Chromosome Instability in Human Lung Cancers: Possible Underlying Mechanisms and Potential Consequences in the Pathogenesis

Chromosome instability in human lung cancers: possible underlying mechanisms and potential consequences in the pathogenesis

Akira Masuda1 and Takashi Takahashi*,1

1Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan

Chromosomal abnormality is one of the hallmarks of isms that ensure genetic stability. Chromosome neoplastic cells, and the persistent presence of chromo- abnormalities can be classified broadly into numerical some instability (CIN) has been demonstrated in human (i.e., aneuploidy) and structural alterations (e.g., cancers, including lung cancer. Recent progress in deletion, translocation, homogenously staining region molecular and cellular biology as well as cytogenetics (HSR), double minutes (DMs)) of chromosomes. Such has shed light on the underlying mechanisms and the chromosomal alterations, which are indeed present in biological and clinical significance of chromosome most human tumors, are especially marked in solid abnormalities and the CIN phenotype. Chromosome tumors, including lung cancer, but the underlying abnormalities can be classified broadly into numerical mechanisms and the biological and clinical significance (i.e., aneuploidy) and structural alterations (e.g., dele- have not been elucidated to any satisfactory degree. tion, translocation, homogenously staining region (HSR), However, recent progress in molecular and cellular double minutes (DMs)). However, both alterations biology as well as technical development in cytoge- usually occur in the same cells, suggesting some overlap netics has shed light on these important issues related in their underlying mechanisms. Missegregation of to cancer development and progression, providing chromosomes may result from various causes, including evidence for the involvement of failures in the cell defects of mitotic spindle checkpoint, abnormal centro- cycle checkpoint control and defects in the DNA some formation and failure of cytokinesis, while double-strand break (DSB) repair system in the genesis structural alterations of chromosomes may be caused of chromosome instability (CIN). especially by failure in the repair of DNA double-strand In the present article, we will give a brief overview of breaks (DSBs) due to the impairment of DNA damage chromosomal alterations thus far reported in lung checkpoints and/or DSB repair systems. Recent studies cancer, and then summarize recent progress in the also suggest that telomere erosion may be involved. The understanding of possible underlying mechanisms of consequential acquisition of the CIN phenotype would CIN and its potential involvement in the development give lung cancer cells an excellent opportunity to of lung cancer, with reference to recent findings in the efficiently alter their characteristics so as to be more analysis of other human cancers as well as in those of malignant and suitable to their microenvironment, there- various organisms such as mice and yeast. by gaining a selective growth advantage. Oncogene (2002) 21, 6884 – 6897. doi:10.1038.sj.onc. 1205566 Numerical and structural chromosome abnormalities in human lung cancer Keywords: chromosome instability; cell cycle checkpoint; DNA double-strand break; telomere; lung Early in the 20th century, Theodor Boveri described cancer abnormal mitosis and chromosomal changes in cancers, and hypothesized that malignancy of the mammalian somatic cells resulted from an abnormal Introduction chromosomal constitution (Boveri, 1914). Later on, Philadelphia chromosome (Ph1) was identified in the High fidelity DNA replication and faithful chromo- majority of chronic myelocytic leukemia (CML), some segregation are fundamentally essential processes supporting Boveri’s hypothesis. To date, a large allowing cells to transmit their genetic information to number of additional non-random chromosomal the descendants. Failures in maintaining genetic translocations have been identified especially in tumors stability may cause their death or such abnormal of hematopoietic lineage (Heim and Mitelman, 1989). phenotypes as malignancy. Cancer cells frequently In contrast, in most malignancies of epithelial origin, exhibit marked chromosome abnormalities, which are including lung cancer, studies on the possible involve- generally considered to reflect defects in the mechan- ment of chromosomal alterations in carcinogenesis were hampered by technical limitations. Conventional karyotyping analysis principally requires the presence *Correspondence: T Takahashi; E-mail: [email protected] of mitotic cells in their preparations, but primary solid Chromosome instability in lung cancer A Masuda and T Takahashi 6885 tumors often provide too few mitotic cells. Further- random losses and gains of particular chromosomal more, the presence of very complex karyotypes in solid portions even in solid tumors. CGH enables precise tumor cells from numerical and structural points of analysis of losses and gains of chromosomal segments view often made it difficult to accomplish the by competitive hydridization between cancer and karyotyping analysis satisfactorily. In human lung normal DNAs on a karyotype spread of normal cells, cancer, nearly all cases show complex karyotypes with and thus does not require the abundant presence of multiple structural and numerical abnormalities (Testa mitotic cells in primary cancer cells (Kallioniemi et al., and Siegfried, 1992; Testa, 1996). Approximately 70 – 1992). CGH studies have revealed novel and histolo- 80% of lung cancer cases carry from a near triploid to gical type-specific gains and losses of chromosome tetraploid ranges of karyotypes with various structural segments in human lung cancers (Schwendel et al., abnormalities including deletion, non-reciprocal trans- 1997; Levin et al., 1994, 1995; Petersen et al., 1997a,b), location and isochromosome. DMs are also observed in addition to those previously reported by conven- in 20 – 30% of small cell lung cancer (SCLC) and 10 – tional cytogenetic approaches. 20% of non-small cell lung cancer (NSCLC). SKY is also a powerful technique, which is based on Fluorescence activated cell sorter (FACS) and image 24-color, whole chromosome-painting assay (Liyanage cytometric analyses can assess any overall increase or et al., 1996; Veldman et al., 1997). SKY makes it decrease in the DNA content of a tumor cell, not only possible to detect subtle karyotypic alterations, which in the mitotic phase, but also in the interphase, even would be otherwise overlooked, as well as to identify with fixed and paraffin-embedded specimens. Numer- the chromosomal origins of abnormalities, which ous studies have been reported on the relationships would have been designated merely as a ‘marker between abnormal DNA content reflecting aneuploidy chromosome’ in conventional karyotyping analysis. and various clinical features including histological type, To date, only a few SKY studies have been reported clinical stage, sensitivity to chemotherapy, and prog- in lung cancers, but these have resulted in the nosis. Although most of such studies have identification of a greater degree of chromosomal demonstrated the presence of markedly abnormal rearrangements than those detected by previous G- DNA contents in 65 – 85% of NSCLC and SCLC, banding and CGH analyses (Luk et al., 2001; Dennis the relationship between aneuploidy and patient and Stock, 1999). survival remains controversial. In this connection, As a consequence, it has become evident that the Choma et al. (2001) recently conducted a meta-analysis losses and gains of chromosomes in lung cancer are not of data obtained with 4033 NSCLC patients in 35 random. It is also notable that although lung cancers previously published studies, demonstrating that do share similar chromosomal abnormalities in patients with tumors with abnormal DNA content common, they also have certain histologic type-specific had a significantly shorter survival duration than those characteristics of chromosomal abnormalities. The with normal DNA content reflecting diploid or short arm of chromosome 3 (3p) was among the first psuedodiploid chromosomes. In contrast, the prognos- to be identified as a non-randomly affected chromoso- tic value of the degree of abnormal DNA ploidy does mal region in lung cancer (Whang-Peng et al., 1982). In not seem to be evident in SCLC (Oud et al., 1989; addition to 3p, it has been reported that chromosomal Jackson-York et al., 1991; Viren et al., 1997). gains are frequent in 5p, the long arm of chromosome Fluorescence in situ hybridization (FISH) analysis 8 (8q), 17q and 19q in NSCLC and 3q, 5p, 8p and Xq with centromeric probes of a specific chromosome(s) in SCLC, whereas chromosome losses are frequent in can obtain information of losses and gains of the 1p, 4q, 5q, 6q, 8p, 9p, 13q and 17p in NSCLC and 5q, specific chromosome in each cell of a whole tissue 13q and 17p in SCLC (Schwendel et al., 1997; Levin et section or cultured cells. Although it has been al., 1994, 1995; Petersen et al., 1997a,b; Michelland et suggested that cancer cells possess persistent CIN al., 1999). based on observations of the karyotypic heterogeneity There is considerable circumstantial evidence for even within the same tumor, it has remained rather the involvement of CIN in the initiation, progression, ambiguous as to whether chromosomes in cancer cells and metastasis of human malignancies. Notable are indeed unstable, which would reflect the presence examples are the existence of hereditary CIN of persistent CIN. Lengauer et al. (1997) demonstrated syndromes including the Ataxia telangiectasia (A-T), by means of FISH analysis that aneuploidy in colo- Nijmegen breakage, Bloom’s and Werner syndromes rectal cancer cell lines is not a result of a few past as well as Fanconi anemia, all of which are known to catastrophic processes of missegregations, but rather be predisposed to various cancers (Wright, 1999; that it reflects the persistence of an unstable karyotypic Vessey et al., 1999). A few mitotic checkpoint genes state. Haruki et al. (2001) have also demonstrated important for proper chromosome segregation have persistent CIN in human lung cancer cell lines with a been found to be mutated in human cancer cells notable association with aneuploid karyotypes. (Cahill et al., 1998; Nomoto et al., 1999; Percy et al., Recently invented novel means of cytogenetic 2000; Hernando et al., 2001; Gemma et al., 2000; analysis, such as comparable genomic hybridization Sato et al., 2000), while mice deficient in the Mad2 (CGH) and spectral karyotyping (SKY), have allowed mitotic checkpoint gene have been shown to a fine mapping of chromosomal losses or gains in specifically develop lung tumors with the CIN cancer cells, and have revealed clear pictures of non- phenotype (Michel et al., 2001). Furthermore, the

Oncogene Chromosome instability in lung cancer A Masuda and T Takahashi 6886 precise assay of chromosome aneuploidy in primary and ensuring a smooth progression through the cell tumors with G-banding, SKY and CGH have cycle (Hartwell and Weinert, 1989). Thus, defects in revealed the presence of specific chromosomal either cell cycle machinery or checkpoints are likely abnormalities, which correlate with distinct tumor candidates for the underlying mechanism of CIN type, suggesting their non-random nature (Seizinger et (Figure 1). In addition, other malfunctions of cells, al., 1991; Rooney et al., 1999). such as telomere erosion, may be involved in the What would then be the consequence of CIN in generation of CIN. In the following sections, these terms of its contributions to tumorigenesis and possible underlying mechanisms of CIN are discussed progression? Several possible explanations may be in reference to recent findings in lung and other human applicable, although it should be noted that these cancers as well as to those in various experimental explanations are not exclusive of one another: (1) CIN systems. alters chromosomal configurations, and increases the gene dosage of growth-promoting genes, such as oncogenes, and decreases the gene dosage of negative Numerical CIN and its potential causes regulators, such as tumor suppressor genes on the affected chromosomes. Cancer cell clones with such Mitosis consists of spatially and temporally organized advantageous changes would eventually develop pre- complex events including nuclear envelope breakdown, dominance during the tumor progression processes. centrosome-mediated nucleation of new microtubules, Gene amplification of members of the myc gene family condensation and movement of chromosomes, and may be one of the typical examples. (2) CIN cytokinesis. Failures in any of these events would accelerates the occurrence of loss of heterozygosity induce CIN (Figure 2). The mitotic spindle checkpoint (LOH) of tumor suppressor genes. In combination with is activated by kinetochores that remain unattached to inactivation of the other allele by genetic alterations, the spindle and delays the cell cycle at prometaphase such as point mutations or gene silencing due to until all chromosomes have established bipolar attach- aberrant hypermethylation or possibly due to the ment. Although the precise signaling mechanism of the naturally implemented genomic imprinting, it may lead mitotic spindle checkpoint remains to be clarified, to complete loss of expression of the affected tumor accumulating evidence points to an important role of suppressor genes, such as p16INK4A, p53 and Rb. (3) the Bub and Mad genes (Amon, 1999; Rundner and Global changes of gene expression may be caused by Murray, 1996). The presence of an unattached CIN, because either loss or gain of a whole or even a kinetochore is detected by a presently unknown portion of a chromosome simultaneously affects mechanism, and a complex of the Bub and Mad gene thousands of genes. Such massive changes in the gene products is then recruited to the unattached kineto- expression profile would provide a force sufficient to chore, transmitting an activating signal to Mad2 and alter the physiological balance of various cellular BubR1 (Shah and Cleveland, 2000; Wassmann and systems, such as cell cycle checkpoints, metabolic and Benezra, 2001; Hoyt, 2001). cell cycle machineries, and cell-to-cell communications. The Bub and Mad genes are conserved well in Since these may in turn lead to relaxation of the precise mammalian genomes as well as in lower eukaryotes control of cell growth and genomic fidelity, it would such as yeast, in which they were originally identified in consequently give cancer cells an excellent opportunity the analyses of mutants that failed to arrest in response to efficiently change their phenotypes so as to be more to microtubule-depolymerizing drugs. The activated malignant and suitable to their microenvironment. Mad2 inhibits anaphase promoting complex/cyclosome

Potential involvement of various malfunctions of cells in the induction of CIN

The consequence of CIN can be reﬂected as numerical and structural alterations of chromosomes, with the former often termed aneuploidy, while the latter includes deletion, translocation, HSR and DMs. These changes are not mutually exclusive, and in fact they are usually detected in the same cells, perhaps because of considerable overlap in the underlying mechanisms as we discuss below. The cells pass through an organized series of controlled events referred to as the cell cycle during proliferation. In addition to the machinery necessary for promoting cell cycle progression, cell cycle checkpoints are built-in as a monitoring system, Figure 1 A simpliﬁed view of signal transduction pathways involved in the cellular responses to DNA damage and mitotic which arrests cells at a particular phase of the cycle in spindle defect. Arrows and bars do not necessarily mean direct response to a lack of appropriate signals for cell cycle interactions between the proteins. Molecules, alterations of which progression, thereby maintaining the genetic stability have been reported in lung cancer, are indicated in bold

Oncogene Chromosome instability in lung cancer A Masuda and T Takahashi 6887

Figure 2 Possible defects of the mitotic machinery involved in the generation of missegregation of chromosomes. Failure of anchoring of mitotic spindle to kinetochore, abnormal centrosome formation, abnormal chromosome separation and failure of cytokinesis can cause missegregation of chromosomes and CIN

(APC/C), which is a multi-subunit E3 uniquitin ligase scarcity of alterations in the mitotic spindle checkpoint associated with proteosome-mediated proteolysis, genes may point to the possibilities of an inactivating through association with p55CDC. Since activation of mechanism other than mutation or an as yet APC/C, which promotes the degradation of Securin by unidentified major target gene responsible for the 26S proteosome, is a prerequisite for the sister- mitotic spindle checkpoint defects. It is also possible chromatid separation, the inhibition of APC/C conse- that there might be a large number of affected genes quently arrests the cell at prometaphase. each playing a role in a small proportion of cases. In Considering the importance of the mitotic spindle this connection, it is interesting to note that haplo- checkpoint in normal chromosome segregation, it is a insufficiency of the MAD2 gene resulted in a defective good candidate for the mechanism, defects of which mitotic spindle checkpoint in both human cancer cells may be involved in the genesis of numerical CIN. and murine primary embryonic fibroblasts (MEFs) and Indeed, Cahill et al. (1998) have reported that the that mad2 (+/7) mice developed lung tumors at presence of the CIN phenotype in colon cancer cell unusually high rates after long latencies (Michel et al., lines was tightly associated with dysfunction of the 2001). Frequent loss of heterozygosity in 4q, where mitotic spindle checkpoint. A significant fraction (up to MAD2 resides, has been reported in lung cancer 40%) of lung cancer cell lines also exhibit mitotic (Petersen et al., 1997a,b; Girard et al., 2000; Ullmann spindle checkpoint defects (Takahashi et al., 1999), but et al., 2001; Pei et al., 2001) as well as in other types of CIN in lung cancer appears to be less closely related to human cancers including Hodgkin’s disease, hepatocel- the presence of mitotic spindle checkpoint impairment lular carcinomas, and breast cancers (Dohner et al., than that in colon cancer (Haruki et al., 2001), 1992; Rashid et al., 1999; Shivapurkar et al., 1999). suggesting the possibility that cancer type-specific Several lines of evidence suggests the possibility that differences may exist in the major underlying causes inactivation of a colon tumor suppressor protein, APC, of CIN between these two common cancers of adults. may also lead to CIN. The C-terminal domain of APC Mutations in the mitotic spindle checkpoint genes binds to microtubules as well as to a protein called have been reported in a small fraction of several types EB1. It has been shown that the EB1 protein associates of human cancers including lung and colon cancers, at the spindle microtubules and centrosomes, and including MAD1 in lung cancer (Nomoto et al., 1999), functions in the cytokinesis checkpoint (Muhua et al., MAD2 in breast and bladder cancers (Percy et al., 1998). Recently, APC was also suggested to play a role 2000; Hernando et al., 2001), BUB1 in lung and in kinetochore-microtubule attachment, whereas trun- colorectal cancers (Gemma et al., 2000; Sato et al., cated mutations of APC, which eliminate microtubule 2000; Cahill et al., 1998), and BUBR1 in colon cancer binding, induce chromosomal missegregation and the (Cahill et al., 1998). Considering the frequent occur- CIN phenotype (Kaplan et al., 2001; Fodde et al., rence of mitotic spindle checkpoint impairment in lung 2001). Tighe et al. (2001) recently reported that (Takahashi et al., 1999), colon (Cahill et al., 1998), and introduction of a mutant APC into a non-CIN nasopharyngeal cancers (Wang et al., 2000) as well as colorectal cancer cell line with the intact mitotic possibly in other types of human cancers, the relative spindle checkpoint impaired mitotic delay following

Oncogene Chromosome instability in lung cancer A Masuda and T Takahashi 6888 spindle damage, providing additional support for its induction of CIN without accompanying centrosome possible involvement. Although p53 was once suggested abnormalities by conditional overexpression of cyclin E to play a role in the mitotic checkpoint, it is now well alone using tet/tTA gene-induction system. It was also accepted that p53 is involved in the post-mitotic reported that introduction of the Aurora-A centro- checkpoint, where it prevents endoreduplication (Lanni some-associated kinase, which is frequently and Jacks, 1998; Khan and Wahl, 1998; Notterman et overexpressed in multiple types of human cancers al., 1998). (Sen et al., 1997; Bischoff et al., 1998; Zhou et al., Scolnick and Halazonetis (2000) recently identified a 1998), induced abnormal centrosomes and transforms gene, named CHFR, which appears to coordinate rodent fibroblastic cell lines (Bischoff et al., 1998; Zhou another important phase of cell cycle progression, et al., 1998). Overexpression of other centrosome- i.e., mitotic prophase. Interestingly, they also identified associated kinases such as Aurora-B and Aurora-C has a loss of CHFR expression in one neuroblastoma and also been reported in human colon cancers (Katayama two colon cancer cell lines, although the molecular et al., 1999; Takahashi et al., 2000). mechanism underlying the inactivation remained The p53 tumor suppressor protein may also unclear. Mizuno et al. (2002) found that CHFR participate in the regulation of centrosome duplication. transcripts were undetectable in a fraction of lung Multiple copies of functionally competent centrosomes cancer cell lines and that this appeared to be due to are generated during a single cell cycle in MEFs aberrant hypermethylation of the promoter region of lacking p53 (Fukasawa et al., 1996), while introduction CHFR, although it remains to be clarified whether loss of p21, a target of p53-dependent transactivation, of CHFR directly contributes to the acquisition of partially restores the centrosome duplication control CIN. in p53-deficient cells (Tarapore et al., 2001). MEFs Defects or abnormal regulation of molecules consti- from mice lacking Gadd45a, another target of p53- tuting the mitotic machinery may also induce dependent transactivation, shows centrosome amplifi- missegregation of chromosomes, and thus they are cation, unequal segregation of chromosomes due to candidates for the causes of CIN in human cancers. multiple spindle poles, and the induction of aneuploidy Centrosomes, which consist of a pair of centrioles (Hollander et al., 1999). Ectopic overexpression of surrounded by a pericentriolar protein matrix, play a Mdm2, which inactivates p53, induces centrosome pivotal role in organizing the microtubule network in hyperamplification and CIN in Swiss 3T3 cells (Carroll interphase and the mitotic spindle at M phase. et al., 1999). Human papilloma virus-E6 (HPV-E6) Inappropriate centrosome duplication and multipolar oncoprotein, which inactivates p53, cooperates with the mitosis was first suggested by Boveri to be related to Rb-inactivating HPV-E7 oncoprotein, resulting in the the chromosome abnormalities seen in cancer (Boveri, induction of abnormal centrosomes, aberrant mitotic 1914). Abnormal centrosomes are commonly seen in a spindle pole formation and genomic instability (Duen- wide range of cancers including lung, breast, prostate, sing et al., 2000). Thus, the most frequently and widely brain, and colon cancers (Lingle et al., 1998; Pihan et inactivated tumor suppressor protein, p53, seems to al., 1998). Centrosomes of tumor cells display diverse have a regulatory role in the process of centrosome structural alterations including an increase in their duplication, and this may be one of the mechanisms by number and size, accumulation of excess pericentriolar which inactivation of p53 function contributes to the material, supernumerary centriole and inappropriate induction of CIN. phosphorylation of centrosome proteins (Salisbury et Changes in other molecules of the mitotic machinery al., 1999; Doxsey, 2001). Normal cells duplicate a may also cause missegregation of chromosomes and centrosome only once at the S phase during the cell consequently confer the CIN phenotype, when over- cycle, while cyclin E and cyclin A have been suggested expressed, mutated or functionally abrogated (Pihan to play a role in centrosome duplication (Meraldi et al., and Doxsey, 1999). While ectopic expression of 1999; Hinchcliffe et al., 1999; Lacey et al., 1999; Securin, a sister-chromatid separation inhibitor, trans- Matsumoto et al., 1999), possibly through nucleophos- forms NIH3T3 (Zou et al., 1999), up-regulation of min/B23 modification (Okuda et al., 2000). Increased Securin has been reported in some human tumors (Saez expression of pericentrin and g-tubulin, the compo- et al., 1999; Heaney et al., 2000). A human colorectal nents of centrosomes, are often detected in tumor cells, cancer cell line engineered to be securin deficient was suggesting their potential contribution to CIN through shown to be abnormal in chromosome segregation, and formation of disorganized mitotic spindles (Pihan et to lose chromosomes at a high frequency (Jallepalli et al., 1998; Shu and Joshi, 1995). al., 2001). Recent experimental evidence suggests relationships Oncogenes may also be involved in the genesis of between various genetic lesions and centrosome CIN. Introduction of ectopic vav-2 and mos oncogenes abnormalities in human cancers. While overexpression have been shown to induce cytokinesis abnormalities of cyclin E is common in human cancers (Keyomarsi by uncoupling of nuclear division and cytokinesis, and and Herliczek, 1997), overexpression of cyclin E and to frequently induce multinucleation and abnormal loss of p53 has been shown to synergistically induce spindles, resulting in CIN (Schuebel et al., 1998; centrosome hyperamplification and CIN in mouse Fukasawa and Vande Woude, 1997). Several lines of embryonic fibroblasts (MEFs) (Mussman et al., evidence also point to a role of mutational activation 2000), whereas Spruck et al. (1999) reported significant of the ras genes, which are known to be present in

Oncogene Chromosome instability in lung cancer A Masuda and T Takahashi 6889 certain types of human cancers including lung involved in the DNA damage checkpoints through adenocarcinomas. Abnormal mitotic figures, mainly various pathways, and the fact that p53 is one of the characterized by lagging chromosomes in prometa- most frequent targets for somatic alterations in various phase, and the resulting various ranges of aneuploidy types of human cancers including lung cancer indicates were frequently observed in NIH-3T3 expressing its important role as a guardian of the genome against activated human K-ras (Hagag et al., 1990; Nigro et various genomic insults. The ATM protein kinase al., 1996). Increased karyotypic alterations were also signals the occurrence of DNA damage to p53 by observed in a chromosomally stable human colorectal phosphorylated serine 15 of p53 as well as threonine 68 cancer cell line, SW480, in proportion to the expression of Chk2, an activated form of which in turn levels of ectopic human c-H-ras (de Vries et al., 1993). phosphorylated p53 at serine 20. These modifications stabilize, activate, and execute the effects of p53 by transcriptional activation of various target genes such Structural instability and its potential causes as p21 and 14-3-3s (Harper et al., 1993; el-Deiry et al., 1993; Hermeking et al., 1997). p21 inhibits various Complex structural aberrations of chromosomes are cyclin-dependent kinase activity including CDK2/cyclin frequently seen in common epithelial tumors of adults E, resulting in the cell cycle arrest at the G1/S including lung cancer, suggesting the presence of boundary. 14-3-3s excludes Cdc2/cyclin B from the persistent structural CIN, although yet to be formally nuclei, leading to cell cycle arrest at G2. p53R2, a novel proven by future experimentation. DSBs can be inducible form of a catalytic subunit of ribonucleotide introduced into the genome by intracellular stresses reductase, was recently identified as a downstream such as oxidative stress as well as by environmental target of p53 and was implicated in DNA repair, stresses such as ionizing irradiation. DSBs are the most providing intriguing evidence for a linkage between cell detrimental form of DNA damage, since they can cycle checkpoints and DNA repair by p53 (Yamaguchi cause erroneous rejoining of the broken genome, et al., 2001). It was recently proposed that cells severely conferring a high risk for gross chromosomal structural damaged to an unrepairable extent may be eliminated abnormalities such as deletion, translocation, HSR and by the p53-dependent activation of p53DINP1 and its DMs. As a consequence, loss of a chromosomal downstream molecule p53AIP1 (Oda et al., 2000; segment may uncover an inactivating mutation occur- Okamura et al., 2001). Among these downstream genes ring in the other allele of a tumor suppressor gene, of p53, inactivation of 14-3-3s due to aberrant whereas significant overrepresentation of a chromoso- hypermethylation of its promoter region has been mal region may lead to overexpression of proto- shown to be frequent in several common cancers of oncogenes and/or the induction of multidrug resistance adults including lung, breast, gastric, and hepatocel- against chemotherapy (Figure 3). lular carcinomas (Ferguson et al., 2000a; Suzuki et al., The DNA damage checkpoints prevent damaged 2000; Iwata et al., 2000; Osada et al., 2002). Although cells from starting DNA replication (the G1/S ATM appears to play a key role in the upstream of p53 checkpoint), from progressing with replication (the in the cellular response to DNA damage, somatic intra S checkpoint) or from going into mitosis (the G2 disruption of both alleles of the ATM gene is rarely checkpoint) (Rotman and Shiloh, 1999; Dasika et al., observed. In this connection, it is of note that somatic 1999). Mounting evidence indicates that the p53 gene is ATM aberrations were recently reported in sporadic B- cell chronic lymphocytic leukemia (Schaffner et al., 1999). In addition, individuals heterozygous for ATM mutations may possibly have an increased risk for cancer susceptibility (Khanna, 2000). The S phase checkpoint is less well understood in terms of its possible contribution to the generation of chromosomal aberrations. A recent study shows that the S phase checkpoint is activated at least in part through the ATM-Chk2-Cdc25A pathway and that overexpression of Cdc25A cancels the S checkpoint signal through inhibition of CDK2 activity, which is needed for DNA synthesis (Falck et al., 2001). Interestingly, overexpression of Cdc25A has been reported in 60% of NSCLC (Wu et al., 1998). The G2 checkpoint prevents cells from entering into mitosis with broken chromosome. If the G2 checkpoint fails to provide sufficient time for repair of DNA Figure 3 Cellular responses to DNA double-strand breaks damage before entering mitosis, cells will have to deal (DSBs). The occurrence of DSBs may be repaired by the activa- with a chromosome broken into the centromeric and tion of cell cycle checkpoints and DNA damage repair systems, whereas cells with extensive damage may be eliminated by the in- the acentric fragments. The fate of broken chromo- duction of cell death. Impairment of these systems can lead to somes in such a situation includes degradation, healing genomic instabilities including chromosomal aberrations as a truncated chromosome, and generation of a

Oncogene Chromosome instability in lung cancer A Masuda and T Takahashi 6890 translocated chromosome due to fusion between two chromosomes. Fusion between two centromere- containing chromosomes results in the formation of a chromosome with two centromeres, i.e., dicentric chromosome, which lacks a considerable portion of genetic material. Such a dicentric chromosome will be broken in the next mitosis, after which it fuses again, thus entering the highly unstable breakage – fusion – bridge (BFB) cycle (McClintock, 1938, 1940; Gisselsson et al., 2000) (Figure 4). BFB cycles are also thought to play a role in the chromosome rearrangement and gene amplification. Significant data has been accumulated during the past few years with regard to the molecules and signal transduction pathways involved in the G2 checkpoint (Taylor and Stark, 2001; Dasika et al., 1999; Khanna, 2000). Of particular relevance in terms of DNA damage response of this checkpoint is phosphorylation and subsequent activation of Chk1 and Chk2, which is thought to be mediated preferentially by ATR and ATM, respectively. Their activation leads to phosphorylation of Cdc25C, a phosphatase that normally activates Cdc2, and results in Cdc25C being bound to the 14-3-3 proteins and consequentially inactivated. It is of interest that Konishi et al. (2002) recently identified the presence of abnormal G2 checkpoint response in a significant fraction of SCLC cell lines as well as a homozygous deletion of 14-3-3e in a SCLC cell line. Another member of the 14-3-3 family, 14-3- 3s, which is transactivated by p53 in response to DNA damage, has been shown to have a role in the maintenance of G2 arrest through nuclear exclusion of the Cdc2/cyclin B1. Somatic knockout cells of 14-3- 3s show a loss of the normal G2 checkpoint response to DNA damage and an accumulation of chromosomal aberrations (Chan et al., 1999; Dhar et al., 2000). In this connection, frequent epigenetic inactivation of 14- 3-3s due to aberrant hypermethylation has been reported in lung cancer (Osada et al., 2002) as well as in a few other types of human cancers including breast, gastric and hepatocellular carcinomas (Suzuki et al., 2000; Ferguson et al., 2000a). Additional evidence for the involvement of molecules implicated Figure 4 The breakage – fusion – bridge (BFB) cycle resulting in the G checkpoint is provided by the demonstration from DNA double-strand breaks. (a), A broken chromosome is 2 replicated, resulting in two chromatids. After fusion of these chro- of germline CHK2 mutations in family members of the matids at their ends by NHEJ, the chromosome breaks again at a Li-Fraumeni syndrome, who do not carry p53 secondary site, and enters the BFB cycle. (b), Two broken chro- mutations in their germline. CHK2 has also been mosomes are fused with one another to form a dicentric chromo- shown to be somatically mutated in a small fraction of some with two centromeres. At mitosis, the chromosome breaks again at a secondary site, if the two centromeres are pulled to op- lung cancer (Haruki et al., 2000; Matsuoka et al., posite sides by mitotic spindles, and enters the BFB cycle. (c), A 2001). chromosome broken at plural sites is fused with itself to form a Two distinct and complementary mechanisms, which ring chromosome. Torsion or chromatid exchange during replica- are termed non-homologous end joining (NHEJ) and tion leads to the formation of a chromosome with two centro- homologous recombination (HR), are installed in cells meres. At mitosis, the chromosome breaks again at two secondary sites, when the two centromeres are pulled to opposite for repair of detrimental DSBs, which can cause sides by mitotic spindles, and enters the BFB cycle chromosomal structural abnormalities resulting from its erroneous rejoining of the broken genome. These two repair pathways differ in their requirement for a homologous template DNA and in the fidelity of DSB sequence (510 bp), termed microhomology (Khanna repair. In contrast to generally error-free HR, NHEJ is and Jackson, 2001; Ferguson and Alt, 2001) (Figure 5). an error-prone pathway that joins DNA ends with no Rad50-Mre11-NBS1 complex plays an important sequence homology or those with a short homologous role in both HR and NHEJ pathways by processing

Oncogene Chromosome instability in lung cancer A Masuda and T Takahashi 6891 breast cancer (Hiramoto et al., 1999; Matsuda et al., 1999). In the process of NHEJ repair, the DNA-dependent protein kinase (DNA-PK), which is a member of PI3- kinase related kinases consisting of a heterodimeric DNA targeting subunit (Ku70 and Ku80) and a catalytic subunit (DNA-PKcs), plays a role as one of the key components. DNA Ligase4 (Lig4) and XRCC4 catalyze the DNA ligation step in the process of NHEJ. Defects in any of Ku70, Ku80, XRCC4 or Lig4 have been shown to generate frequent chromosome and chromatid breaks, premature cellular senescence and apoptosis in their respective knockout mice (Bailey et al., 1999; Difilippantonio et al., 2000; Ferguson et al., 2000b; Gao et al., 2000; Karanjawala et al., 1999). Embryonic lethality and premature senescence of Figure 5 The double-strand break (DSB) repair by either non- fibroblasts in NHEJ-deficient mice can be rescued by homologous end-joining (NHEJ) and homologous recombination simultaneous deficiency of p53, and they develop (HR). NHEJ pathway (left). The Ku heterodimer binds to DNA ends and recruits DNA-PKcs, while Mer11/Rad50/NBS1 are progenitor B cell lymphomas (Difilippantonio et al., thought to have enzymatic and/or structural roles. The XRCC4/ 2000; Guidos et al., 1996; Vanasse et al., 1999; Frank Ligase IV complex joins the DNA ends. HR pathway (right). et al., 2000). LIG4 mutation was recently reported in a DSBs are initially processed by the Mre11/Rad50/NBS1 nuclease leukemic patient, who was highly radiosensitive complex. Rad52 protects DNA ends and RAD51 facilities DNA strand invasion. Intact DNA from the sister chromatid or homo- (Riballo et al., 1999). logous chromosome is used as a template Gene amplification of certain oncogenes, including members of the ERBB and MYC gene families, is occasionally present in various human cancers including lung cancer (Brison, 1993), where it is often DSBs prior to its repair. While NBS1 is phosphory- reflected as HSR and DMs in karyotyping analysis. lated by ATM in response to DNA damage, germline The frequencies of gene amplification increase in NBS1 mutations leads to Nijmegen breakage syndrome association with increase in tumor stage, suggesting (NBS), an A-T like hereditary cancer prone disorder that the event may be involved in tumor progression with CIN, developmental defects, and radiosensitivity rather than in initiation. Although the molecular (Petrini, 2000; Carney, 1999). MRE11 mutations have mechanisms of gene amplification are not fully under- been found in patients of another A-T-like disorder stood, it has been suggested that DSBs near the AT-LD as well as in a small fraction of breast and amplified gene, incorrect repair and the resulting BFB lymphoid tumors (Stewart et al., 1999; Fukuda et al., cycle may be involved in the genesis of gene 2001). ATM-dependent, c-Abl-mediated phosphoryla- amplification (Singer et al., 2000). Quantitative tion of Rad51 increases the association between Rad51 measurements have indicated that gene amplification and Rad52, which play a role in HR repair. It is of the CAD gene can be induced in response to N- notable that other proteins implicated in the orchestra- (phosphonoacetyl)-L-aspartate (PALA) treatment at a tion of a proper Rad51 response include the breast higher rate (1073 –1075) in cancer cells than that tumor suppressor Brca1 and Brca2. It seems that Brca1 (51079) in normal cells (Tlsty et al., 1989; Tlsty, interacts indirectly with Rad51, whereas Brca2 binds 1990). Notably, p53-deficient human fibroblasts have directly to Rad51 (Chen et al., 1998; Davies et al., been shown to be more vulnerable to CAD gene 2001). Both Brca1 and Brca2 co-localize with the Rad amplification than the proficient cells (Livingstone et proteins in radiation-induced foci (Scully and Living- al., 1992; Yin et al., 1992), suggesting that p53 ston, 2000). Embryonic lethality in Brca1- or Brca2- inactivation may play a role in this type of genetic deficient mice is attenuated by deficiency of p53 instability. (Hakem et al., 1997; Sharan et al., 1997). Loss of Another important clue for a better understanding of either Brca1 or Brca2 leads to aneuploidy in associa- the underlying mechanisms of CIN came from recent tion with centrosome amplification and chromosomal progress in the studies on telomere and telomerase, i.e., missegregation (Xu et al., 1999b; Lee et al., 1999). possible involvement of progressive telomere erosion in While tumors in Brca2-deficient mice have been shown the acquisition of CIN in cancer cells (Harley and to carry mutations in the mitotic spindle checkpoint Sherwood, 1997; Artandi and DePinho, 2000). Even genes Bub1 or Mad3L (Lee et al., 1999), loss of p53 though telomeres are essential for normal chromosome accelerates the formation of mammary tumors in mice structure and function, they cannot be fully replicated carrying conditional mutation of Brca1 (Xu et al., by the conventional DNA polymerase complex. 1999a). A DNA-dependent helicase Rad54, which plays Normal somatic cells show a progressive loss of their a role in a later step of HR in association with Rad51, telomere DNA during proliferation, whereupon they as well as its homologue RAD54B have been shown to enter replicative senescence, a non-proliferating, but be mutated in human lymphoma, colon cancer and metabolically active stable state, which often accom-

Oncogene Chromosome instability in lung cancer A Masuda and T Takahashi 6892 panies generation of polyploid cells (Sherwood et al., mation and tumors in nude mice (Hahn et al., 1999), in 1988). In contrast, it is presumed that premalignant/ contrast to the failure of anchorage-independent cancer cells, which have lost normal control of growth of fibroblasts, which were serially transduced proliferation due to the activation of oncogenes and with hTERT, E6/E7 and then activated H-ras (Morales inactivation of tumor suppressor genes, continue to et al., 1999). These findings suggest that the ordered proliferate, resulting in the extensive telomere short- occurrence of telomere crisis and subsequent re- ening. This may consequently lead to telomere expression of telomerase may be a requisite for association and subsequent formation of structurally malignant transformation. The precise molecular abnormal chromosomes, such as dicentric and ring mechanism involved in the re-expression of telomerase chromosomes. The resultant structurally abnormal in cancer cells remains to be elucidated, but several chromosomes are notably unstable by nature and possible candidates have recently been proposed. undergo the BFB cycle, providing the driving force Members of the Myc gene family directly transactivate behind continuous generation of structurally abnormal hTERT (Wang et al., 1998; Wu et al., 1999), while chromosomes (Counter et al., 1992). Mad, an antagonizing binding partner of Myc, Several lines of evidence suggest the occurrence of represses hTERT gene expression (Oh et al., 2000). telomere dysfunction in carcinogenesis. Human fibro- High hTERT mRNA levels have been suggested to be blasts senesce and cease proliferation, which is linked to myc gene overexpression in various human accompanied by activation of p53 as well as increased cancers (Latil et al., 2000; Bieche et al., 2000; Sagawa expression of p21, p16INK4A and p14ARF (Atadja et al., et al., 2001). Introduction of HPV-E6 also stimulates 1995; Kulju and Lehman, 1995; Alcorta et al., 1996; the expression of hTERT through activation of Dimri et al., 2000). p53- and Rb-inactivating oncopro- unidentified transcription factors that share the binding teins of viral origin such as SV40 large T, HPV-E6/E7 site with Myc and Sp1 (Klingelhutz et al., 1996; Oh et and adenovirus E1A/E1B drive presenescent cells into al., 2001). Interestingly, microcell transfer experiments extended proliferation and result in the crisis (Shay et have revealed that a gene or genes that represses al., 1991; Counter et al., 1992) (Figure 6). Lack of the expression of hTERT may be present on human p21 gene allows continued cell division despite chromosome 3p, which is among the most frequently increasing telomere dysfunction, leading to telomere altered regions in lung cancer (Ohmura et al., 1995; crisis in p21-deficient, otherwise normal human fibro- Cuthbert et al., 1999). blasts (Brown et al., 1997). Notably, telomerase- deficient mice of successive generational matings not only exhibit a subset of premature aging phenotypes Current understanding and perspective on future studies but also develop spontaneous tumors at a high rate, of the molecular basis of CIN in lung cancer suggesting a role of telomere erosion due to its extensive shortening (Rudolph et al., 1999; Artandi et There are many theoretically possible candidates for al., 2000). It is notable that serial retroviral transduc- the underlying mechanisms that would cause CIN in tion of primary human fibroblast with SV40 large T, lung cancer, if one takes experimental data in yeast activated H-ras, and then hTERT resulted in transfor- into consideration of human cancers. In this section, we will discuss the current status and perspective on the studies of potential underlying mechanisms of CIN in lung cancer as well as the direction of future studies, based on the molecular genetic and cytogenetic findings thus far identified in this tumor type. Many molecules or pathways can be nominated as a potential candidate for lesions, alterations of which may directly contribute to the acquisition of the CIN phenotype in human lung cancer. One such example would be an abnormal centrosome. It is well recognized that centrosomes are frequently over- represented in lung cancer with significant atypia in shape, size, and composition (Pihan et al., 1998). A fairly good correlation has been reported between centrosome abnormalities and the CIN phenotype in lung cancer cell lines (Haruki et al., 2001). As discussed Figure 6 Excessive shortening (erosion) of telomere and the oc- above, orderly duplication of centrosomes requires currence of cancer cells with CIN. Telomere shortening in primary human cells leads to p53 and Rb-dependent replicative regulated expression of cyclin E and cyclin A, and senescence, whereas cells with inactivated p53 and Rb continue overexpression of cyclin E has been shown to induce to divide, entering telomere crisis due to telomere erosion. Reac- centrosome abnormalities and numerical CIN in tivation of telomerase, which is observed in 80 – 90% of lung can- combination with loss of p53 in rodent embryonic cers, allows cells to escape from telomere crisis. Note that telomere erosion may lead to the formation of unstable chromo- fibroblasts (Spruck et al., 1999; Mussman et al., 2000). somes such as dicentric and ring chromosomes, which are suscep- In this context, frequent overexpression of cyclin E and tible to entering the BFB cycle (See text and Figure 4) cyclin A has been shown in lung cancer (Volm et al.,

Oncogene Chromosome instability in lung cancer A Masuda and T Takahashi 6893 1997; Dobashi et al., 1998; Mishina et al., 2000; additional molecule(s) that may account for the high Fukuse et al., 2000). In addition, frequent genetic and frequency of G2 checkpoint aberrations in SCLC as epigenetic alterations present in lung cancer may be well as to examine the contribution of the perturbed G2 associated with the induction of CIN in this scheme. checkpoint in the progression of lung cancer. Mutations in the RB gene, which are frequent Structural abnormalities in chromosomes are especially in SCLC (Harbour et al., 1988), may commonly seen in lung cancer, suggesting the existence conceivably result in the deregulated expression of of defects in the DSB repair systems. Although the cyclin E and cyclin A through the consequential E2F- potential involvement of defects in the DSB repair mediated transactivation (Harbour and Dean, 2000), system is conceivable, no direct evidence has yet been which would in turn confer CIN to lung cancer cells. obtained, and this is obviously an area requiring Inactivation of p16INK4A due to aberrant hypermethy- thorough future investigation. In addition, human lation, which is present almost exclusively in NSCLC, orthologues of various genes that, when altered, can may have similar consequences in terms of the result in CIN phenotypes in lower eukaryotes, such as generation of CIN, because loss of p16INK4A impairs yeast, would be excellent candidates for such as yet its negative regulatory function on the CDK4 activity unidentified genetic lesions (Chen and Kolodner, 1999). that inhibits Rb function. Accumulating evidence suggests that telomere Another important candidate is defects in the mitotic erosion and consequential formation of abnormal spindle checkpoint. Lung cancer cells lines have been chromosomes, such as dicentric and ring chromosomes shown to carry mitotic checkpoint defects up to 40% may also be involved in the acquisition of CIN as (Takahashi et al., 1999) and those with such defects discussed above. In this connection, telomere lengths of indeed exhibited persistent ongoing CIN (Haruki et al., lung cancer are often significantly shorter than the 2001). It is notable that despite relatively frequent normal case, and telomerase is re-activated in more mitotic spindle checkpoint impairment, mutations and than 80% of lung cancers (Hiyama et al., 1995a,b). It down-regulations of the mitotic spindle checkpoint should prove interesting to investigate the timing of genes are infrequent in human lung cancer (Nomoto et these changes in premalignant lesions, since the order al., 1999; Gemma et al., 2000; Sato et al., 2000). of occurrence of telomere crisis and re-expression of Collectively, these findings suggest that although the telomerase has been suggested to be crucial in terms of impaired mitotic spindle checkpoint may have a certain neoplastic transformation (Morales et al., 1999). role as a CIN-inducing lesion in lung cancer, we seem p53 mutations are among the most frequent genetic to have a missing piece(s) allowing a better under- lesions thus far identified in lung cancer (Takahashi et standing of how it is introduced. In this connection, al., 1989), and it has been shown that both inactivation Tighe et al. (2001) recently showed that abrogation of of p53 and aneuploidy are detectable even in the a colon tumor suppressor APC’s function in a non- dysplastic lesions of the lung (Sundaresan et al., 1992; CIN colon cancer cell line induced chromosomal Nuorva et al., 1993; Hirano et al., 1994). In addition, missegregation and the CIN phenotype despite reten- up-regulation of MDM2 and down-regulation of tion of a robust mitotic spindle checkpoint. They also p14ARF, both of which inactivate p53 function, are reported the presence of APC mutations correlated also detectable in a fraction of NSCLC (Higashiyama well with the CIN phenotype in colon cancer cell lines. et al., 1997; Vonlanthen et al., 1998). Accumulating Although APC mutations have not been reported in evidence, however, suggests that inactivation of p53 lung cancer, it would be an intriguing possibility that may be indirectly involved in the induction of CIN in alterations of the signaling pathway involving APC lung cancer by permitting cells to proliferate in the might play a part in the genesis of CIN in lung cancer. presence of defects, which directly lead to CIN. Haruki The recent finding of frequent G2 checkpoint et al (2001) recently reported that inactivation of p53 impairment in SCLC is of considerable interest in allowed lung cancer cells to go through an inappropri- terms of CIN as well as other aspects (Konishi et al., ate second division cycle under mitotic stresses, 2002). Cells with such a defect may well be susceptible resulting in the induction of the CIN phenotype in to loss of chromosomes in mitosis or initiation of the conjunction with the generation of aneuploidy. BFB cycles as discussed above, while up to 90% of Furthermore a similar permissive role of abrogation SCLCs carry p53 mutations. It has been shown that G2 of p53 function has been shown to exist in diverse checkpoint-overriding agents such as caffeine and settings, which might have relevance to the generation pentoxyphylline enhance the sensitivity of cells that of CIN in lung cancer. For example, p53 inactivation are defective in the G1/S delay to DNA damaging rescues knockout mice that have defects in DSB repair agents (Fan et al., 1995; Powell et al., 1995; Russell et machineries from their lethality (Difilippantonio et al., al., 1995). These findings may therefore be consistent 2000; Guidos et al., 1996; Vanasse et al., 1999; Frank with the notion that SCLC is sensitive to chemo- and et al., 2000), while telomerase deficient MEFs are more radiotherapy but often recurs after the initial successful readily transformable by cellular oncogene in coopera- treatment, showing aggressive clinical behavior. tion with p53 deficiency (Chin et al., 1999). It is Tr/Tr Although a few genes related to the G2 checkpoint interesting that growth defects in MEF from Brca2 have been found to be inactivated in lung cancer mice can be overcome and their cellular transformation (Haruki et al., 2000; Konishi et al., 2002; Osada et al., promoted by the expression of dominant negative 2002), further studies are necessary to identify an mutants of p53 or Bub1 (Lee et al., 1999). These

Oncogene Chromosome instability in lung cancer A Masuda and T Takahashi 6894 findings also reflect well the presence of p53 mutations carcinogenesis and progression. To answer these as well as either Bub1 or Mad3L mutations in tumors important questions, systematic studies with regard to arising in the Brca2Tr/Tr mice, suggesting a strong the molecular lesions involved will certainly be selective pressure to remove DNA damage and mitotic important. In addition, molecular correlative studies spindle checkpoints during the transformation on the relationship of clinical characteristics of lung processes, which in turn consequently leads to the cancer patients with the presence of CIN and/or the induction of CIN. Overexpression of anti-apoptotic lesions involved should give us important clues to a genes such as BCL-2 and Survivin in lung cancer might better understanding. Recent advances in the cutting provide the opportunity to resist apoptotic signals in a edge technology for molecular cytogenetic and cellular p53-independent manner (Pezzella et al., 1993; Monzo biological studies as well as the enormous amount of et al., 1999). Finally, but no less importantly, once information about the human genome should make it alterations of chromosomes are imposed in lung cells possible to further elucidate the molecular basis and by the above mentioned mechanisms, the resultant clinical importance of CIN, eventually leading us to the chromosomal defects themselves may provide the development of new therapeutic approaches for this driving force for maintaining persistent CIN because highly prevalent and aggressive form of cancer. of the BFB cycle and potentially abnormal number of chromosomes itself (Gisselsson et al., 2000; Duesberg et al., 1998; Matzke et al., 1999). Growing evidence suggests the possible biological and clinical impact of CIN in the pathogenesis of lung cancer. However, currently available information is far Acknowledgements from sufficient to envisage the precise roles of CIN. We We apologize for the omission of many important original papers because of limited space. We are grateful to all have yet to identify which are the major underlying members of our division for helpful discussion and mechanisms of CIN as well as when CIN is introduced comments on the manuscript as well as to surgical during the process of initiation and progression of this pathologists, clinicians and surgeons of our lung cancer fatal disease. Also, it is important to elucidate how the research group of the Aichi Cancer Center for continuing presence of CIN in lung cancer cells contributes to the fruitful collaborations for many years.

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