Common and Distinct Genomic Events in Sporadic Colorectal Cancer and Diverse Cancer Types

Research Article

Eric S. Martin,1,3 Giovanni Tonon,1,3 Raktim Sinha,1 Yonghong Xiao,2 Bin Feng,2 Alec C. Kimmelman,1,4 Alexei Protopopov,2 Elena Ivanova,2 Cameron Brennan,8 Kate Montgomery,7 Raju Kucherlapati,7 Gerald Bailey,6 Mark Redston,6 Lynda Chin,1,2,5 and Ronald A. DePinho1,2,3

1Department of Medical Oncology and 2Center for Applied Cancer Science, Belfer Institute for Innovative Cancer Science, Dana-Farber Cancer Institute, 3Department of Genetics and Medicine, 4Harvard Radiation Oncology Program, and 5Department of Dermatology, Harvard Medical School, 6Department of Pathology, Brigham and Women’s Hospital, and 7Harvard Partners Center for Genetics and Genomics, Boston, Massachusetts; and 8Neurosurgery Service, Memorial Sloan-Kettering Cancer Center, Department of Neurosurgery, Weill Cornell Medical College, New York, New York

Abstract alone.9 Extensive genetic and genomic analysis of human CRC has Colorectal cancer (CRC) is a major cause of cancer morbidity uncovered germ line and somatic mutations relevant to CRC and mortality, and elucidation of its underlying genetics has biology and malignant transformation. These mutations have been advanced diagnostic screening, early detection, and treat- linked to well-defined disease stages from aberrant crypt prolife- ment. Because CRC genomes are characterized by numerous ration or hyperplastic lesions to benign adenomas, to carcinoma non-random chromosomal structural alterations, we sought in situ, and finally to invasive and metastatic disease, thereby to delimit regions of recurrent amplifications and deletions establishing a genetic paradigm for cancer initiation and prog- in a collection of 42 primary specimens and 37 tumor cell ression (1). lines derived from chromosomal instability neoplasia and Genetic and genomic instability are catalysts for colon microsatellite instability neoplasia CRC subtypes and carcinogenesis (2). CRC can present with two distinct genomic to compare the pattern of genomic aberrations in CRC with profiles termed (a) chromosomal instability neoplasia (CIN), those in other cancers. Application of oligomer-based array- characterized by rampant structural and numerical chromo- comparative genome hybridization and custom analytic tools somal aberrations driven in part by telomere dysfunction (3) and identified 50 minimal common regions (MCRs) of copy mitotic aberrations (2) and (b) microsatellite instability neo- number alterations, 28 amplifications, and 22 deletions. plasia (MIN), characterized by near-diploid karyotypes with alter- Fifteen were highly recurrent and focal (<12 genes) MCRs, ations at the nucleotide level due to mutations in mismatch five of them harboring known CRC genes including EGFR and repair (MMR) genes (4). Germ line MMR mutations are highly MYC with the remaining 10 containing a total of 65 resident penetrant lesions that drive the MIN phenotype in hereditary genes with established links to cancer. Furthermore, compar- nonpolyposis CRCs, accounting for 1% to 5% of CRC cases (4). isons of these delimited genomic profiles revealed that 22 of Although CIN and MIN are mechanistically distinct, their geno- the 50 CRC MCRs are also present in lung cancer, glioblasto- mic and genetic consequences emphasize the requirement of ma, and/or multiple myeloma. Among 22 shared MCRs, nine dominant mutator mechanisms to drive intestinal epithelial cells do not contain genes previously shown genetically altered in toward a threshold of oncogenic changes needed for malignant cancer, whereas the remaining 13 harbor 35 known cancer transformation. genes, of which only 14have been linked to CRC pathogenesis. A growing number of genetic mutations have been identified Together, these observations point to the existence of many and functionally validated in CRC pathogenesis. Activation of the yet-to-be discovered cancer genes driving CRC development, WNT signaling pathway is an early requisite event for adenoma as well as other human cancers, and show the utility of high- formation. Somatic alterations are present in APC in >70% of non- resolution copy number analysis in the identification of familial sporadic cases and seem to contribute to genomic ins- genetic events common and specific to the development of tability and induce the expression of MYC and CCND1 (5), whereas various tumor types. [Cancer Res 2007;67(22):10736–43] activating CTNNB1 mutations represent an alternative means of WNT pathway deregulation in CRC (6). KRAS mutations occur f Introduction early in neoplastic progression and are present in 50% of large adenomas (7). The BRAF serine/threonine kinase and Colorectal cancer (CRC) is the third most commonly diagnosed PIK3CA lipid kinase are mutated in 5% to 18% and 26% of spo- f cancer and ranks second in cancer mortality, with 106,680 new radic CRCs, respectively (8, 9). BRAF and KRAS mutations are cases and an estimated 55,170 deaths in the United States in 2006 mutually exclusive in CRC, suggesting overlapping oncogenic acti- vities (10). Mutations associated with CRC progression, specifically the Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). adenoma-to-carcinoma transition, target the TP53 and the trans- Requests for reprints: Ronald A. DePinho, Dana-Farber Cancer Institute, Harvard forming growth factor-h (TGF-h) pathways. Greater than 50% Medical School, 44 Binney Street, M413, Boston, MA 02115. Phone: 617-632-6085; Fax: 617-632-6069; E-mail: [email protected] and Lynda Chin, Dana-Farber of CRCs harbor TP53 inactivating mutations (7), and 30% of Cancer Institute, Harvard Medical School, 44 Binney Street, M446, Boston, MA 02115. Phone: 617-632-6472; Fax: 617-582-8169; E-mail: [email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-2742 9 http://www.cancer.org/

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Genomic Events in Sporadic Colorectal Cancer cases possess TGFh-RII mutations (11). MIN cancers consistently human CRC genomic profiles and integrated these profiles with inactivate TGFh-RII by frameshift mutations, whereas CIN cancers those from other cancers in an effort to identify common genes target the pathway via inactivating somatic mutations in the TGFh- and loci driving the pathogenesis of CRC and other human cancer RII kinase domain (15%) or in the downstream signaling types. components of the pathway, including SMAD4 (15%) or SMAD2 (5%) transcription factors (12). Numerous molecular, cytogenetic, copy number analyses, and re- Materials and Methods sequencing efforts have pointed to a large number of genetic and Cell lines and primary tumors. All of the primary tumors were genomic events that may underlie CRC pathogenesis. Recent re- acquired from the Brigham and Women’s Hospital tissue bank (Boston) sequencing of >13,000 coding sequences in breast cancer and CRC under an approved institutional protocol. The tumor histology was identified 189 genes with somatically acquired, nonsynonymous confirmed by pathologists (M.R., G.B.) before inclusion in this study. All mutations (so-called can-genes), the majority of which were not of the cell lines were obtained from the American Type Culture Collection. The characteristics of the primary tumors and cell lines are detailed in previously implicated in the neoplastic process (13). Similarly, high- Supplementary Table S1. resolution copy number alteration (CNA) analyses employing aCGH profiling on oligonucleotide microarrays. Genomic DNAs from bacterial artificial chromosome (BAC)–based array-comparative cell lines and primary tumors were extracted according to the manufac- genome hybridization (aCGH) have defined focal events, frequent turer’s instructions (Gentra Systems). Genomic DNA was fragmented and gains of 8q, 13q, and 20q, and losses of 5q, 8p, 17p, and 18q (14–19). random-prime labeled as described (20, 22, 23) and hybridized to human The pathogenetic relevance of these amplifications and deletions oligonucleotide microarrays. The oligonucleotide array contains 22,500 is inferred by their recurrence, presence of known cancer genes at elements designed for expression profiling (Human 1A V2, Agilent Tech- these loci, and alternative mechanisms targeting resident genes by nologies), for which 16,097 unique map positions were defined (National mutation or epigenetic means (13, 14, 16, 18). Center for Biotechnology Information (NCBI) Build 35). The median Together, these observations point to the possible existence of interval between mapped elements is 54.8 kb, 96.7% of intervals are <1 Mb, many genetic aberrations driving CRC development, the majority of and 99.5% are <3 Mb. Fluorescence ratios of scanned images of the arrays were calculated as the average of two paired arrays, and the raw aCGH which have yet to be defined. Establishment of robust oligomer- profiles were processed to identify statistically significant transitions in based aCGH and computational methods has enabled high- copy number by using a segmentation algorithm (20, 22, 23). In this study, resolution genome-wide analysis of CNAs using full complexity significant copy-number changes are determined on the basis of seg- genomic DNA (20–22). This approach has identified a large number mented profiles only. of highly focal recurrent amplifications and deletions in diverse Automated minimal common region definition. Loci of amplification human cancers, including non–small cell lung cancer, glioblastoma, and deletion are evaluated across samples with an effort to define minimal and multiple myeloma. Here, we have generated comparable common regions (MCRs) targeted by overlapping events in two or more

Figure 1. Recurrence of genomic alterations and chromosomal segment length distribution in sporadic CRC. Genome-wide percent recurrence of 37 tumor-derived colorectal cell lines (top) and 42 primary colorectal cancers (bottom). Integer value of percent recurrence of CNAs in segmented data (y-axis)is plotted for each probe aligned along the x-axis in chromosome order. Dark red bars, gain of chromosomal material; bright red bars, probes within regions of amplification. Dark green bars, loss of chromosome material; bright green bars, probes within regions of deletion.

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Table 1. List of high-priority MCRs in the CRC oncogenome

Cytogenetic band MCRs MCR recurrence HRF locus

Position (Mb)Size (Mb)Maximum/minimum Genes Gain (Amp)/loss (Del) value % T C

Gain and amplification 1q21.1-1q25.2 143.18–158.37 15.19 2.89 437 28 11 (5) 11 (5) 3q12.1-3q12.3 101.56–101.95 0.38 2.22 5 30 10 (3) 14 (4) + 6p21.2-6q12 39.26–41.10 1.84 1.96 11 22 12 (1) 5 (2) + 6p21.2-6q12 41.72–43.00 1.27 1.96 25 23 13 (1) 5 (2) 6p21.2-6q12 50.91–54.32 3.40 1.15 33 23 13 (2) 5 (2) 6q22.32-6q23.3 133.13–135.58 2.45 2.18 18 28 12 (1) 10 (4) 7p15.2-7p11.2 44.99–48.46 3.47 1.74 22 54 25 (5) 18 (4) 7p15.2-7p11.2 54.41–55.31 0.90 0.81 7 56 25 (4) 19 (5) + 7q11.23-7q31.1 105.26–107.15 1.89 1.18 16 48 22 (2) 16 (6) 8q22.2-8q22.3 100.03–103.64 3.61 0.93 26 43 23 (8) 11 (5) 8q24.12-8q24.12 120.92–121.53 0.61 5.55 5 48 25 (8) 13 (7) + 8q24.13-8q24.21 128.31–129.14 0.84 5.22 4 54 25 (9) 18 (13) + 8q24.21-8q24.23 130.92–136.68 5.77 1.15 20 47 25 (9) 12 (7) 12p13.33-12p13.31 0.17–6.67 6.50 0.93 80 24 7 (1) 12 (6) 12p12.1-12q13.12 24.61–27.46 2.85 1.53 25 28 7 (1) 15 (9) 13q12.11-13q14.12 19.14–33.30 14.16 5.08 120 65 28 (12) 23 (16) 13q21.33-13q22.3 72.22–73.15 0.93 1.56 7 62 28 (12) 21 (13) + 13q32.1-13q34 97.47–114.11 16.64 1.37 101 67 30 (10) 23 (14) 14q32.2-14q32.2 100.06–100.27 0.21 1.50 4 23 10 (5) 8 (1) + 16q11.2-16q12.1 45.52–47.13 1.61 1.24 11 28 11 (2) 11 (1) 16q21-16q23.3 63.53–65.35 1.81 0.95 15 23 10 (1) 8 (2) 17p11.2-17q12 21.83–23.84 2.01 1.17 30 25 8 (1) 12 (4) 19p13.3-19p13.3 0.94–0.98 0.04 0.92 4 16 9 (3) 4 (0) 20p13-20p12.1 2.26–3.84 1.58 0.89 40 42 19 (4) 14 (8) 20p11.23-20q11.23 25.22–29.52 4.30 0.97 21 63 24 (15) 26 (16) 20p11.23-20q11.23 30.08–30.49 0.41 0.97 9 65 25 (15) 26 (14) + 20q12-20q13.33 55.35–62.37 7.03 1.39 113 66 26 (15) 26 (17) 22q11.21-22q12.3 21.54–21.72 0.18 0.91 16 6 2 (1) 3 (1) Loss and deletion 2p22.1-2p21 42.46–47.52 5.06 1.40 45 8 4 (2) 2 (2) 4q12-4q13.1 57.51–57.73 0.22 1.22 5 34 14 (2) 13 (7) + 4q27-4q28.2 121.97–129.40 7.43 1.10 34 34 13 (2) 14 (8) 5q15-5q15 92.95–94.05 1.10 0.91 7 20 8 (1) 8 (2) + 6p25.3-6p24.3 0.29–0.35 0.06 0.88 2 19 4 (1) 11 (4) 8p23.3-8p21.3 0.18–13.99 13.80 1.09 152 49 19 (6) 20 (9) 8p21.3-8p12 21.60–38.38 16.78 1.04 134 46 19 (6) 17 (7) 8p12-8p11.21 39.08–41.23 2.16 1.31 11 25 7 (2) 13 (6) + 8p11.21-8p11.21 42.99–43.17 0.18 0.90 5 23 5 (2) 13 (5) + 8q22.3-8q23.1 104.51–108.33 3.82 0.83 11 6 1 (1) 4 (1) 10q25.2-10q26.11 114.82–115.79 0.97 0.86 9 11 4 (1) 5 (1) + 10q26.13-10q26.3 132.78–133.61 0.84 1.24 5 10 4 (1) 4 (2) 12q21.1-12q21.2 73.73–75.24 1.51 1.11 11 8 3 (1) 3 (2) 14q21.2-14q21.2 44.04–44.65 0.61 1.57 7 28 13 (1) 9 (2) + 14q32.2-14q32.2 98.93–99.26 0.33 0.80 5 28 12 (1) 10 (1) + 15q21.3-15q24.1 53.90–72.97 19.07 0.91 188 24 10 (1) 9 (1) 17q11.2-17q21.1 30.70–30.92 0.23 3.40 7 9 3 (1) 4 (1) 18p11.32-18q11.2 0.66–2.79 2.13 1.09 11 46 22 (5) 14 (9) 18p11.32-18q11.2 5.22–14.67 9.44 1.09 77 48 23 (5) 15 (9) 18q23-18q23 75.32–76.01 0.69 1.17 15 61 25 (6) 23 (16) 19q13.2-19q13.31 47.62–48.55 0.93 0.90 26 11 4 (2) 5 (0) 21p11.2-21q21.3 9.93–16.17 6.24 1.36 35 30 10 (2) 14 (3)

samples. An algorithmic approach is applied to the segmented data, as were PCR amplified, purified, and sequenced using standard protocols described in Supplementary Methods. at the Harvard Partners Center for Genetics and Genomics (primers avail- Sequencing, mutation, and immunohistochemical analysis. For able upon request). Chromatograms were assembled using the program mutation analysis of the KRAS,BRAF , and PIK3CA genes, coding exons SEQUENCHER (Gene Codes) and manually compared with the NCBI

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Table 2. CRC MCR candidate gene list and cross-tumor MCR overlap of CRC, lung adenocarcinoma, glioblastoma, and multiple myeloma MCRs

c b Chromosome MCR position HRF locus Known cancer Can-genes Candidate miRNA Lung MCR GBM MCRx MM MCRk (Mb) genes* cancer (hsa-mir-X) genes

Amplification 1 143.18–158.37 BCL9, ARNT, THBS3, IRTA2 9-1{ 150.95–151.92 142.60–152.10 AF1Q, TPM3, MUC1, PRCC, NTRK1, IRTA1, FCGR2B 3 101.56–101.95 + TFG 95.26–103.06 6 39.26–41.10 + CCND3 DAAM2 6 41.72–43.00 TFEB 6 50.91–54.32 PKHD1 ICK, 206, 133b49.91–52.16, 45.99–53.24 FBXO9 53.47–55.73 6 133.13–135.58 MYB EYA4 7 44.99–48.46 IGFBP3 7 54.41–55.31 + EGFR 54.41–55.94 54.41–56.27 7 105.26–107.15 PIK3CG 101.7–126.26 8 100.03–103.64 COX6C 8 120.92–121.53 + MTBP 120.50–126.52 8 128.31–129.14 + MYC 128.5–129.02 128.50–146.20 8 130.92–136.68 30b, 30d{ 128.50–146.20 12 0.17–6.67 CCND2, ELKS, 0.17–0.81 3.81–5.48 ZNF384 12 24.61–27.46 KRAS KRAS 24.61–26.95 15.02–37.98 13 19.14–33.30 ZNF198, CDX2, FLT3 13 72.22–73.15 + KLF5 13 97.47–114.11 FGF14, IRS2 111.77–112.97, 113.25–114.11 14 100.06–100.27 + DLK1 16 45.52–47.13 PHKB 16 63.53–65.35 CDH11 CDH5 17 21.83–23.84 19 0.94–0.98 0.24–11.19 20 2.26–3.84 PTPRA, CDC25B 103-2{ 20 25.22–29.52 20 30.08–30.49 + PLAGL2 20 55.35–62.37 GNAS, SS18L1 GNAS 296, 1-1, 57.04–60.21 133a-2, 124a-3 22 21.54–21.72 Deletion 2 42.46–47.52 MSH2 4 57.51–57.73 + REST 52.587–68.443 4 121.97–129.40 PRDM5, SPYR1 5 92.95–94.05 + 6 0.29–0.35 0.295–0.356 8 0.18–13.99 LOC157697 DLC1 124a-1 0.187–20.123 8 21.60–38.38 WRN PPP2CB, 320 28.74–33.48 22.078–33.573 DUSP4, DUSP26 8 39.08–41.23 + SFRP1 8 42.99–43.17 + 8 104.51–108.33 10 114.82–115.79 + TCF7L2, CASP7 TCF7L2

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Table 2. CRC MCR candidate gene list and cross-tumor MCR overlap of CRC, lung adenocarcinoma, glioblastoma, and multiple myeloma MCRs (Cont’d)

c b Chromosome MCR position HRF locus Known cancer Can-genes Candidate miRNA Lung MCR GBM MCRx MM MCRk (Mb) genes* cancer (hsa-mir-X) genes

10 132.78–133.61 BNIP3 TCERG1L, 128.9–135.241 PPP2R2D 12 73.73–75.24 PHLDA1 14 44.04–44.65 + C14orf155 BTBD5 14 98.93–99.26 + CCNK 90.27–101.47 15 53.90–72.97 TCF12 SMAD3, NEDD4 190 CYP1A1 17 30.70–30.92 18 0.66–2.79 18 5.22–14.67 PTPRM, 6.95–10.67 PTPN2 18 75.32–76.01 19 47.62–48.55 CEACAM1 47.78–48.56 21 9.93–16.17 TPTE 9.93–10.08 14.4–15.26 9.932–10.167

NOTE: Known cancer genes were derived from the Cancer Gene Census and/or have been previously implicated in CRC. Can-genes are from Sjoblom et al. (13), whereas non-CRC MCRs have been previously reported (20, 22, 23). *http://www.sanger.ac.uk/genetics/CGP/Census/. cSjoblom et al. (13). bTonon et al. (22). xMaher et al. (21). kCarrasco et al. (20). {Volinia et al. (34). reference sequence for each gene for the identification of possible muta- tumors [five of six BRAF mutant primary tumors show MSI, and all tions. Immunohistochemical analysis and evaluation of hMLH1,MSH2 , are MLH1 deficient (data not shown)]. and TP53 were done as described (24). A prominent feature of the CRC genomic profiles was the large number of complex CNAs (n = 251). The presence of CNAs Results targeting the same locus across different tumor samples and cell Recurrent CNAs in sporadic CRC. Forty-two primary CRC lines enabled the definition of a minimal common region (MCR) of tumors and 37 CRC cell lines were subjected to copy number gain/amplification or loss/deletion. To identify those MCRs with analysis employing a well-established oligomer-based aCGH potentially greater pathogenic relevance, MCRs were further platform and a modified circular binary segmentation methodol- selected based on the occurrence of at least one CNA in a primary ogy (see Supplementary Methods). The clinical and histopatho- tumor and at least one high-amplitude event at that locus (see logic characteristics of these samples, including microsatellite Supplementary Methods; ref. 22). This approach identified and instability (MSI) status, are summarized in Supplementary Table delimited 50 MCRs consisting of 28 recurrent amplifications with a S1, and the aCGH data are available online.10 The overall pattern median size of 1.86 Mb(range, 0.04–16.64 Mb)containing a total of genomic aberrations defined in our sample set agrees well with of 1,225 known genes (median of 19 per MCR) and 22 recurrent the frequencies of previously reported CRC amplifications and deletions with a median size of 1.31 Mb(range, 0.06–19.07 Mb) deletion (14, 15, 17–19, 25–29), many of which are consistent containing a total of 802 known genes (median of 11 per MCR; with patterns of allelic changes often observed in CRC (ref. 30; Table 1). A total of 8 of 42 (20%) primary tumors were determined Fig. 1). In addition, the analysis of KRAS,BRAF,TP53 , and PIK3CA to be MMR deficient (see Materials and Methods; Supplementary mutations indicated that mutation frequencies in our primary Table S1; data not shown), and their aCGH profiles were classically tumor and cell line sample set are in line with those of published MIN, containing fewer gross genomic changes and/or focal ampli- databases (Supplementary Table S2), although the PIK3CA fications and deletions relative to CIN samples (refs. 16, 18, 32; mutation frequency of 5% in our primary tumors is significantly Supplementary Fig. S1). less than that of 28% in our cell lines (P = 0.0095, Fisher’s exact test) The veracity of the aCGH data was confirmed by quantitative and 32% in a published report (31). Also, consistent with previous PCR, fluorescence in situ hybridization (FISH), and/or spectral reports (8, 10), we observed a mutually exclusive pattern of BRAF karyotyping analysis of randomly selected MCRs (n = 25; Sup- and KRAS mutations with BRAF mutations predominant in MIN plementary Fig. S2, data not shown) and by the presence of all previously identified human CRC loci (refs. 14, 15, 17–19; Table 1), including those targeting signature CRC genes like KRAS,MYC , and EGFR. Moreover, other known cancer genes or their homologues 10 http://www.ncbi.nlm.nih.gov/geo/; GEO Accession # GSE7604 not previously implicated in CRC resided within our CRC MCRs

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(Table 2). Additionally, 11 of 50 (22%) MCRs contain common MYC, and KRAS loci (Table 2). Eight of these CRC MCRs (6 of 28 proviral integration sites (data not shown), and 8 MCRs encompass amplifications; 2 of 22 deletions) overlapped with an MCR present 13 microRNAs (Table 2), several of which, hsa-mir-9-1,hsa-mir-30d , in two of the other tumor types (Table 2). The overlap of our novel and hsa-mir-103-2, are known to be aberrantly expressed in CRC MCRs with an MCR in other cancer types supports their multiple cancer types (33, 34), suggesting that these genomic cancer relevance. events may promote deregulated expression of these dominant Given the shared cross-tumor type MCRs, we asked whether this and recessive microRNAs in CRC. overlap might delimit further the CRC MCRs, using information We identified 15 highly recurrent (>10%) and focal (V12 MCR from other cancers, and select a more limited list of genes with resident genes) events designated high-recurrence-focal (HRF) potential cancer relevance to be enlisted in functional validation. MCRs: 8 amplicons and 7 deletions. The HRF MCRs show a mean Examination of Table 2 reveals that this approach allows for the recurrence of 35% among primary tumors and cell lines and reduction in size of several MCRs. For example, the chromosome 6 contain 101 known genes (Table 2). Several HRF MCRs contain CRC amplicon (50.91–54.32 Mb) is targeted in non–small cell lung known CRC genes, including SFRP1 (35), MYC (36), and EGFR (37), cancer (49.91–52.16 Mb) and multiple myeloma (45.99–53.24 Mb), as well as REST, a tumor suppressor gene identified in a genetic generating a common MCR (50.91–52.16 Mb) that contains the screen and found to be deleted and/or mutated in CRC (38). In CRC can-gene,PKHD1 (13), and the somatically altered ICK (43). addition, the 10q25.2-10q26.11 HRF MCR spans nine genes, includ- Such data integration not only strengthens the case for PKHD1 ing the WNT signaling mediator TCF7L2/TCF4 (39) and the effector and/or ICK in CRC pathogenesis, but also points to its potential CASP7 (40). Among the remaining 10 HRF MCRs with a total of 65 role in non–small cell lung cancer and multiple myeloma. A second genes (Tables 1 and 2), there are genes encoding cancer-relevant case is a focal deletion of chromosome 10 in CRC (132.78–133.61 functions. For example, the focal 13q21.33-13q22.3 amplification Mb), which enabled refinement of a large MCR (128.9–135.24 Mb) that contains the intestinal-enriched kruppel-like factor (KLF5), in multiple myeloma that was delimited to a region with only three a positive regulator of cellular proliferation and transformation genes: PPP2R2D,BNIP3 , and TCERG1L. Thus, cross-tumor compa- (41), and the focal 6p21.2-6q12 amplification, which contains risons can be useful in delimiting regions of potential interest for DAAM2, a homologue of DAAM1, a mediator of the WNT induced additional in-depth analysis. Dishevelled and Rho complex, a key regulator of cytoskeletal archi- tecture and cell polarity (42). Relationship of CRC MRCs to somatic can-gene mutations. Discussion In addition to known classic CRC mutations, we also examined the In this study, we defined regions of recurrent CNAs in a collection extent to which the CRC MCR gene list (Table 2) concurs with the of sporadic primary CRC specimens and tumor cell lines. The 50 list of 69 CRC can-gene somatic mutations identified in the recent CRC MCRs identified were notable for the presence of well-known re-sequencing study of 13,023 CCDS genes (13). Of the 2,027 CRC CRC genes (KRAS,EGFR , and MYC) and several CRC can-genes (13) MCR genes, 1,082 (53%) were part of the CCDS gene set. Notably, with a modest enrichment for CRC can-genes over breast can-genes, only 7 of 69 CRC can-genes are present on the list of 1,082 CRC presence of known cancer-relevant microRNA genes (hsa-mir-9-1, MCR genes (Table 2) with no enrichment for can-genes in the CRC hsa-mir-30d, and hsa-mir-103-2), and overlap with MCRs in other MCRs relative to the CCDS gene set (P = 0.249, Fisher’s exact test). cancer types (22 of 50 CRC MCRs). As a whole, these data indicate This observation suggests that the majority of cancer genes that many additional loci drive CRC pathogenesis and suggest that identified in these two data sets preferentially use distinct both common and distinct loci drive the biological processes mutational mechanisms, or alternatively, that the majority of can- needed for various cancer types to achieve their malignant end genes or MCR resident genes are not driving the tumorigenic point. process. Arguing against the latter, at least for the aCGH analysis, Chromosomal gains and losses previously identified using con- is the presence of known cancer genes within the MCRs identified ventional and lower resolution aCGH were further resolved in our in our data set previously linked to colon and other cancers, study. Frequent gains of chromosomes 7p, 7q, 8q, 13q, 20p, and cancer-relevant microRNAs, and common proviral integration sites 20q have each been previously documented at resolutions nearing in our data set as noted above. Next, we asked whether there was f1 Mbusing a BAC aCGH platform (15) and appear with any enrichment of CRC can-genes versus breast cancer can-genes in frequencies ranging between 43% and 67% in our sample set our CRC MCR resident gene list. The Sjoblom et al. re-sequencing (Table 1). The most frequent chromosomal gain, 13q (>62%), study identified 122 can-genes in breast cancer, of which only 2 contained an MCR (13q21-q22, 72.22–73.15 Mb) with only seven were also CRC can-genes (13). Subsequently, 4 of 122 (3.3%) breast resident genes, including the transcription factor KLF5. KLF5 is can-genes mapped to the CRC MCRs versus 7 of 69 (10.1%) CRC highly expressed in epithelia in regions of active proliferation can-genes (P = 0.0588, Fisher’s exact test). The modest enrichment and has been shown to promote cellular proliferation (44). KLF5 for CRC versus breast-specific can-genes within genomic regions binds directly to the 5¶ regulatory region of EGFR, which leads of alterations defined in CRC suggests that these mutation pat- to the transcriptional up-regulation of EGFR and the subsequent terns, and, by inference, our MCRs, may reflect distinct biologically activation of MEK/ERK signaling (44). Furthermore, the regulation relevant processes in these specific cancer types. of proliferation by KLF5 is dependent on EGFR and MEK/ERK Comparison of CNAs in CRC and other human cancer types. signaling because the proliferative response to KLF5 is blocked Finally, we asked whether any of our CRC MCRs overlap with, or by pharmacologic inhibition of EGFR or MEK. Inhibition of EGFR are distinct from, similarly derived MCR lists of other cancer types. or MEK also decreases KLF5 expression. Thus, KLF5 regulates Comparison with non–small cell lung cancer, glioblastoma, and MEK/ERK signaling via EGFR and is also downstream of multiple myeloma (20–22) MCRs revealed that 22 (13 amplifica- MAPK signaling, providing a novel mechanism for signal ampli- tions and 9 deletions) of the 50 CRC MCRs (44%) matched with one fication or suppression and control of proliferation in epithelial MCR in at least one of the other tumor types and include the EGFR, cells (44). www.aacrjournals.org 10741 Cancer Res 2007; 67:(22). November 15, 2007

An additional event of potential strong relevance to CRC patho- expression in CRC cells to attenuate WNT signaling even in the genesis is a highly focal (0.41 Mb) and highly recurrent (65%) MCR presence of downstream mutations (50). on chromosome 20 (20q12-20q13.33, 30.08–30.39) that contains Because most bona fide cancer genes are subject to alterations only nine resident genes including the putative oncogene PLAGL2. by multiple mechanisms, a priori selection of genes residing within PLAGL2 has been shown to cooperate with the CBFB-MYH11 regions of CNAs for focused re-sequencing represents a plausible fusion gene product in vivo in a mouse model of acute myelo- strategy. Subsequently, we determined the extent of convergence of genous leukemia (AML) and to promote S-phase entry and expan- somatically mutated candidate cancer genes (can-genes) identified sion of hematopoietic progenitors and increased cell renewal by Sjoblom et al. (13) and recurrent CNAs defined by our analysis. in vitro (45). PLAGL2, and its family member PLAG1, are over- We found that the majority of their can-genes (13) did not reside expressed in 20% of human AML samples (45). within our MCRs. However, the failure to establish a statistically Amplifications targeting chromosome 8q24 was also a very significant enrichment, or lack thereof, of somatically mutated recurrent event (>48%) in our data set and could be additionally genes in regions of genomic alterations may be due the use of small, resolved into two smaller regions (<0.85 Mb) of high-amplitude gain exploratory data sets and/or a limited number of genes sequenced. at 120.92–121.53 and 128.31–129.14 Mb. One region of amplification Interestingly, among the MCR-resident somatically mutated genes contains the MYC proto-oncogene whose deregulated expression identified by Sjoblom et al. (13), they were more likely to be CRC and activity in CRC has been linked variously to the aberrant can-genes when compared with breast-specific can-genes. activation of WNT signaling (46) and/or genomic amplification. Finally, our comparison of CRC MCRs with those similarly The other, more centromeric, 8q24 MCR contains five genes defined in lung adenocarcinoma, glioblastoma, and multiple including MTBP, which encodes a protein capable of binding to and myeloma identified 23 CRC MCRs common to at least one of stabilizing MDM2 and promoting MDM2-mediated degradation of these tumor types (20–22). The use of cross-tumor comparisons TP53 (47). Amplification and/or overexpression of MTBP locus may in defining highly informative MCRs can provide a useful means provide a cooperative or alternative mechanism to the inactivation in narrowing a list of candidate genes and in identifying genes of TP53, a hallmark of CRC pathogenesis. that, when targeted by either CNA and/or somatic mutation, may Complex cytogenetic and genomic rearrangements of distal impact a broad spectrum of tumors. Furthermore, the prioritiza- chromosome 8p that include allelic loss via mitotic recombination/ tion of these MCRs may provide a high-yield entry point for the loss of heterozygosity, translocation, and/or copy number loss are discovery of novel genes important in the development of a wide one of the most frequent events across a wide spectrum of epi- spectrum of cancers. thelial tumors (48). Recently, high-resolution aCGH analysis of several tumor types including colon carcinomas revealed several highly resolved, complex CNAs along chromosome 8p (17, 48). Acknowledgments Collectively, FISH, breakpoint mapping, and aCGH studies (48) Received 7/19/2007; revised 8/23/2007; accepted 9/25/2007. have implicated many possible candidate tumor suppressor genes Grant support: NIH grants to R.A. DePinho (U01 CA84313 and R01CA84628) and along distal 8p, including the WRN helicase on 8p12 (31 Mb). In our to L. Chin (RO1 CA099041 and RO1 CA93947) and NCI grant P50 CA127003-01 (R.A. DePinho and L. Chin). Genome-wide aCGH profiles were generated at the Arthur analysis, copy number loss of distal chromosome 8p ranged from and Rochelle Belfer Cancer Genomics Center at the Dana-Farber Cancer Institute. 23% to 49%, with two MCRs surviving the HRF criteria (Table 1). E.S. Martin is supported by National Cancer Institute 5T32CA09361. G. Tonon is supported by the International Myeloma Foundation, Brian D. Novis Research Award, Although the informative deletion of 8p12-p11 (39.08–41.23) in and by a Special Fellow Award from the Leukemia and Lymphoma Society. A.C. our data lies centromeric to the previously defined breakpoint Kimmelman is a recipient of the Leonard B. Holman Research Pathway fellowship. cluster of 8p12 (48), only 11 genes reside in this 2-MbMCR. One of R.A. DePinho is an American Cancer Society Research Professor. The costs of publication of this article were defrayed in part by the payment of page these genes, SFRP1, is of particular interest due to its preferential charges. This article must therefore be hereby marked advertisement in accordance hypermethylation in CRC (49) and the capacity of enforced SFRP1 with 18 U.S.C. Section 1734 solely to indicate this fact.

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