[CANCER RESEARCH 64, 2998–3001, May 1, 2004] Advances in Brief Three Classes of Genes Mutated In Colorectal Cancers with Chromosomal Instability Zhenghe Wang,1 Jordan M. Cummins,1 Dong Shen,1 Daniel P. Cahill,1 Prasad V. Jallepalli,1 Tian-Li Wang,1 D. Williams Parsons,1 Giovanni Traverso,1 Mark Awad,1 Natalie Silliman,1 Janine Ptak,1 Steve Szabo,1 James K. V. Willson,2 Sanford D. Markowitz,2 Michael L. Goldberg,3 Roger Karess,4 Kenneth W. Kinzler,1 Bert Vogelstein,1 Victor E. Velculescu,1 and Christoph Lengauer1 1Sidney Kimmel Comprehensive Cancer Center and Howard Hughes Medical Institute at Johns Hopkins University School of Medicine, Baltimore, Maryland; 2Howard Hughes Medical Institute, Department of Medicine and Ireland Cancer Center, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, Ohio; 3Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York; and 4Centre National de la Recherche Scientifique, Centre de Ge´ne´tique Mole´culaire, Gif-sur-Yvette, France Abstract occur in a higher fraction of cancers (9–12), but no definitive muta- tions of these genes have been identified, and their contribution to Although most colorectal cancers are chromosomally unstable, the CIN remains conjectural.5 basis for this instability has not been defined. To determine whether genes In contrast, a large number of genes have been identified that shown to cause chromosomal instability in model systems were mutated in Saccharomyces cerevisiae colorectal cancers, we identified their human homologues and determined trigger CIN when mutated in (13, 14). their sequence in a panel of colorectal cancers. We found 19 somatic These genes are involved in a variety of cellular pathways including mutations in five genes representing three distinct instability pathways. chromosome condensation, sister-chromatid cohesion, kinetochore Seven mutations were found in MRE11, whose product is involved in structure and function, microtubule formation, and cell cycle control. double-strand break repair. Four mutations were found among hZw10, Similarly, several genes can cause CIN in Drosophila melanogaster hZwilch/FLJ10036, and hRod/KNTC, whose products bind to one another when genetically altered (15, 16). Based on these observations, we in a complex that localizes to kinetochores and controls chromosome wondered whether the previous failures to detect mutations in poten- segregation. Eight mutations were found in Ding, a previously uncharac- tial CIN genes in human cancers were simply due to the fact that there terized gene with sequence similarity to the Saccharomyces cerevisiae are a large number of such genes, only a small number of which have Pds1, whose product is essential for proper chromosome disjunction. This been analyzed. analysis buttresses the evidence that chromosomal instability has a genetic basis and provides clues to the mechanistic basis of instability in cancers. Materials and Methods Introduction Gene Identification. Yeast genes that can cause an instability phenotype were identified in the Saccharomyces cerevisiae genome.6 The corresponding A very large fraction of cancers consists of cells with an abnormal protein sequences were used to search for human homologues in the Celera chromosomal content, called aneuploidy (1). Aneuploidy is often draft human genome sequence. In addition, several genes were selected by associated with chromosomal instability (CIN), a condition in which close homology to identified human genes and Celera hCTs or by membership cancer cells gain and lose whole chromosomes or large parts thereof to the same Panther protein family. All exons and adjacent intronic sequence at elevated rates compared with normal cells (2). The molecular basis of these genes were extracted from the Celera draft human genome sequence. PCR and Sequencing. Primers for PCR amplification and sequencing of CIN has remained mysterious. Many mechanisms have been pos- were designed using the Primer 3 program7 and were synthesized by MWG tulated to be responsible for CIN (3, 4). Like other phenotypes (High Point, NC) or IDT (Coralville, IA). PCR amplification and sequencing characteristic of cancer, it is possible that mutations in genes that were performed on tumor DNA from 24 early-passage cell lines as described control chromosome stability are responsible for CIN. However, only previously (17) using a 384 capillary automated sequencing apparatus (Spec- a small number of human cancers with mutations in genes known to trumedix, State College, PA). Of the 1351 exons extracted, 1282 were suc- cause experimental forms of CIN have been identified. These genes cessfully analyzed in an average of 23 tumor samples. Sequences of all primers include hBUB1, ATM, ATR, BRCA1, and BRCA2, each of which is used for PCR amplification and sequencing are available in Supplementary very infrequently mutated in nonfamilial cancers (5–8). Increased Table 1. For the five genes identified in the initial screen, coding exons were copy numbers of aurora2/STK15 and PLK1 have been reported to analyzed in tumor DNA from an additional 168 early-passage aneuploid colorectal cancer cell lines passaged in vitro or as xenografts in nude mice Analysis of Mutations. Sequence traces were assembled and analyzed to Received 2/18/04; accepted 2/27/04. identify potential genomic alterations using the Mutation Explorer software Grant support: The Virginia and D.K. Ludwig Fund for Cancer Research, The Benjamin Baker Scholarship Fund, The Clayton Fund, and NIH Grants CA 43460, CA package (SoftGenetics, State College, PA). 57345, and CA 62924. The costs of publication of this article were defrayed in part by the payment of page Results and Discussion charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We compiled a list of more than 1000 genes by computational Note: Z. Wang, J. Cummins, and D. Shen contributed equally to this work. D. Cahill identification of human homologues of “instability” genes of yeast is currently at Massachusetts General Hospital, Department of Surgery, Boston, MA; P. Jallepalli is currently at Molecular Biology Program, Memorial Sloan-Kettering Cancer and D. melanogaster. From this list, 100 candidate genes were se- Center, New York, NY; G. Traverso is currently at Trinity College, Cambridge, United lected based on the strength of the phenotypes observed in yeast or D. Kingdom; and M. Awad is currently at Department of Pediatrics, Johns Hopkins Univer- sity, Baltimore, MD. Supplementary data for this article can be found at Cancer Research Online (http://cancerres.aacrjournals.org). 5 While this paper was under review, it was shown that hCDC4 is frequently mutated Requests for reprints: Christoph Lengauer, Sidney Kimmel Comprehensive Cancer in aneuploid colorectal cancers, and that its inactivation causes CIN. (Rajagopalan et al. Center, Johns Hopkins University School of Medicine, CRB, Room 585, 1650 Orleans Nature 2004;428:77–81.) Street, Baltimore, MD 21231. Phone: (410) 955-8878; Fax: (410) 955-0548; E-mail: 6 http://ncbi.nlm.nih.gov/PMGifs/Genomes/yc.html. [email protected]. 7 http://www-genome.wi.mit.edu/cgibin/primer/primer3_www.cgi. 2998 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. THREE CLASSES OF GENES MUTATED IN COLORECTAL CANCERS Table 1 Chromosomal instability (CIN) candidate genes analyzed Table 1 Continued No. Celera accession Genbank accession Gene Nucleotides Exons No. Celera accession Genbank accession Gene Nucleotides Exons 1 hCT10388 NM_016374 BCAA 5,568 22 84 hCT32914 NM_021076 NEFH 2,102 3 2 hCT11285 SIRT2 1,917 15 85 hCT32971 NM_007068 DMC1 2,147 14 3 hCT11790 NM_012415 FSBP 3,021 15 86 hCT401149 NM_014586 HUNK 7,385 11 4 hCT12352 319 2 87 hCT6634 NM_007027 TOPBP1 5,255 28 5 hCT12678 NM_005883 APCL 10,749 17 88 hCT6664 PIK3C2A 5,061 32 6 hCT13183 NM_003579 RAD54L 2,533 18 89 hCT7084 NM_006219 PIK3CB 3,213 22 7 hCT13660 NM_002647 PIK3C3 2,975 25 90 hCT7133 NM_014840 ARK5 6,821 7 8 hCT14027 NM_002691 POLD1 3,271 26 91 hCT7448 NM_002646 PIK3C2B 7,610 34 9 hCT14094 NM_020439 CAMK1G 1,535 12 92 hCT7976 NM_002649 PIK3CG 5,309 11 10 hCT14327 1,502 1 93 hCT87379 742 5 11 hCT14628 NM_001348 DAPK3 2,157 9 94 hCT87385 6,078 3 12 hCT14647 NM_016539 SIRT6 1,636 8 95 hCT87415 NM_006231 POLE 1,560 13 13 hCT14856 NM_016195 MPHOSPH1 6,276 33 96 hCT8974 HCA127 2,156 5 14 hCT15239 NM_005732 RAD50 4,049 25 97 hCT9089 NM_002914 RFC2 1,517 11 15 hCT15320 2,308 15 98 hCT9098 NM_152619 MGC45428 3,222 14 16 hCT16364 NM_014915 KIAA1074 5,360 34 99 hCT9356 NM_172080 CAMK2B 1,441 16 17 hCT1642589 486 2 100 hCT9836 NM_006904 PRKDC 7,710 43 18 hCT1643619 1,031 2 19 hCT1643963 NM_001254 CDC6 1,961 12 20 hCT1644019 876 3 21 hCT1646711 NM_001340 CYLC2 2,088 7 melanogaster and the extent of similarity to the human homologue 22 hCT1657158 641 2 23 hCT16627 NM_005432 XRCC3 2,528 9 (Table 1). The complete sequence of these genes was then determined 24 hCT1686440 NM_134422 RAD52 2,691 12 in a panel of colorectal cancers. 25 hCT1686635 NM_058216 RAD51C 1,252 9 Public and private genomic databases were used to extract the 1351 26 hCT173001 4,889 26 27 hCT1766645 1,267 3 exons that encode the 100 candidate genes, and 5022 primers were 28 hCT1767458 NM_058216 RAD51C 849 2/9 designed for PCR amplification and sequencing (Supplementary Ta- 29 hCT1770914 1,750 14 30 hCT1775724 1,141 8 ble 1). Using these primers, each exon was then individually amplified 31 hCT17786 NM_014635 GCC185 4,498 18 and sequenced from DNA of 24 colorectal cancers. This analysis 32 hCT1783089 NM_003550 MAD1L1 936 7 revealed the presence of 373 variations not present in current human 33 hCT1786284 NM_016538 SIRT7 1,714 10 34 hCT1787138 NM_005026 PIK3CD 3,538 21 genomic databases. To find out whether these variations were somatic 35 hCT1788172 LATS1 3,723 5 (i.e., tumor specific), we determined whether any of them were 36 hCT17934 AA447812 SNRK 1,902 2 present in DNA from matching normal tissues of the patients in whom 37 hCT1816212 NM_001813 CENPE 8,263 50 38 hCT1817706 3,107 13 the mutations were originally detected.