Three Classes of Genes Mutated in Colorectal Cancers with Chromosomal Instability

[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 mutations 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

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. We thereby discovered so- 39 hCT1817729 NM_012291 2,632 12 matic mutations in five genes. All five genes were then analyzed for 40 hCT1823014 NM_002916 RFC4 1,346 11 41 hCT1824077 466 2 mutations in a larger panel of tumors including 168 additional colo- 42 hCT1826039 NM_001184 ATR 1,071 7 rectal cancers. Altogether, more than 10 Mb of DNA was sequenced, 43 hCT1826040 NM_001184 ATR 1,644 8 allowing us to identify 19 somatic mutations distributed among three 44 hCT1829493 330 2 45 hCT1829782 618 2 classes of genes (Table 2; Fig. 1). 46 hCT18305 NM_022909 CENPH 744 9 MRE11. Eight somatic mutations in seven different CIN cancers 47 hCT1834200 603 3 were found in the MRE11 gene (Table 2). Four mutations generated 48 hCT18373 NM_004628 XPC 2,204 11 49 hCT18816 NM_133627 RAD51L3 1,567 9 premature translational stop signals within exons 7, 8, or 13 (Fig. 1), 50 hCT18916 NM_176827 SIRT4 1,154 3 whereas the others were missense mutations; all but one of these were 51 hCT1961597 NM_017975 FLJ10036 1,776 18 52 hCT19876 NM_002945 RPA1 2,393 17 heterozygous. MRE11 is known to be involved in DNA double-strand 53 hCT201497 1,603 5 break repair and participates in exonuclease and endonuclease activ- 54 hCT20446 NM_015070 DING 6,584 20 ities (18). Hypomorphic mutations in MRE11 have been described in 55 hCT20952 NM_000123 ERCC5 4,055 15 56 hCT21449 NM_018225 SMU-1 1,703 12 two families with ataxiatelangiectasia-like disorder (19). The cellular 57 hCT22552 NM_014791 MELK 2,470 18 features resulting from these mutations are similar to those seen in 58 hCT2308143 NM_014708 KNTC1 6,630 64 ataxiatelangiectasia as well as in patients with the Nijmegen breakage 59 hCT2334792 NM_005591 MRE11A 2,927 20 60 hCT23382 NM_002915 RFC3 1,477 9 syndrome and include hypersensitivity to ionizing radiation, radiore- 61 hCT23387 NM_004734 DCAMKL1 5,703 18 sistant DNA synthesis, and abrogation of ATM-dependent events. The 62 hCT23494 NM_012238 SIRT1 4,086 9 63 hCT23655 NM_006231 POLE 4,355 21 mutations listed in Table 1 represent the first report of MRE11 64 hCT23665 NM_004153 ORC1L 3,153 17 mutations in human cancers. MRE11 is known to form complexes 65 hCT24254 NM_002485 NBS1 4,388 16 with the hRad50 and NBS1 proteins, whose role in genomic stability 66 hCT28290 NM_133338 RAD17 3,164 17 67 hCT28652 NM_012241 SIRT5 1,012 5 is well documented. Recently published homozygous knockouts of 68 hCT28965 NM_133377 RAD1 1,870 8 MRE11 in vertebrate cells showed that this gene is essential for cell 69 hCT29050 NM_012239 SIRT3 1,862 7 proliferation and the maintenance of a normal chromosomal content 70 hCT29475 NM_032430 KIAA1811 2,109 18 71 hCT29790 NM_002913 RFC1 4,882 25 (20, 21). 72 hCT30161 NM_001274 CHK1 2,004 13 hZw10, hZwilch, and hRod. We found four somatic mutations in 73 hCT30207 NM_133487 RAD51 809 6 74 hCT30362 NM_002592 PCNA 1,300 6 the hZw10, hZwilch/FLJ10036, and hRod/KNTC1 genes (Table 2). 75 hCT30596 NM_006297 XRCC1 2,087 17 The mutation in hRod was a homozygous missense change (Fig. 1), 76 hCT30844 NM_004724 ZW10 2,340 16 whereas the heterozygous mutation in Zwilch affected a splice accep- 77 hCT30866 NM_177990 PAK7 3,546 9 78 hCT30904 NM_007370 RFC5 557 5 tor site predicted to result in a premature truncation of the Zwilch 79 hCT31391 NM_007057 ZWINT 834 9 RNA in its fifth exon. The other two mutations were heterozygous 80 hCT31470 NM_006768 BRAP 2,036 12 missense changes in hZw10 (Fig. 1). These three genes act in the same 81 hCT32115 NM_004111 FEN1 1,761 2 82 hCT32245 NM_007194 CHK2 1,741 14 pathway and cause a severe CIN phenotype when mutated in D. 83 hCT32452 NM_003292 TPR 7,500 51 melanogaster. Both Zw10 and Rod (rough deal) mutations disrupt the 2999

Table 2 Somatic mutations in chromosomal instability (CIN) cancers Nonsynonymous and splice site mutations observed in a panel of 192 colorectal cancers. Gene Celera accession Genbank accession Exon Nucleotidea Amino acidb MRE11 hCT2334792 NM_005591 7 G629G/A W210Xc 8 T747T/A C249Xc 13 C1375C/T Q459Xc 13 A1419A/C; G1378G/T L473F; E460Xc 15 T1568T/A M523K 17 C1885A Q629K 19 G2041G/A M675I ZW10 hCT30844 NM_004724 4 A368A/C N123T 13 A1867A/G S623G ZWILCH (FLJ10036) hCT1961597 NM_017975 4 Ϫ1G Ͼ G/A (pos. 202-1) 3Ј splice sitec ROD (KNTC1) hCT2308143 NM_014708 63 A6597C E2199D DING hCT20446 NP_055885.1 7 ϩ2T Ͼ C/T (pos. 1554ϩ2) 5Ј splice site 7 G647G/A S216N 9 G1243G/A E415K 13 A2529A/C E843D 14 C2605C/T R869Xc 14 T3202C S1068P 14 G3259G/A A1087T 15 C3766C/T R1254C a Nucleotide change resulting from mutation. b Amino acid change resulting from mutation. c Mutation results in a truncated protein.

Fig. 1. Somatic mutations in genes representing three distinct instability pathways. Position within gene and amino acid change resulting from the mutation are indicated below name of the gene (left column). Sequencing histograms and nucleotide sequences of normal and corresponding tumor DNA are shown in the middle and right columns, respectively. Affected triplets are boxed. 3000

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