious effects of ERCC1 deficiency and become sensitive to certain therapies. Stalled RNA polymerase II recruits Cockayne syndrome B (CSB) to the site of damage. mutant mice defective in NER also accumulate DNA damage.4.part discovered by mutation in the genetic syndromes xeroderma pigmentosa (XPA-XPG) and Cockayne syndrome (CSA and CSB). whereas no benefit was seen in tumors in which normal ERCC1expression was maintained.87 (Fig.91 Two separate NER pathways have been identified. for example. and it has been hypothesized that the lack of cancer in Cockayne syndrome may be related to a particular sensitivity of cells to apoptosis or impairment of transcription.92 A subgroup analysis of the International Adjuvant Lung Cancer Trial (IALT) demonstrated that ERCC1 deficiency was observed in tumor samples from 56% of patients and correlated with a significant improvement in survival following cisplatin-based chemotherapy. XP genes are involved in the recognition and repair of lesions in global genome NER. Nucleotide excision repair and base excision repair. while CS genes play a specific role in transcription coupled repair. Thus.4). Subsequent stages of NER are similar and involve ERCC1. Nucleotide excision repair (NER) is activated in response to bulky lesions that are generated. Global genome (GG) repair involves proteins identified by complementation groups in patients with xeroderma pigmentosa (XP proteins). and occurs when RNA polymerase II stalls at the site of lesions. this provides a general model for targeted therapy based on fixation of mutations that compromise genomic integrity during tumorigenesis.89 Mutation of XP genes can also be seen in the related disorder trichothiodystrophy. Whereas all three disorders exhibit dramatic sun sensitivity. DNA is locally unwound around the injured site by a TFIIH complex containing . Initial recognition of lesions occurs by a complex containing xeroderma pigmentosa C (XPC). reduced expression of ERCC1 in NSCLC has been associated with response to cisplatin-based adjuvant chemotherapy. followed by DNA replication to complete the repair process. Transcription coupled repair (TCR) also involves proteins identified by mutation in Cockayne syndrome (CS proteins). while reduced ERCC1 expression may promote genetic instability and facilitate NSCLC development in a significant fraction of patients. 2. Potentially. one that involves scanning the entire genome for lesions (global genome NER) and another that detects lesions that interfere with elongating RNA polymerases (transcription coupled repair or TCR)9. only xeroderma pigmentosa is associated with a marked incidence of sun-induced skin cancer.

DNA polymerase and ligase. This may be because of partial redundancy of DNA glycosylases. and XRCC1. which has been estimated to occur on the order of 104 times per day per cell.94 DNA Damage Response to Double-Strand Breaks DNA DSBs represent a significant threat to genomic integrity. and other proteins for TCR. Subsequent repair is influenced by PARPmediated ADP ribosylation of histones and other proteins. 2. or simply to the need for additional investigation. as well as spontaneous reactions such as base loss from hydrolysis of glycosyl DNA bonds. including histone H2AX. amplifications. These latter enzymes catalyze nucleotide reinsertion and ligation into the injured strand as part of the short patch repair pathway (major BER pathway). The initial detection and activation of signal transduction pathways mediating repair of DNA DSBs involves the PI(3)K-like kinases. CSA.93.9. action of DNA glycosylases.95 DSBs induced by DNA damage activate ATM. Key downstream targets of ATM and ATR include the checkpoint mediator proteins . human disorders or inherited cancer susceptibility syndromes due to mutation of components of the BER machinery have yet to be described. As described later. This process also involves XPG. Even a single DSB in budding yeast can trigger a DNA damage response checkpoint. or after ionizing radiation or oxidative damage. a finding that is not surprising as DSBs can promote major cytogenetic abnormalities such as chromosome translocations. Subsequent steps involve DNA synthesis and ligation to complete the repair. In base excision repair (BER) (lower panels).24 In addition. Disruption of any of these genes in the BER pathway results in the cellular accumulation of oxidative DNA damage.24. Defects in multiple components of this process have been linked to genomic instability and cancer predisposition.XPB and XPD. DSBs are detected and repaired by an intricate cascade of proteins. XPA and replication protein A (RPA) contribute to stabilization of an open intermediate and recruitment of the ERCC1 and XPF endonucleases that excise the lesion. poly(ADP-ribose) polymerase (PARP).4.11. BER is primarily involved in the response to damage caused by small chemical alterations and x-rays. while regions of single-stranded DNA at stalled replication forks recruit and activate ATR. abasic sites generated by spontaneous hydrolysis. ATM and ATR kinase activity results in phosphorylation of multiple targets. ATM and ATR. the fact that mutation of core BER components (in mice) results in embryonic lethality. while XRCC1 serves as a scaffold for recruitment of DNA polymerase β and DNA ligase 3.4). single-strand nicks can be converted into DSBs by DNA replication.10. and deletions. Once activated. Despite being integrally important to the maintenance of genome stability. and abasic sites are recognized by a complex that includes the APEX1 endonuclease. resulting in the local alteration of chromatin structure. pharmacologic inhibition of PARP may selectively sensitize cancer cells with a pre-existing defect in another repair pathway to death by DNA damage. Once unwound. ultimately involving the processes of homologous recombination (HR) or NHEJ.9 They can occur during DNA replication at sites of stalled replication forks.24 Damaged bases are removed by DNA glycosylases. as well as PARP and XRCC1. or x-ray–induced single-strand breaks are recognized by the APE1 endonuclease. a scaffolding protein that interacts with most of the core components (Fig.

and FANCN/PALB2.10 Inactivating mutations in ATR (AT and Rad3related) result in embryonic lethality in mice and are not observed in familial human cancer syndromes. congenital abnormalities. ATM/CHK2 and ATR/CHK1 have also been shown to slow progression through S phase by down-regulating CDC25A. at least in yeast. In response to DNA damage.104 . and a high frequency of malignancies. and in multiple sporadic tumor types. similar to ATR. cellular sensitivity to ionizing radiation. and. As previously described.103 Interestingly.102. with ATM principally activating CHK2 and ATR activating CHK1. ATR/CHK1 signaling is linked with activation of the Fanconi anemia pathway and BRCA2. while NBS1 is mutated in the Nijmegen breakage syndrome.99. the MRN proteins have important roles in telomere maintenance. which results in dwarfism. which. and these proteins further interact with the downstream FA proteins. FANCJ.9 DSBs are recognized by the MRN complex. could reflect strongly disadvantageous effects of CHK1 loss on viability. The initiation of the repair process itself involves recruitment of multiple other proteins to sites of DSBs in conjunction with ATM and ATR. and the encoded FA proteins cooperate in a common DNA repair pathway. in NHEJ. hypomorphic alleles of ATR that result in low levels of expression have been associated with the Seckel syndrome. DNA Repair of Insterstrand DNA Crosslinks Fanconi anemia is another autosomal recessive human disease characterized by bone marrow failure. and chromosome instability in cells treated with mitomycin C. However. the important caretaker function of CHK2 is evidenced by its mutation in a subset of patients with Li Fraumeni syndrome. Eight of the FA proteins are assembled in a multisubunit ubiquitin ligase complex.97 Mutation of CHK1 is observed less frequently in tumors. mediating downstream checkpoint activation.98 Mutations in MRE11 result in an ataxia telangiectasia-like disorder. in Seckel syndrome the disadvantages of compromised ATR for cell viability may outweigh the potential for an increase in cancercausing mutations. consisting of MRE11 (meiotic recombination 11)/RAD50/NBS1 (Nimjen breakage syndrome 1). cellular hypersensitivity to DNA cross-linking agents such as mitomycin C and cisplatin. this ligase monoubiquitinates two additional FA proteins. Monoubiquitination is activated by the ATR/CHK1 pathway. and predisposition to malignancies such as acute leukemia and squamous cell carcinomas. ATM/CHK2 as well as ATR/CHK1 can phosphorylate and activate p53.96 Patients with Seckel syndrome are not significantly predisposed to cancer development.4. As previously described.(CHK).9.28 ATM was identified by virtue of its association with the neurodegenerative disorder ataxia telangiectasia.101 Monoubiquitinated FANCD2 and FANCI are required for normal homologous recombination repair. in which patients are also predisposed to malignancies such as acute lymphoblastic leukemia and lymphoma. chromosomal instability. perhaps reflecting the general balancing of fitness effects and oncogenic potential. CHK2 mutation has also been observed in familial breast cancer. FANCD1. In addition to their role in DSB repair by HR. and both diseases are characterized by immunodeficiency. FANCD2 and FANCI. or during normal S phase progression. FANCD1 is identical to the BRCA2 gene. microcephaly. presumably reflecting the key role of this kinase in normal DNA replication.100 There are 13 known FA genes.

sequences from a homologous DNA duplex are used to provide a template for reconstruction of the damaged DNA segment.g.107 During BIR a broken chromosome end invades a homologous site and replication proceeds to copy the entire sequence of the template chromosome. Mediator proteins such as BRCA2 and RAD52 stimulate assembly of a RAD51 nucleoprotein filament complex that guides subsequent homology search and strand invasion into the homologous strand (e.9 The template for repair can either be the identical sister chromatid (the preferred substrate in mitotic cells) or the homologous chromosome (the preferred substrate during meiosis). this complex was required for nucleolytic incisions near an interstrand cross-link and for translesion DNA synthesis past the cross-link. in conjunction with topoisomerase 3 α. with subsequent recruitment of DNA-dependent protein kinase. ―Mediator‖ proteins such as BRCA2 or Rad52 then facilitate the recruitment of Rad51-related proteins. In a cell-free system.The three downstream FA genes are breast/ovarian cancer susceptibility genes. Subsequent DNA synthesis and ligation results in the formation of recombination intermediates that contain double Holliday junctions. Biochemical functions of monoubiquitinated FANCD2/FANCI were recently elucidated.106 Figure 2. the identical sister chromatid in late S/G2 phase and mitosis).5).5. The process of NHEJ involves recognition of DSB ends by the Ku70-Ku80 heterodimer. among other proteins. replacing RPA. DSBs are recognized by the MRN complex and by checkpoint proteins as previously described. A 5′-3 exonuclease generates 3′ overhangs.105 Homologous Recombination In repair by HR. which are then coated with replication protein A (RPA). A homology search ensues.4. The links between DNA strands (double Holliday junctions) can be resolved to produce exchange between chromosomes (crossovers) or no exchange (non-crossovers). FANCD1. Double-strand break (DSB) repair by homologous recombination and nonhomologous end-joining (NHEJ). DSBs are recognized by the MRN complex. followed by strand invasion and DNA synthesis. the FA pathway was shown to be required for the generation of a new. with mutations in a single copy of FANCJ. Thus. These are resolved by resolving enzymes such as the RecQ helicase BLM. in conjunction with topoisomerase IIIα can resolve these double Holliday junctions. DNA ends are then ligated following recruitment of XRCC4 and DNA ligase 4. Several other HR pathways also exist. . 2. This is relevant to cancer because it can result in large-scale LOH. 5′-3′ exonuclease activity results in the generation of single-strand overhangs that are coated with RPA. Enzymes such as the RecQ helicase BLM. and heterozygote carriers. partially processed DNA substrate that can be further repaired by the downstream homologous recombination machinery. The classic HR pathway involves the following basic steps (Fig. which form filaments on the singlestranded DNA.. or FANCN have an increased cancer risk. A potentially important mechanism for cancer development is break-induced replication (BIR). In homologous recombination (HR).

117 .110. often suboptimal. mutation in PALB2. and in the organization of heterochromatin. has also been described in familial breast cancer. with a presumed role in the global sensing and coordinated response to DNA damage. Another large. which encodes a BRCA2-binding partner. Rothmund-Thomson syndrome is associated with mutation in the related RecQ helicase RECQ4. The combination of aging and cancer predisposition phenotypes associated with RecQ lesions provides yet another example of the balance between the deleterious and growth-promoting effects of genomic instability on developing cancer cells. and may contribute to inherited prostate cancer as well. with mutations in CHK2 and p53 responsible for an additional 5% and 1% of cases.9 These are then channeled into alternate. Recently. In addition.112– 114 BRCA1 forms a heterodimer with the structurally related protein BARD1. Cancer-causing mutations have been associated with multiple steps in HR. and BLM. and age-associated malignancies. RAD51. and an elevated frequency of malignancies such as osteogenic sarcomas. interact with components of the MMR and BER machinery. containing tumor suppressors. male sterility.108 Finally. mismatch repair proteins. Given that RecQ helicases are particularly important resolving enzymes in this process. Mutations in BRCA1 and BRCA2 are associated with a 60% to 85% lifetime risk of developing breast cancer and a 15% to 40% lifetime risk of developing ovarian cancer.113 They account for approximately 40% of cases of familial breast cancer. RecQ helicases have also been shown to facilitate NHEJ.4. early alopecia and hair graying. osteoporosis. skin disorders. juvenile cataracts. BRCA1 and BRCA2 are perhaps the most extensively studied cancer susceptibility genes required for HR. They are mutated in familial breast and ovarian cancer syndromes and represent key components of the response to DSBs and subsequent repair by HR. growth deficiency. and other proteins involved in the regulation of repair by HR. a disorder characterized by premature aging. respectively. aging is accompanied by a switchlike increase in mutagenesis and BIR after a certain number of generations. and as previously described. BRCA1 has also been implicated in S phase and G2/M checkpoint control. the WRN helicase was identified by virtue of its mutation in Werner syndrome. a disease characterized by immunodeficiency. forms a complex together with BRCA2. with early atherosclerosis. MRN.111 Mutation of BLM results in Bloom syndrome.109 Disruption of recombination pathways produces complex effects that pose the danger of chromosomal rearrangement due to the accumulation of recombination intermediates. and play an important role in telomere maintenance. dwarfism. and a high incidence of both leukemia and solid tumors. ATM. multiprotein complex termed BASC (BRCA1-associated genome surveillance complex) has been identified.115 PALB2 is also identical to the Fanconi anemia gene FANCN. and affected patients exhibit characteristic photosensitivity with poikilodermatous skin changes.106. BIR is an important mechanism for healing breaks at chromosome ends resulting from telomere attrition. in yeast. repair pathways. 116 Germ line biallelic mutations in PALB2 or BRCA2 (FANCD1) result in Fanconi anemia.Furthermore. increasing the potential for errors and rearrangements. type 2 diabetes. although only 2% to 3% of all breast cancer cases are associated with mutation in one of these genes. their inactivation results in widespread accumulation of recombination intermediates.

Although these sporadic triple-negative breast cancers appear to have normal BRCA1. where the genome lacks extensive repetitive sequences. . Thus. During V(D)J recombination and certain other physiologic settings. NHEJ is integrally involved in the V(D)J recombination and class switching that occurs during normal lymphocyte maturation. A subset of sporadic breast cancer. the small-scale errors generated by NHEJ are less detrimental than the potential large-scale errors (deletions or translocations) that could arise from HR. Somatic inactivation of BRCA1 and BRCA2 has been reported in other tumor types such as colorectal cancer. NHEJ can occur during any phase of the cell cycle.119. NHEJ involves direct end-joining of the broken double-strand DNA ends.95. in principle it should be error-free. as have defects in other components of DSB repair such as ATM and FANC genes.Although it is clear that mutations in BRCA1 and BRCA2 can destabilize the genome and promote cancer susceptibility. HR predominantly occurs when a homologous template sequence is held in close physical proximity to the break by the cohesion between sister chromatids. Thus. without a template for repair. DNA-PK–mediated phosphorylation of Artemis facilitates its activation and results in the processing of DNA ends in a subset of DSBs. One factor that prevents this type of error is that repair by HR is limited to late S and G2 phases of the cell cycle. Another possibility is that breast tissue selectively accumulates genotoxins that induce a heightened requirement for BRCA1 and BRCA2. In humans and other higher eukaryotes it appears that NHEJ is relatively more important than in other organisms such as fungi. HR is indeed mostly error-free. HR poses the danger of repeat sequence recombination resulting in gross chromosomal rearrangements. after DNA replication. at least in G1 cells. However. Nonhomologous End-Joining Given that HR uses an identical sister chromatid as template to guide repair. which recruits the catalytic subunit of DNA-dependent protein kinase (DNA-PK) and the Artemis nuclease120–122 (Fig. cleavage induced by the RAG nuclease may generate regions of microhomology that allow for relatively precise end-joining.5). broken DNA ends are recognized by a heterodimer of Ku70/Ku80. These sporadic basal-like cancers also exhibit defects in X chromosome inactivation. DNA ligation is subsequently mediated by a complex that contains XRCC4 and DNA ligase IV. It seems that.120 In yeast. although it primarily occurs during G1 phase. contributing to the error-prone nature of NHEJ. Further study is needed to elucidate the mechanism behind the tissue-specific nature of BRCA1 and BRCA2mutant cancer. in humans.2. it remains poorly understood which roles of these proteins are specifically involved in tumor suppression and why patients with germ line defects primarily develop breast and ovarian cancer. In the process of NHEJ. where the genome contains extensive repetitive sequences.4.118 and co-cluster with BRCA10-deficient breast cancers on transcriptional arrays. defined by lack of expression of the estrogen and progesterone receptors and absence of HER2 amplification (termed triple-negative breast cancer) and a ―basal-like‖ phenotype shares strong similarities with the tumors that develop in patients with germ line BRCA1 and BRCA2 mutations. these similarities have led to the suggestion that they may be defective at another point within the BRCA1 pathway.

While mutations in these core NHEJ components in mice cause severe immunologic defects. Consistent with this idea. telomerase is expressed at low to undetectable levels in somatic tissues. it may also be the case that more human tumors need to be carefully characterized for subtle mutations. NHEJ deficiency has been linked to impaired telomere capping and end-to-end fusions. and epigenetic silencing. Although there is abundant evidence that NHEJ defects can promote tumorigenesis in mouse models—at least in the setting of concomitant p53 deficiency—there are few reports that implicate NHEJ deficiency in human cancer. the structures at the ends of chromosomes composed of repetitive sequences and a 3′ G-strand overhang. resulting in stabilization of telomere length and restoration of capping function. concomitant p53 inactivation results in a high frequency of lymphomas with recurrent. consistent with the observation that CIN arises early during colorectal tumorigenesis. at a time of short telomere length. and premature senescence of cultured fibroblasts.125 In humans. haploinsufficiency.124However. the lack of complex karyotypes in many lymphomas may be related to early activation of telomerase in the setting of the frequent dysregulation of Myc. Conversely. The reasons for this are unclear. a positive regulator of telomerase expression. The activation of telomerase expression during cancer progression supports the idea that telomere dysfunction contributes to chromosome instability during a specific window in oncogenesis. such as the c-myc and the immunoglobulin heavy chain (IgH) loci. telomerase is expressed in many tumors. telomere shortening as a consequence of cell turnover in the setting of chronic inflammatory states such as hepatocellular cirrhosis may contribute to chromosome instability and carcinogenesis in these settings. Artemis deficiency.126. and high in late adenomas and colorectal carcinomas. Furthermore. These regions may contain specific sites that are recognized by the RAG nuclease.128 Studies of mice deficient in telomerase activity have yielded powerful evidence that telomere dysfunction can drive chromosomal instability and epithelial carcinogenesis.127 For example. there is a report of a ligase IV mutation in a leukemia patient. similar to common translocations observed human lymphomas. telomerase activity is low in small and intermediate-sized colon polyps. and the initial cleavage combined with defective NHEJ may be responsible for these aberrant chromosome fusions. leading to an agedependent compromise of telomere integrity and resultant telomere dysfunction. Strongly reminiscent of the shift in tumor spectrum to epithelial malignancies on aging humans. is observed in rare lymphoma-prone patients. Similarly. aging mice deficient in telomerase and heterozygous for mutant p53 exhibit carcinomas of the . Alternatively. with so-called breakage-fusion-bridge cycles (see later discussion) promoting translocations and gene amplifications. are targeted in a recurrent fashion in these lymphomas. are key mediators of genomic instability. By contrast. apoptosis. clonal rearrangements.123 Certain chromosome regions. Loss of NHEJ may be cell-lethal in humans. which results in a very restricted NHEJ defect. It is also possible that fragile sites within these chromosomal loci and elsewhere throughout the genome may account for the particular susceptibility of certain chromosome regions to breakage and rearrangement. Telomere Maintenance Telomeres.

Breakage-fusion-bridge cycle. and has been shown to interact with a number of DNA repair proteins. severe telomere shortening. skin. Moreover. the WRN and BLM helicases. In addition to generating translocations. Moreover. which primarily develop sarcomas and hematopoietic malignancies. the fused chromosome ends form anaphase bridges as sister centromeres are pulled to opposite centrosomes. A breakage-fusion-bridge cycle is believed to be responsible for chromosomal fragmentation and the nonreciprocal translocations observed in such tumors (Fig. With progressive erosion of telomeres. including lung. Figure 2. and chromosomal instability. with TRF2 promoting recombination at telomeres and derepression of pathways that lead to alternative lengthening of telomeres (ALT). During mitosis. is overexpressed in a variety of epithelial malignancies.breast.129. the fused chromosome ends are placed under tension and form anaphase bridges. with the formation of a dicentric chromosome. These pulling forces result in chromosome breaks that contribute to deletions. such as the MRN complex. The further generation of atelomeric chromosomes by this process can result in propagation of breakage-fusion-bridge mechanisms and continued chromosome instability. this form of genetic instability can also result in amplifications and deletions.6.130 The observation that mouse epithelial tumors in this model exhibit amplified and deleted regions syntenic to those seen in human carcinomas lends further support to the notion of chromosomal fragile sites that may be conserved between species. PARP. an important regulator of telomere protection and telomere length. as sister chromatids are pulled to opposite poles. with UV-induced skin cancer. More recently.132 TRF2. and translocations.131. DNA-PK. key regulatory elements of telomere structure have been implicated in genomic instability and tumorigenesis. During anaphase. mice develop an XP-like syndrome when TRF2 is expressed in the skin at high levels. telomeric fusions may occur between identical sister chromatids or between different chromosomes (dicentrics). resulting in chromosome breakage.6).128 Such tumors are exceedingly rare in wild type mice. colon.133 Concomitant telomerase inactivation in these mice dramatically accelerates carcinogenesis. the cycle may be repeated. because of the further generation of unprotected chromosome ends. 2. the presence of cytogenetic profiles similar to human carcinomas supports a role for telomere dysfunction in epithelial carcinogenesis. amplifications. In the setting of telomere dysfunction and uncapping of chromosome ends. and breast cancer. and skin.131 ALT involves recombination between telomeres as an alternative means of telomere extension . and ERCC1/XPF. unprotected chromatid ends can undergo end-to-end fusion. In addition.

they lend further support to the idea that stem cells. Subsequent work demonstrated that a compromised spindle assembly checkpoint could result in CIN. What Causes Chromosomal Instability and Whole-Chromosome Aneuploidy? Chromosomal instability (CIN). are protected from genomic injury. some tumors are aneuploid with a uniform. which express higher levels of telomerase than their differentiated progeny. the catalytic subunit of telomerase. and solid tumors. however. but the aneuploid colon carcinoma cells exhibited deviations from the modal chromosome number. that telomerase-deficient mice are viable and do not exhibit significant phenotypic defects until later generations. or the ―rate‖ of karyotypic change. Although CIN leads to aneuploidy.136–138 In addition. the colon cancer cell lines with MIN maintained a stable chromosome content. patients with DC are also prone to develop myelodysplastic syndrome. In all cases.and is operative in a small minority of tumors that are telomerase-negative. TERC. or the telomere binding protein TRF1-interacting nuclear factor 2. and the reason it is down-regulated in association with cell differentiation remains to be determined. and components of DNA damage response. Autosomal recessive DC can be caused by mutations in the dyskerin-associated proteins NHP2 and NOP10. It is important to note. indicating the presence of CIN. telomere-shortening syndromes in humans recapitulate the impaired maintenance of proliferative tissues and the tumor predisposition seen in telomerase-knockout mice. The fusion of MIN and CIN cells resulted in hybrid cells that retained the CIN phenotype. is a common characteristic of many cancers. leukoplakia. which leads to reduced levels of TERC and telomerase activity. leukemia. not all aneuploid tumors exhibit CIN. or the ―state‖ of the karyotype. has been shown to impair heterochromatin formation on a global level and to disrupt the response to DNA damage in normal human fibroblasts. Taken together. dyskeratosis congenita (DC). The specific role of telomerase in chromatin maintenance and the DNA damage response remains to be elucidated. Autosomal dominant DC can be caused by mutations in TERT. defined as a persistently high rate of loss and gain of whole chromosomes.135 These results are consistent with the emerging ties between regulators of telomere maintenance. Thus. these studies identify a fundamental role for telomeres and their regulatory proteins in the genesis of chromosome abnormalities and epithelial malignancies. The first breakthrough in understanding the mechanisms of CIN came from studying different colon cancer cell lines that exhibited either a CIN or MIN phenotype.134 It is expressed at low levels during S phase in normal cells. X-linked DC is caused by mutations in dyskerin. In addition to bone marrow failure. heterochromatin structure. suggesting that the mechanisms that cause CIN act in dominant fashion. CIN. The role of telomere dysfunction in promoting cancer is further supported by findings in a rare genetic disorder. and nail dystrophy.139 In a single-cell cloning assay. and targeted disruption of hTERT.140 .134. should be distinguished from aneuploidy. stable karyotype. patients have very short germ line telomeres. Telomerase may also play important roles beyond telomere maintenance in the regulation of genome stability. DC is a progressive bone marrow failure syndrome that is classically associated with the clinical triad of abnormal skin pigmentation.

141. and CDC20 activates the anaphase-promoting complex to ubiquitinate substrates such as cyclin B and securin.145–147 Although. mutations in genes involved in sister chromatid cohesion.7). the significance of this mechanism in generation of CIN has not been definitively established. BUBR1.Chromosome Cohesion Defects Accurate chromosome segregation is achieved through carefully orchestrated interactions between the mitotic spindle. Sister chromatids are held together by a cohesin. as discussed in the next section. excessive separase could cause premature chromatid disjunction and subsequent chromosome missegregation events. 2. Spindle checkpoint. and include MAD1. a protein ring that physically links them together. organelles that are assembled onto centromeric chromatin. Recently. Once the final kinetochore is occupied by the spindle. BUB3. Additional evidence suggesting a role for the disruption of cohesion in the development of CIN comes from data demonstrating that some breast cancer tumors. were identified in colorectal tumors through the sequencing of human homologues of genes known to cause CIN in budding yeast. replicated sister chromatids are held together by cohesin. including subunits of the cohesion complex. in theory. and cohesin. Figure 2.148.150 To prevent chromosome mis-segregation. BUB1.144 The frequency and functional consequences of these mutations has not yet been tested experimentally. Kinetochores that remain unattached to the spindle catalyze the formation of an active MAD2 complex (―wait‖ anaphase signal) that binds and inhibits CDC20. The resultant proteasomal degradation of securin releases the enzyme separase to cleave cohesin and allow for sister chromatid separation under the tension of the mitotic spindle.7).149A number of spindle checkpoint proteins have been identified. MAD2. Although mutations in these spindle assembly checkpoint proteins do occur in human tumors it appears to be a relatively rare event. as well as osteosarcoma and prostate tumors.11. During metaphase.29 Replicated chromosomes attach to the spindle microtubules via the kinetochore. an organelle that is assembled onto centromeric chromatin.143 Most cohesin is lost from chromosome arms prior to metaphase in a manner that requires the Polo and Aurora B kinase. a protein ring that physically links the sisters. express high levels of separase. the spindle checkpoint proteins bind to kinetochores that are improperly attached to the spindle and form a ―stop‖ or ―wait‖ . and a BUB3related protein RAE1. 2. Prior to anaphase.7.142 The detailed molecular mechanism of how cohesin holds sisters together is a topic of much current research. kinetochores. Centromere cohesion is then lost after anaphase onset. the wait anaphase signal is lost. Spindle Assembly Checkpoint Defects Attachment of kinetochores to microtubules is monitored by the spindle checkpoint (Fig. initially via screens in budding yeast.29. paired sister chromatids attach to the bipolar mitotic spindle apparatus at kinetochores. a direct consequence of the protease separase cleaving a cohesin subunit (Fig.

Conversely. which results in three or more aneuploid .155.anaphase signal. Once all kinetochores are attached to the spindle. the wait anaphase signal is lost and CDC20 is released.158 Furthermore.156 Recent work has shown that phosphorylation of an Aurora B substrate at the kinetochore depends on its distance from the kinase at the inner centromere. the APC. an indication of successful biorientation. increasing the stability of kinetochore-microtubule attachments in the diploid cell line was sufficient to cause chromosome segregation defects at levels comparable to those in cancer cells with CIN. recent work demonstrated that kinetochore-microtubule attachments were more stable in cancer cells with CIN than in a noncancerous. diffusion of the activated signal throughout the cell. Activated APC then triggers mitotic cyclin degradation. It is important to note that although some CIN cell lines have a defective spindle assembly checkpoint (SAC). How this checkpoint signal is rapidly reversed is poorly understood. Thus. Early theories hypothesized that extra centrosomes generate CIN by promoting a multipolar anaphase. overexpression of proteins that cause increased kinetochore-microtubule dynamics was sufficient to restore stability to chromosomally unstable tumor cell lines. which reduces phosphorylation and stabilizes kinetochore microtubules.151 Kinetochore-Microtubule Attachment Defects Attachment of kinetochores to microtubules is necessary but not sufficient for proper chromosome segregation.157 The efficient correction of microtubule-kinetochore attachment errors requires the release of incorrectly attached microtubules.159 This work demonstrates that the temporal control of microtubule attachments to chromosomes during mitosis is central to genome stability in human cells. Supernumerary Centrosomes CIN is also known to be correlated with extra centrosomes. Ultimately the wait anaphase signal prevents cleavage of cohesin by separase. several live-cell imaging studies have demonstrated that most CIN cells actually have an intact and functional checkpoint. separase activation. In fact.151–153 Likewise. it was not until recently that a mechanism was proposed to link these two common characteristics of cancer cells. and binding of spindle checkpoint proteins to CDC20. both diploid and CIN cells underwent mitotic arrest in response to spindle poisons with equal efficiency. Although this relationship was long-standing. Repositioning Aurora B closer to the kinetochore prevents stabilization of bioriented attachments and activates the spindle checkpoint. interactions that inappropriately stabilize microtubule attachments might be expected to increase chromosome mis-segregation errors and generate CIN. and cohesin cleavage.154 Improper kinetochore-microtubule attachments that are not under normal tension are disassembled by a mechanism involving phosphorylation of kinetochore proteins by the Aurora B kinase. Thus. This involves a cascade of events that include activation of spindle checkpoint proteins at the kinetochore. centromere tension can be sensed by increased spatial separation of Aurora B from kinetochore substrates. diploid cell line. which is the key activator of the E3 ubiquitin ligase complex. in another study. The spindle checkpoint is also activated if kinetochores are attached but not under proper tension.

some changes are recurrent in a given tumor type. a causal role for aneuploidy in cancer progression has been controversial. that these cells with extra centrosomes had a significantly increased frequency of lagging chromosomes during anaphase. The second cell illustrates the clustering of centrosomes. ―CIN geometry‖ may be as important as ―CIN genes. 2. Note that the geometry predisposes to ―merotelic‖ attachments (see chromosome in the middle) where a single chromatid forms attachments to different spindle poles.168. and multiple childhood malignancies. Does Whole-Chromosome Aneuploidy Cause Cancer? Whole chromosome losses or gains are very common in human cancer.160–164 The imaging also demonstrated. Further analysis revealed that these segregation errors were a result of the cells with supernumerary centrosomes passing through a transient ―multipolar spindle intermediate‖ in which merotelic kinetochore-microtubule attachment errors accumulated before centrosome clustering and anaphase (Fig. live-cell imaging experiments.8). microcephaly.169 The identification of germ line mutations in the gene encoding BUBR1 in the disease mosaic variegated aneuploidy has provided the strongest evidence for a causal link between aneuploidy and cancer in humans because patients with this disorder exhibit growth retardation. though. Merotely. Premature chromatid separation is frequently seen in more than 50% of lymphocytes from patients with mosaic variegated aneuploidy and many tissues exhibit more than 25% aneuploid cells. Note that the merotelic attachment persists and does not activate the spindle assembly checkpoint. two common characteristics of solid tumors. which is required for cancer cell survival. for chromosome segregation. glioblastomas frequently exhibit loss of chromosome 10 and melanomas often show gain of chromosome 7. Deregulated expression of spindle .‖ Figure 2. revealed that cells with extra chromosomes typically cluster the extra chromosomes during mitosis to ensure that anaphase occurs with a bipolar spindle. was previously known to generate lagging chromosomes during anaphase and cause chromosome segregation errors. daughter cells are illustrated with resulting aneuploidy. however. have also been described in sporadic cancers and cell lines. Mutations in other components of the spindle assembly checkpoint. The first cell illustrates a cancer cell with extra centrosomes undergoing a multipolar metaphase. Cell three has undergone anaphase and the merotelic attachment results in a lagging chromosome. the gain of chromosome 3 or 3q is reportedly as common in cervical cancer as the Philadelphia chromosome is in chronic myelogenous leukemia. a conformation where a single kinetochore is attached to microtubules arising from opposite spindle poles.8.daughter cells. Lastly. consequently. including MAD1 and MAD2. Recent long-term. Thus. A mechanism linking extra centrosomes to chromosomal instability.167 Likewise. For example. Despite the fact that whole chromosome loss or gain is frequently observed in both solid and hematologic malignancies. provide a direct mechanistic link between extra centrosomes and CIN.73 Although the specific gains or losses vary from tumor to tumor.166 The results of these live-cell imaging experiments.165.

the cells switch to a state resembling G1 in which the DNA replication inhibitor geminin. making it difficult to isolate a primary mitotic defect as the cause of tumor formation in these mice. but cells eventually recover and fail cytokinesis in the face of persistent chromosome segregation errors. fibrosarcomas. Mitotic defects lead to spindle checkpoint activation. is re-expressed. This issue has been addressed by targeted deletion of CENP-E. This phenomenon is known as mitotic slippage. and infertility. Recent work has also identified a pathway linking telomere damage to tetrapoloidy.174 Over 50% of mice engineered to overexpress MAD2 exhibit tumors including hepatocellular carcinomas. including MAD2. but it is always difficult to know which cells to compare with the tumor cells. muscle atrophy. which is required for origin licensing. as restoration of normal MAD2 levels had no effect on subsequent tumor progression. or errors in spindle function and chromosome segregation. facile way to accumulate whole-chromosome aneuploidy is via a tetraploid intermediate. many models display an increase in tumors after carcinogen exposure.173. at least in theory.checkpoint proteins is potentially more common in tumors. Heterozygous loss of CENP-E results in an age-dependent increase in aneuploidy in mice. is degraded and Cdt1. Heterozygous wild type/null animals are viable. Eventually. MAD2 is not required for tumor maintenance in this setting. This observation is consistent with the idea that MAD2 overexpression triggers genetic instability during an early phase of tumor development.177 Consistent with this idea. but display increased aneuploidy. Although heterozygous checkpoint mouse models do not display a dramatic increase in spontaneous tumors. CENP-E heterozygosity also inhibits tumor formation in the setting of p19ARF loss. cataracts.178–180 Interestingly. may have additional functions outside mitosis. causes extensive chromosomal abnormalities associated with a wide variety of tumors in mice. This illustrates an important point that partial loss of spindle checkpoint function is biologically significant. they also illustrate that there is no simple one-to-one correspondence between checkpoint defects and cancer. Thus. spindle checkpoint genes. and lymphomas. many studies have linked tetraploidy to tumorigenesis. promoting subsequent self-sufficiency in tumor growth.181 Persistent telomere dysfunction in p53-deficient cells activates an ATM/ATR and Chk1/Chk2 signaling cascade that blocks entry into mitosis and extends G2. Mouse models support the notion that spindle checkpoint misregulation can contribute to tumorigenesis. cachectic dwarfism. with associated formation of splenic lymphomas and benign lung tumors.175 Nonetheless. mice with significantly reduced BUBR1 display an array of early aging phenotypes: reduced lifespan. a kinetochore-associated motor protein that is specifically expressed during mitosis. lung adenomas. which prevents re-replication in G2.170 An additional. suggesting that aneuploidy can act both oncogenically and as a tumor suppressor depending on the genetic context.150. Homozygous null mutations in spindle checkpoint genes are early embryonic-lethal.170–172 However. Furthermore. The tumor-suppressing effect of aneuploidy likely reflects the fitness cost from gene expression imbalance. which is commonly observed in sporadic tumors and can result from RB inactivation. However. Overexpression of MAD2. A number of cell-division defects can lead to cytokinesis failure and tetraploidy: errors in DNA replication or repair. in the face of persistent telomere or other DNA damage. .176.

work has shown that extra centrosomes promote CIN and chromosome mis-segregation through a transient multipolar spindle intermediate.162 Further evidence for this tetraploid intermediate model has also come from the study of progressive dysplasia in Barrett‘s esophagus. in this model. the mechanism by whichMAD2 overexpression generates aneuploidy appears to involve mitotic slippage. which reveals that early loss ofp53 is correlated with the development of tetraploidy and subsequent aneuploidy. generates additional karyotypic evolution by destabilizing the proteins that segregate. such as . and repair chromosomes. such evolutions generate new cancer-causing karyotypes. It has been hypothesized that the presence of an additional complement of normal chromosomes may enhance fitness by buffering against the effects of deleterious mutations. Extra copies of chromosomes could provide an advantage under certain selective pressures by increasing the expression of a single gene. Thus. Sporadically. resulting in the formation of tetraploid cells. or a combination of multiple genes on the aneuploid chromosome. this remains an open issue because little genomic instability has been detected at early stages in APC-deficient mouse models. synthesize. Another theory proposes that the driving force of tumorigenesis is the inherent instability of aneuploid karyotypes. recent studies suggest that genetic inhibition of cytokinesis or viral induction of cell fusion can promote tumorigenesis.86 However. as well as the acquisition of drug resistance by Candida albicans. aneuploidy associated with APC loss has been shown to result from a combination of defects in mitosis and apoptosis that results in an early stage of tetraploidy and polyploidy.186.169 According to this theory. which are stabilized by selection for their oncogenic function. non–whole-chromosome genomic instability. What is the Mechanism of Tumorigenesis? There are multiple theories of how whole-chromosome aneuploidy could promote tumorigenesis.184 Given that APC loss occurs early in colorectal cancer development and is associated with the majority of the 85% of sporadic cancers with CIN. it is possible that associated up-regulation of MAD2 promotes aneuploidy more generally in cancer cells via the production of unstable tetraploid cells.cells skip mitosis entirely and ―endocycle‖ between G1 and S phase. it is possible that genomic instability in this setting is related to the formation of a tetraploid intermediates. A variation on this theme suggests that aneuploidy can cause protein imbalances that generate additional.185 Finally. Cell fusion is also an additional mechanism by which tetraploidy is generated. which is initiated by either a carcinogen or spontaneously. CIN is generated in an autocatalytic fashion. Finally. tetraploidy itself may enhance genomic instability by the presence of extra centrosomes.179 Finally. a chromosome mis-segregation event.182 Moreover. producing tetraploid cells. chromosome rearrangements.161.183. and rapid tumor formation in a mouse breast cancer model.187 An alternative explanation for the tumor-promoting activity of aneuploidy is that the extra chromosomes buffer cancer cells against the effects of deleterious mutations in essential and haploinsufficient genes.173 Because RB pathway inactivation is fundamental to tumorigenesis. This type of mechanism has been described for budding yeast to adapt to defects in cytokinesis. As previously described. experimental inhibition of cytokinesis in p53-null cells results in the generation of whole-chromosome aneuploidy. In addition.

The possible roles for LOH in tumorigenesis include the duplication of oncogenes. once transformed. the fitness of the tumor cells may be compromised by the ongoing mutator phenotype. but this potentially comes with the cost of acquiring deleterious mutations that can impair fitness. At one end of the spectrum. As detailed in this chapter. resulting in a tumor with a stable genome.179 It is unclear. . result in normal development but a tissue-specific predisposition to cancer. later in tumorigenesis.the acquisition of transforming mutations. and BRCA. result in predominantly degenerative disease. Genomic instability can facilitate tumor development by accelerating the accumulation of such growth-promoting mutations. cell death. inherited cancer syndrome mutations such as mismatch repair gene defects in HNPCC can speed up the acquisition of critical growth-promoting mutations. recent studies suggest the ERCC1deficient lung tumors are more sensitive to the cytotoxic agent cisplatin. Aneuploidy may also contribute to tumorigenesis through loss of heterozygosity.92 However. the outcome of genomic instability can be cancer. or acquisition of improper epigenetic patterns.78 Perspectives and Implications for Cancer Therapeutics Oncogenesis represents an evolutionary process by which cells acquire successive genetic alterations that facilitate growth. Thus. For example. leading to genomic instability. some genome-destabilizing events are transient. and ultimately properties that allow for dissemination to distant sites. but it can also be tissue degeneration. whether these structural rearrangements are directly linked to the missegregation events. these genes may be reactivated.188 However. Another important consideration is whether conditions leading to genomic instability are present in cancers at diagnosis or are transient. are often noted alongside numerical chromosome abnormalities. which have more limited and overlapping roles in the DNA damage response. Likewise.77 As discussed earlier. Similarly. A major challenge for the field now is to elucidate the specific mechanisms and genetic interactions that tip the balance in one direction or the other. a wide variety of mechanisms can result in the generation of genomic instability. dicentric chromosomes. and aging. and doubleminute chromosomes. On the other hand. MSH2. which play critical roles in normal genome maintenance. such as nonreciprocal translocations. mutations in proteins such as the RecQ helicases. For instance. rampant aneuploidy is suppressed by telomerase re-expression. in sporadic tumors. Also. loss of tumor suppressors. manifesting premature aging phenotypes in addition to cancer development. although short telomeres can produce a crisis accompanied by gross chromosomal rearrangements. Thus. such as MLH1. but. destabilization of the genome can vary in the degree to which cancer promotion or tissue degeneration is favored. mutations in mismatch repair genes. it has been demonstrated in mice that Bub1 insufficiency can drive tumor formation through tumor suppressor gene LOH. though. structural chromosome abnormalities. hit-and-run events. loss of repair proteins such as ERCC1 in NSCLC may initially promote tumorigenesis. some FANC genes are methylated and silenced early in cancer progression. but the presence of these defects in mature tumors may provide a point of attack for certain chemotherapeutic agents. survival. In support of this model. For instance.

such as temozolomide and irinotecan. with a therapeutic window that is relatively narrow. Many cytotoxic agents. induce cancer cell killing through DNA damage. or prostate cancer. Tumor cell killing is at least in part correlated with p53 expression and the ability of cancer cells to undergo apoptosis in response to damaging agents. metastases and recurrences can have the same abnormal karyotype as the primary tumor. An elegant example of this approach has been demonstrated in vitro for BRCA1. Traditional cytotoxic chemotherapy combinations have largely been derived empirically. . or alternatively.194 Originally defined in yeast. Indeed.195 A recent phase 1 study reported that the orally active PARP inhibitor olaparib (AZD2281) is well tolerated and has few of the adverse effects associated with conventional chemotherapy. The profound sensitivity of BRCA mutant cells to PARP inhibition has led to the development of a number of clinical trials to test the efficiency of this approach.92 Moreover.190 Similarly. Indeed.189 However. the recent observation that restoration of wild type p53 function in mouse models of oncogenesis induces spontaneous tumor regression highlights the fact that some tumors become ―addicted‖ to p53 loss. extension of this concept to targeted cancer therapy may ultimately result in improved selective cancer cell killing with a wider therapeutic window. topoisomerase I inhibitors such as the camptothecins have been shown to have enhanced sensitivity in the setting of defects in the multiple protein components that respond to DSBs. with potential efficacy in other contexts in which HR or even other types of DNA damage responses are impaired. The apparent sensitivity of ERCC1-deficient NSCLC to platinum-based chemotherapy highlights this point.94 Deficiency or inhibition of PARP1 in normal cells results in impairment of the BER response. Exposure of cells lacking BRCA1 and BRCA2 to PARP inhibition results in the lethal accumulation of DNA damage. Ill-defined adaptations may enable many tumors to tolerate their altered genomes. the heterogeneity of response within tumor types also suggests that genome-destabilizing mutations present in the cancer genome may sensitize certain subtypes to specific cytotoxic agents. relates to the concept of synthetic lethality. cancer cells may be aneuploid. predispose to cell death in response to targeted inhibition of another pathway.196 Furthermore.18. If the major genome destabilization is transient.and BRCA2-deficient cells.93. causing lesions that would normally be repaired by BER to be channeled into the HR pathway. spindle checkpoint defects. as inhibition of PARP can potentiate the effects of numerous DNAdamaging agents.191. all of whom had ovarian.cytokinesis failure and tetraploidy may be transient early events. but stably aneuploid. objective antitumor activity was reported in patients with BRCA1 or BRCA2 mutations. Thus. if they become fixed in cancer cell populations.192 This idea. such as platinum chemotherapies. PARP inhibition appears to be selectively toxic for BRCA-deficient cancer cells. it is fairly common for every cell in an aneuploid tumor to have the same abnormal karyotype. PARP inhibitors are also being used in combination regimens.193. in which defects in one pathway facilitate sensitivity to DNA-damaging agents. may modulate the response to microtubule-based agents such as taxanes and vinca alkaloids. breast. Understanding the mechanisms of genome destabilization that are operative in specific tumors will likely have important consequences for cancer therapeutics.

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