Oncogene (2010) 29, 647–661 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Characterization of a major colon cancer susceptibility (Ccs3) on mouse 3

C Meunier1, J Cai1, A Fortin1,2, T Kwan3, J-F Marquis1, C Turbide4, L Van Der Kraak1, S Jothy5, N Beauchemin1,4 and P Gros1,4

1Department of Biochemistry, McGill University, Montreal, Quebec, Canada; 2Dafra Pharma International, Turnhout, Belgium; 3McGill University and Genome Que´bec Innovation Centre, McGill University, Montreal, Quebec, Canada; 4Goodman Cancer Centre, McGill University, Montreal, Quebec, Canada and 5Department of Laboratory Medicine and Pathobiology, St Michael’s Hospital and University of Toronto, Toronto, Ontario, Canada

Treatment of mice with the carcinogen azoxymethane Introduction (AOM) induces a number of lesions in the colon, including hyperplastic lesions, as well adenomas and carcinomas Colorectal cancer (CRC) is the second most common in situ. Inbred strains of mice show different responses to cause of cancer in Western societies. In the United AOM-induced carcinogenesis. A/J mice are highly suscep- States, there are an estimated 108 000 new cases of colon tible and develop a greater number of hyperplastic lesions and 41 000 cases of rectal cancer every year, with an and tumors (15–70 tumors per mouse) than resistant estimated fatality rate of 30% (www.cancer.gov/ C57BL/6J mice (0–6 tumors per mouse). Susceptibility to cancertopics/types/colon-and-rectal). CRC arises from AOM-induced tumors segregates as a co-dominant trait in a deregulation in the organization, proliferation, differ- (A  B6)F1 hybrids. Using a set of 23 AcB and BcA entiation and apoptosis of colonic epithelial cells. recombinant congenic mouse strains derived from A/J Pathogenesis is a progressive multistage process, which (susceptible) and B6 (resistant) parents, we observed that includes histological landmarks associated with altera- the number of hyperplastic lesions and tumors induced by tions in specific and biochemical pathways AOM was under different genetic controls in AcB/BcA (Rajagopalan et al., 2003; Wood et al., 2007). The strains. The multiplicity of AOM-induced tumors is earliest manifestation of colorectal neoplasia is the controlled by a major locus that we have mapped on the appearance of aberrant crypt foci (ACF), where a few distal portion of chromosome 3, to which we have given the colonic crypts may show presence of dysplastic cells. temporary designation colon cancer susceptibility locus 3 The ACF will progress to either hyperplastic or (Ccs3). B6 and A/J alleles at Ccs3 are associated with adenomatous polyps that represent benign tumors resistance and susceptibility, respectively. Haplotype ana- protruding into the lumen. Adenomas eventually pro- lysis in key informative AcB/BcA strains restricts the size gress to carcinomas that show major structural dis- of the Ccs3 locus to a 14 Mb segment that contains 94 organization and invasiveness. This sequence of events annotated genes. The expression level of all these genes in in most cases is due to mutations first in the normal colon has been established by transcript profiling Adenomatous polyposis coli (APC) whereby with microarrays, and has led to the identification of a normal glands form ACF. Additional mutations in the subset of positional candidates that are expressed at high RAS gene underlie progression of ACF to polyps and levels in this tissue. The 4q and 1p human chromosomal further alterations in SMAD2, SMAD4, DCC, p53 and segments sharing syntenic homology with the mouse Ccs3 other oncogenes or tumor suppressor genes support segment are known to be associated with inflammatory progression to invasive carcinoma (Rajagopalan et al., bowel diseases and colorectal tumors in humans, suggesting 2003; Wood et al., 2007). that the study of the mouse Ccs3 locus may help further the The onset, progression and ultimate outcome of CRC pathogenesis of these human conditions. is influenced by a combination of intrinsic predisposing Oncogene (2010) 29, 647–661; doi:10.1038/onc.2009.369; genetic factors, and extrinsic environmental factors published online 16 November 2009 including diet, lifestyle, chronic inflammation and others (Demant, 2003; de la Chapelle, 2004). A small propor- Keywords: colon cancer; Ccs3; genetics; azoxymethane; tion (o10%) of CRC cases show a hereditary and gene mapping highly penetrant genetic component. Patients with familial adenomatous polyposis (FAP) develop thou- sands of adenomas in the colon and rectum from late Correspondence: Dr P Gros, Department of Biochemistry, McGill childhood into adulthood, which if left untreated University, McGill Life Sciences Complex, Bellini Pavilion, Room 366, progress to aggressive carcinomas. Most FAP tumors 3649 Promenade Sir William Osler, Montreal, Quebec, Canada, result from germ-line mutations in the APC gene (de la H3G 0B1. E-mail: [email protected] Chapelle, 2004). Another hereditary syndrome asso- Received 24 December 2008; revised 23 August 2009; accepted 21 ciated with CRC is hereditary nonpolyposis colon September 2009; published online 16 November 2009 cancer (also called Lynch syndrome), which is caused Colon cancer susceptibility locus Ccs3 C Meunier et al 648 by germ-line mutations in the mismatch repair genes To investigate the genetic control of differential MSH2, MLH1, MSH6 and PMS2. In these hereditary susceptibility of susceptible A/J (A) and resistant syndromes, progression to CRCs often implicates C57BL/6 (B) to AOM-induced colon carcinogenesis, additional alterations in the APC, K-RAS, LKB1, we have taken advantage of a set of 35 reciprocal AcB/ SMAD4, AXIN2, BMPR1, CTNNB1, CCND1 and BcA recombinant congenic strains (RCS) derived in our p53 (de la Chapelle, 2004). Conversely, most cases laboratory from A/J and B6 parents (Fortin et al., (>90%) of CRC are sporadic with no prior family 2001). The AcB/BcA set (13 AcB and 22BcA) was history. Predisposition in these cases is thought to derived by systematic inbreeding from a double back- involve a complex and heterogeneous genetic compo- cross (N3), and each strain contains a small amount nent with a plurality of low penetrance genetic effects (12.5%) of DNA from one parent fixed as a set of further modified by environmental factors (Demant, discrete congenic segments on the background (87.5%) 2003). Indeed, recent whole-genome association studies of the other parent. The AcB/BcA set has been in thousands of familial and sporadic cases of CRC genotyped for 625 informative markers (average spacing from different populations have pointed to a complex 2.6 cM) and the position of all congenic fragments has genetic component with contributions from chromoso- been established. Individual resistance/susceptibility loci mal regions 8q24, 15q13, 18q21, 10p14, 8q23.3, 11q23, contributing to a simple or to a complex trait may have 14q22.2, 16q22.1, 19q13.1 and 20p12.3 (Tomlinson segregated in individual RCS and can be studied in et al., 2007, 2008; Zanke et al., 2007; Jaeger et al., isolation, both for gene-mapping and gene identification 2008; Study, 2008; Tenesa et al., 2008). experiments, but also for elucidating the contributions Although the identification of individual causative of single genes to the overall phenotype. The relatively genes in such complex traits is difficult in humans, the small size of the congenic segments fixed in individual genetic component of CRC susceptibility has been RCS facilitates the search and testing of candidate studied in mice, where CRC can be induced by chronic genes. exposure to the carcinogen 1,2-dimethylhydrazine We have tested 23 of the 35 AcB/BcA strains for (DMH) or its metabolite azoxymethane (AOM) susceptibility to AOM-induced CRC, as measured by (Dragani, 2003). These carcinogens induce different the multiplicity of tumors scored 19 weeks following types of lesions over time, including ACF, adenomas, initiation of AOM exposure. The AcB/BcA strains carcinoma in situ and adenocarcinomas (Suzui et al., segregated into two groups being either susceptible or 2002). Colorectal tumors induced by these carcinogens resistant, suggesting a simple genetic control of suscept- harbor genetic lesions similar to those found in human ibility. An analysis of the genotype/phenotype correla- CRCs, including mutations in Apc, K-ras and b-catenin tion indicates that a major gene on the distal part of (Takahashi and Wakabayashi, 2004). Inbred mouse chromosome 3 is responsible for the effect. strains vary dramatically in susceptibility to carcinogen- induced CRC, as determined by the number of tumors (>1 mm diameter) arising over time: strains such as A/J, FVB/J, ICR/Ha, SWR and STS/A being susceptible Results and strains such as C57BL/6, BALB/c, AKR and DBA/ 2J being resistant to DMH and/or AOM-induced CRC AOM-induced colorectal tumors in A/J mice (Evans et al., 1977; Deschner et al., 1988; Papanikolaou The AOM treatment used in our study (eight weekly et al., 1998; Nambiar et al., 2003). In F1 hybrids, injections, 10 mg/kg intraperitoneally) induced a num- susceptibility tends to be co-dominant, and segregation ber of microscopic and macroscopic lesions (Figure 1), analyses and whole-genome scanning have pointed to loci which were defined according to clinical standards. on 3 (CBA  C57BL/6 cross) (Angel et al., When colons were examined 19 weeks following 2000), 4 (B10.O20  O20 cross) (Fijneman and Demant, initiation of treatment, two types of macroscopic lesions 1995) and 12 (ICR/Ha  C57BL/6Ha cross) (Jacoby were detected (Figures 1A and B). These lesions were et al., 1994), as contributing in a strain-specific manner restricted to the distal portion of the colon and were to susceptibility to DMH-induced CRC. Additional rarely seen in the proximal portion. In addition, we did studies in congenic strains derived from BALB/cHeA not observe metastatic lesions to secondary sites. Flat and STS/A have suggested that as many as 15 loci (Scc1- lesions with clearly defined circular boundaries were Scc15, on chromosomes 1, 2, 4, 10, 11 and 18) may identified (Figure 1B), and these were scored as contribute to differential susceptibility to CRC in these hyperplastic lesions in a first quantitative measure of two strains (Moen et al.,1996;VanWezelet al.,1996; susceptibility (see below). Tumors (>1 mm diameter) Ruivenkamp et al., 2003), highlighting the complex protruding into the lumen of the colon, and showing nature of CRC susceptibility in mice. One such locus, different degrees of vascularization, were also detected Scc1, was identified by positional cloning as the Ptprj (Figure 1A). The multiplicity of these tumors was also tyrosine phosphatase (Moen et al., 1996). The human used as a second quantitative trait in our genetic PTPRJ gene was subsequently shown to undergo loss analyses. of heterozygosity in sporadic CRC (Ruivenkamp et al., Microscopic examination of hyperplastic lesions 2003), indicating that genes causing CRC in mice revealed the presence of ACF characterized by the may also be of clinical relevance for human CRC absence of mucin and large nuclei in the epithelial cells (Dragani, 2003). (Figure 1C,b). These lesions exhibited low-grade dys-

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Figure 1 Histological analysis of colon lesions induced by azoxymethane. A/J mice were exposed to azoxymethane (Materials and methods) and 19 weeks after initiation of treatment, colons were recovered, fixed in formaldehyde, sectioned, and stained with hematoxylin and eosin. Macroscopic views of the mucosal side of a longitudinal colon section showing hyperplastic lesions (B) and adenomas protruding in the intestinal lumen and showing different degrees of vascularization (A). (C) Microscopic view of mouse colon (a) normal colon showing vertically oriented epithelial glands with tubular morphology; epithelial cells in the upper segment of the glands have abundant mucin in their cytoplasm and a small basal nucleus. An example of aberrant crypt foci is shown in (b): epithelial cells lack strong mucin staining and show enlarged nuclei. An example of low-grade dysplasia of the colon is shown in (c): dysplastic glands have nontubular morphologies and are lined by epithelium with depletion of mucin and large, irregular nuclei (arrows). An example of high-grade dysplasia is shown in (d): the lesions are composed of markedly crowded and irregular cryptic cells with depleted mucin and pleomorphic nuclei. plasia with nontubular morphologies that are lined by conducted to optimize the efficacy of the AOM epithelium with depletion of mucin and large, irregular carcinogenesis protocol, and to establish conditions to nuclei (Figure 1C,c), or high-grade dysplasia with unambiguously discriminate between susceptible and neoplastic cells with depleted mucin and pleomorphic resistant mice, while minimizing discomfort to the nuclei (Figure 1C,d). On the other hand, the large experimental animals. tumors consisted mainly of adenomas and intramucosal In a first experiment, A/J and B6 mice were injected carcinomas. Adenomas were defined as proliferation of i.p. with AOM (10 mg/kg) once per week for 8 the colonic epithelium with low- or high-grade dysplasia consecutive weeks, and groups of animals (n ¼ 5) were (degree of cytological atypia and architectural complex- killed at weeks 10, 14 and 20. Colons were washed with ity). The designation of intramucosal carcinomas phosphate-buffered saline (PBS), dissected and exam- was used when there was dense crowding evidence of ined for the presence of either hyperplastic lesions and/ high-grade dysplastic epithelial cells packed in the or tumors that were scored (Figure 2a). AOM caused lamina propria, and characterized by prominent back- the appearance of hyperplastic lesions in both A/J and to-back glands and a cribriform architecture, but B6 colons, but their numbers were greater (by twofold) without invasion into the submucosa. Overall, the in A/J at 14 and 20 weeks (14 weeks, average of 3.2 for tumors we observed rarely breached the muscularis B6 vs 7.8 for A/J; Po0.05). AOM-treated A/J mice mucosa. developed tumors starting at 10 weeks (average 1.2 per mouse), and their numbers progressively increased at 14 weeks (average 6.6 per mouse; Po0.05) and up to 20 Differential response of C57BL/6J and A/J mice to AOM weeks (average 21.8 per mouse; Po0.01). At that time chemical carcinogenesis point, the distal colons of A/J were dotted with 20–30 Differential response of A/J (susceptible, S) and C57BL/ clearly identifiable tumors, an example of which is 6J (resistant, R) to AOM (Papanikolaou et al., 1998; shown in Figure 3. Over the same 20 weeks observation Nambiar et al., 2003) or DMH-induced (Evans et al., period, B6 mice did not develop tumors, and only a 1977) colon cancer has been known for many years. single tumor was detected in a B6 mouse at week 20 in However, the nature of the genetic control, about the this experiment. These results indicate that under these number and identity of the genes responsible, has treatment conditions, A/J mice are susceptible to AOM- remained unknown. A number of experiments were induced colon tumors whereas B6 mice are resistant.

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Figure 2 Differential response of C57BL/6J and A/J mice to azoxymethane (AOM) chemical carcinogenesis. Mice were treated with AOM (8 weekly intraperitoneal injections, 10 mg/kg), and at different time points, their colons were recovered and examined for the presence and numbers of hyperplastic lesions and tumors. (a) Incidence of hyperplastic lesions (left panel, empty symbols) and tumors (right panel, filled symbols) in colons from A/J (A, triangles) and C57BL/6J (B6, circles) at 10, 14 and 20 weeks following the first AOM injection. (b) A/J (triangles), B6 (circles) and (A Â B6) F1 hybrids (diamonds) were treated with AOM as described in a, and the numbers of hyperplastic lesions (empty symbols, left panel) and tumors (filled symbols, right panel) were recorded in individual animals 19 weeks following the first AOM injection. The presence of statistically significant intergroup differences (Po0.01) is indicated (**).

In a second experiment, A/J, B6 and (A/J Â B6)F1 Between 5 and 15 animals of each strain were treated hybrids were similarly treated and the presence of with AOM, and their colon were scored for the presence lesions was monitored at 19 weeks (Figure 2b) to of hyperplastic lesions and tumors. All strains were determine the mode of inheritance of the susceptibility tested at least twice. Representative examples of trait. On average, the number of hyperplastic lesions macroscopic images of treated colons are shown in found in A/J mice (average 7.0 per mouse) was greater Figure 3, and the entire data set for the 23 strains is than in B6 (average 3.6 per mouse; Po0.01), whereas F1 tabulated in Figure 4. hybrids showed numbers that were statistically similar When scoring for hyperplastic lesions, we segregated to A/J and distinct from B6 (average 6.6 per mouse; AcB/BcA strains into two groups according to the Po0.01) (Figure 2b, left panel), suggesting that strains genetic background, with all BcA strains showing susceptibility to AOM-induced hyperplastic lesions is numbers similar to the B6 parent (3ono6), and all AcB inherited as a dominant trait in (A Â B6)F1. A similar strains similar to the A/J parent (7ono11) (Figure 4a), analysis of tumor multiplicity at week 19 revealed very with no discordant strains. This suggests that the genetic few tumors in B6 (average 2.4 per mouse), high basis of the differential induction of hyperplastic lesions prevalence in A/J (average 41 per mouse; Po0.01) in A/J and B6 mice is probably complex, with multiple and somewhat intermediate tumor multiplicity in genes involved and with no single congenic fragment (A/J Â B6)F1 (average 21 per mouse), which was transferred from the donor strain capable of influencing statistically different from numbers recorded in either the phenotype of any of the AcB or BcA strains tested. parents (Po0.01). This suggests that susceptibility As expected from their genetic background (87.5% from to AOM-induced colon tumors is inherited in a co- susceptible A/J), most AcB strains tested (five of six) dominant manner in (A/J Â B6)F1 hybrids. developed tumors in numbers (12ono30) that were similar to parental A/J controls (Figures 4b and c), with the exception of AcB52 that had very few tumors (closer Response of AcB/BcA recombinant congenic strains to B6 controls). Conversely, BcA strains (87.5% from to AOM-induced colon carcinogenesis resistant B6) developed very few tumors, with the To study the genetic control of differential susceptibility exception of BcA71, BcA72 and BcA87 that developed of A/J and B6 mice to AOM-induced colon carcinogen- tumors in numbers (22ono35) similar to those detected esis, we phenotyped 23 of the 35 available AcB/BcA in parental A/J controls (Figures 3 and 4c). The resistant RCS. By virtue of the breeding scheme used in their phenotype observed in AcB52 implies the inheritance of derivation, the AcB/BcA strain set constitutes a power- B6 genomic segment(s) that suppresses the tumorigen- ful tool to characterize the genetic control of a A/J vs B6 esis otherwise observed on the A/J background. On the interstrain difference in a given phenotype, allowing (1) other hand, a reciprocal set of RCS, BcA71, BcA72 and efficient and accurate mapping of a major monogenic BcA87 showed susceptibility discordant with their effect (Fortin et al., 2001), and (2) dissection of a parental B6 control due to an A/J-derived segment, complex genetic trait into a series of simple genetic suggesting the segregation of a major cancer suscept- effects (Fortin et al., 2007; Min-Oo et al., 2007). ibility gene in those strains. In addition, strains BcA78,

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Figure 3 Azoxymethane (AOM)-induced colorectal tumors in AcB/BcA recombinant congenic mouse lines. Mice were treated with AOM (8 weekly intraperitoneal injections, 10 mg/kg), and 19 weeks following the first AOM injection, their colons were recovered, washed and photographed (n ¼ 3). Susceptible (S) A/J and resistant (R) B6 controls are shown along with resistant BcA68, and susceptible AcB62 and intermediate AcB58. The critical recombinant strains that delineate the Ccs3 interval, namely AcB52 (R), BcA71 (S), BcA72 (S) and BcA87 (S), are also shown.

BcA85 and BcA86 as well as AcB58 showed tumor treatment are under distinct genetic controls. Two scores (nB9–11) that were somewhat intermediate additional phenotypic variants were noted: (1) when between the two parental extremes, suggesting addi- calculating total tumor volumes per mouse strains tional genetic effects in these strains (Figure 4c). The (Figure 4d), BcA71 and BcA72 were clearly superior large tumor numbers noted in strains BcA71, BcA72 to the other strains with very large tumors protruding in and BcA87 and low tumor numbers in AcB52, despite the intestinal lumen (Figure 3); (2) although most sharing hyperplastic lesions similar in number to other tumors in AcB/BcA strains were found in the distal members of the respective BcA and AcB set and to end of the colon, AcB57 mice harbored tumors in the parental controls, together suggest that induction of proximal portion of the cecum (not shown). These hyperplastic lesions and colonic tumors after AOM results suggest rich phenotypic diversity in the AcB/BcA

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Figure 4 Response of AcB/BcA strains to azoxymethane (AOM)-induced colon carcinogenesis. A/J and C57BL/6J controls, and 23 AcB/BcA recombinant congenic strains were treated with AOM, and 19 weeks later their colons were examined for the presence of hyperplastic lesions and tumors that were enumerated. The average number of hyperplastic lesions (a) and tumors (c) per mouse strain is shown together with raw data for individual animal of each strain (b). The diameter of each tumor in each mouse was measured and used to calculate the total tumor volume per strain (d). For the purpose of establishing a bimodal strain distribution pattern (SDP) for linkage studies, a color code was used in (c) to stratify AcB/BcA lines with respect to tumor multiplicity. Relatively resistant BcA/AcB lines (resistant, o10 tumors per mouse) are shown as empty histograms, while relatively susceptible BcA/AcB (susceptible, >15 tumors) are shown as black histograms. The intermediate strains AcB58 strain is shown as a shaded histogram. The error bars correspond to the standard deviation of the mean.

Oncogene Colon cancer susceptibility locus Ccs3 C Meunier et al 653 set about the type and number of AOM-induced boundaries of the Ccs3 locus are defined by a tumors. recombination event on the proximal side in AcB52, occurring between markers D3Mit254 (pst. 131.99 Mb) rs30453708 Genetic mapping of a major colon tumor susceptibility and (pst. 133.81 Mb) and another one on the locus in AcB/BcA strains distal side in BcA72, occurring between markers D3Mit258 D3Mit292 We undertook to map the major locus controlling tumor (pst. 143.19 Mb) and (pst. 146.25 Mb). multiplicity in the AcB/BcA strains set. For linkage Ccs3 studies, we stratified AcB/BcA strains into two cate- The maximal physical interval of the locus stands at B14 Mb, (D3Mit254 to D3Mit292), and gories based on tumor numbers (Figure 5), being either low or intermediate ( 10 tumors) or high (>15 contains 94 annotated genes (NCBI Database). Among o those, there are three gene families, including a group of tumors). We left out of this stratification AcB58 that seven Adh genes (alcohol dehydrogenase; pst. 137.8– behaved as a true intermediate (nB9–11). This grouping produces a simple bimodal strain distribution pattern 138.2 Mb) that includes two pseudogenes, a cluster of five Gbp genes (guanylate-binding ; pst. 142.1– (SDP) that, when compared to SDPs of genetic markers 142.3 Mb) and a set of five Clca (calcium-activated tested in these mice, can localize the major gene effect chloride channels; 144.3–144.7 Mb). All other genes are controlling tumor numbers. Using this approach, we present at single copy in the interval. identified a perfect match between resistance and susceptibility to AOM-induced tumors and the haplo- type combination on the distal portion of chromosome Transcript-profiling studies 3, with the A/J haplotype associated with susceptibility As a first step in the identification of possible positional and the B6 haplotype associated with resistance candidates for Ccs3, we carried out transcript profiling (Figure 5). Importantly, the three discordant BcA with microarrays to detect which of the genes in the strains that show high tumor multiplicity, namely interval are expressed in the colon (Figure 6). Of the 94 Bc71, BcA72 and BcA87, all show A/J haplotypes in annotated genes in the interval, 48 showed detectable this region, whereas the only discordant AcB strain mRNA expression levels in colon, for at least one of the displaying low tumor multiplicity (AcB52) harbors a B6 probe sets on the array (Affymetrix, Santa Clara, CA, haplotype in this region (Figure 5, see labeled strains USA; mouse genome 430, version 2.0). Of these, 25 (*)). These results show that tumor multiplicity is were expressed at robust levels and represent strong controlled by a single locus in AcB/BcA strains, which positional candidates. Supplementary Table 1 presents has been given the temporary designation Ccs3, for the list of these genes, grouped in five functional colon cancer susceptibility locus 3. At present, the categories (transport, cell cycle/apoptosis, cell signaling,

Figure 5 Association of chromosome 3 haplotypes with susceptibility to azoxymethane (AOM)-induced colon tumors in AcB/BcA recombinant congenic strains. The haplotypes of AcB/BcA recombinant congenic strains for the distal portion of chromosome 3 region are shown along with the proposed location of the major Ccs3 locus. The informative markers used for genotyping are listed on the left together with the position of chromosome 3 (in Mb) and with the size of polymorphic fragments (for dinucleotide repeat markers) generated in A/J and B6 parental strains (http://genome.ucsc.edu/, February 2006 assembly). Alleles at each locus are identified as ‘1’ (A/J) and ‘2’ (B6). The maximum genetic interval for Ccs3 is identified. The phenotype of each strain with respect to tumor multiplicity is indicated at the bottom using the color code described in Figure 4. AcB/BcA RCS for which the phenotypic outcome is discordant compared to the genetic background have been identified (*). The recombinant inbred strain BXA12 was also phenotyped and found to be ‘resistant’; This strain shows an informative recombination event on the distal portion of chromosome 3, and was genotyped only for a subset of informative markers, which are shown.

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Figure 6 Transcript map of the Ccs3 interval. (a) Schematic representation of chromosome 3 that includes the position of the Ccs3 interval. (b) A compilation of all annotated genes along their physical position in the Ccs3 interval is shown. The level of expression of the corresponding mRNAs in normal B6 intestine, as determined by transcript profiling with microarrays, is shown as histograms (averages and standard deviation from three independent arrays from three individual mice; total three measurements). Histograms represent the signal intensity detected per probe set (log2 transformed). The dashed line across the histograms identifies genes that are strongly expressed in intestine, and that are considered positional candidates for Ccs3.

metabolism and structural) and including a summary (Gbp2, Gbp3 and Rap1gds1) and one cytokine regulat- annotation of the known function of the encoded ing activation of the monocyte/macrophage lineage proteins. Several of these proteins can be identified as (Scye1). strong positional candidates for Ccs3, based on We have also compared the level of expression of all their known function. These include two proteins, genes in the interval in normal colon from susceptible Bcl10 and NFkB1, which have been associated directly A/J vs resistant B6 mice (Figure 7a). Using a statistical with tumor initiation and/or progression, three cutoff of two-fold difference in expression (see Materials proteins involved in guanylate nucleoside metabolism and methods), we identified three differentially

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Figure 7 Expression differences of genes in the Ccs3 interval. Histograms of expression differences for all annotated genes on the exon array in the Ccs3 interval ordered by coordinate position for (a) A/J vs B6 strains and (b) A/J tumor-induced vs A/J normal strains. Expression difference is represented as log2-transformed fold change between the mean expression scores of the two strains (n ¼ 2 for each strain). In each comparative data set, genes containing probe sets showing differential expression suggesting alternative splicing (27 genes total) were identified by a & beside the gene symbol. expressed genes, namely Slc39a8 (solute carrier family levels of all genes in the interval in colon tumors 39 member 8; metal ion transporter) and Ddah1 (dimethy- vs normal intestinal tissue (both from A/J) was also larginine dimethylaminohydrolase 1; enzyme) that show carried out (Figure 7b). This analysis identified seven reduced expression in A/J whereas Gbp1 shows dramatic genes (9130221D24Rik, similar to guanyl nucleotide increased expression (>8-fold) in A/J compared to B6 exchange factors; Slc39a8, metal ion transporter; Adh1, normal colon (Figure 7a). A comparison of expression alcohol dehydrogenase; Gbp1/Gbp2, guadenylate binding

Oncogene Colon cancer susceptibility locus Ccs3 C Meunier et al 656 proteins; Clca3, calcium activated chloride channel; Finally, to look for the presence of alternatively Mcoln2, cation channel), expression of which is down- spliced exons in the interval (quantitative differences in regulated in tumors, and five genes (Dkk2, negative expression of normal mRNA variants from the same regulator of Wnt signaling; Dapp1, adaptor for phos- gene; genetic lesions abrogating normal splicing), we photyrosine and phosphoinositide; Tspan5,integral used exon arrays to measure expression of individual membrane ; Hs2st1, heparan sulfate 2-o-sulfo- exons from all the genes. Two large data sets were transferase-1 and Cyr61, cysteine-rich extracellular adhe- compared: normal A/J colon vs normal B6 colon, and sion protein), expression of which is increased in tumors A/J colon tumors vs normal A/J colon. These data are compared to normal tissue (Figure 7b). presented in Figure 8 and include statistical evaluation

Figure 8 Overview of probe set level (exon) expression in Ccs3 interval. Graphical representation of probe set expression levels for all core probe sets in the Ccs3 interval. (a) The DABG (detected above background) is a P-value indicating whether a probe set is expressed at a significant level above background. A P-value p0.05 (very short red bar) suggests the probe set is expressed, whereas P-values >0.05 (tall red bars) indicate the probe set is not expressed. Mean DABG P-values are shown for A/J, B6 and tumor A/J strains. (b) Expression scores for each probe set are shown in blue and represented as expression score (log2), again for A/J, B6 and tumor A/J strains. (c) Fold change expression scores for each probe set are shown in black and are represented as fold change (log2). Shown are fold changes for comparisons of A/J vs B6, as well as, tumor A/J vs normal A/J. (d) Relative locations of all known annotated RefSeq genes in the Ccs3 interval.

Oncogene Colon cancer susceptibility locus Ccs3 C Meunier et al 657 of individual exon expression (a), expression level (log2) AOM induced tumors almost exclusively in A/J mice of individual exons in normal B6 colon, normal A/J with very few detected in B6 (Figures 2 and 3). colon and A/J tumors (b), difference in expression of We have used AcB/BcA RCS to analyse the genetic individual exons (fold change (log2)) between normal basis of the differential response to AOM in A/J and B6. colon from B6 vs A/J and between colon tumors vs By virtue of the breeding scheme used in their normal A/J tissue (c) and the position of the genes in the derivation, AcB/BcA strains contain on average 12.5% interval (d). The entire data for all the probe sets genomic DNA from one parent (B6 in AcBs and A/J in (corresponding to core exons) on the arrays have been BcAs) that has been transferred to the other parental included as Supplementary Table 2. To identify poten- genetic background (A for AcBs and B6 for BcAs). This tial alternatively spliced transcripts in both comparative 12.5% ‘donor DNA’ is present on specific sets of small data sets, we selected individual probe sets showing congenic segments that are unique for every strain, and differential expression above three standard deviations the position of which has been delineated by genotyping (s.d.) from the mean fold change for all probe sets with (Fortin et al., 2001). RCS strains are extremely useful to each gene, as well as a minimum log2 ratio value of 1 study simple and complex traits. Indeed, if a phenotype (equivalent to two-fold change in expression). Genes in distinguishing A/J and B6 is controlled by a single gene, which >75% of probe sets showed differential expres- then its location will be easily determined by examining sion with these criteria were not included, as they most the haplotype of AcB and BcA strains showing a likely represent a whole-gene expression change rather discordant phenotype about parental genetic back- than an alternative splicing event. Overall, 27 unique ground, (for example, a BcA strain that behaves like genes (n ¼ 4 in B6 vs A/J, n ¼ 24 in tumor vs A/J) within A/J, and an AcB strain that behaves like B6), and the interval, including Rap1gds1, Gbp3, Dapp1 and identifying the syntenic segments commonly inherited in Dkk2 (Figure 7), contain probe sets showing differential these strains (Fortin et al., 2001; Tuite et al., 2005). This expression suggesting alternative splicing. is clearly the case in this study. In the case of monogenic The identification of genes in the Ccs3 interval that traits, the RCS strain set is more powerful than standard are (1) expressed in normal colon, and/or (2) differen- backcross or F2 mapping panels, to quickly and tially expressed in normal A/J vs B6 colon, and/or (3) reproducibly delineate the chromosomal boundaries of differentially expressed in normal colon vs colon tumors the major locus, as multiple animals from the key strains and/or (4) which show evidence of alternate splicing in (bearing informative recombinant haplotypes) can be any of these combinations, now provides guidance for sampled at will. In the case of multigenic control, single prioritization of positional candidates for further study. gene effects contributing to a polygenic trait may have segregated from one another and become fixed in individual strains, allowing one to study their contribu- tion and mechanism of action in isolation (Min-Oo Discussion et al., 2007). In addition, simple examination of the SDP of the RCS set for multiple subphenotypes contributing Chemical carcinogenesis in mouse models provides a to a trait distinguishing A/J and B6 parental strain may powerful tool to study initiation and progression of not only reveal the functional architecture of these CRC. Both DMH and its active metabolite AOM have subtraits, but also help determine which ones are been used to induce colon tumors (Dragani, 2003). determined by the same or different genetic control. Although DMH treatment by i.p. injections induces Finally, unique reassortment of parental haplotypes and tumors at a number of anatomical sites, AOM is more emergence of novel mutations during the breeding of the specific and causes lesions only in the colon. Our RCS (Van Wezel et al., 1996; Min-Oo et al., 2003; treatment protocol induced two types of lesions, namely Fortin et al., 2007) may provide additional phenotypic hyperplastic lesions that were detected as early as 10 diversity in the study of a complex trait. weeks after initiation of treatment, and tumors that were In this study, we observed that all AcB strains tested found in permissive animals starting around 14 weeks had similar number of hyperplastic lesions and that after initiation of treatment. These tumors consist in these numbers were approximately twice as high as the adenomas and in situ carcinomas, and a proportion of number of such lesions found in BcA strains, with no AOM-induced colon tumors have been shown to carry obvious discordant strains in either group. The situation loss-of-function mutations in Apc and K-ras (Suzui was different for the number of AOM-induced tumors. et al., 2002; Takahashi and Wakabayashi, 2004; data Indeed, the majority of BcA were B6-like (low tumor not shown), highlighting the relevance of this model in multiplicity) with the notable exception of BcA71, the study of human tumors. Inbred strains of mice differ BcA72 and BcA87 that were discordant (high tumor drastically in susceptibility to DMH or AOM chemical multiplicity). Similarly, the tested AcBs were A/J-like carcinogenesis (Evans et al., 1977; Deschner et al., 1988; with the notable exception of AcB52 that was discordant Papanikolaou et al., 1998; Nambiar et al., 2003). In this and B6-like. A number of conclusions can be reached study, we carried out a systematic analysis of the genetic from this data set. First, the different SDPs of AcB/BcA control of differential response to AOM chemical mice for the number of AOM-induced hyperplastic carcinogenesis in A/J and C57BL/6J mice. We observed lesions vs number of colorectal tumors indicate that the that AOM induced hyperplastic lesions in both mouse two traits are under different genetic controls in A/J and strains, but these were more numerous in A/J. However, B6. The different genetic control of AOM-induced

Oncogene Colon cancer susceptibility locus Ccs3 C Meunier et al 658 preneoplastic lesions and colon adenomas noted herein the Bcl10/Malt1 complex is involved in NF-kB activa- in the AcB/BcA RCS has also been observed in parallel tion for cytokine production (Gross et al., 2008). NF-kB studies of DMH-treated CcS/Dem RCS strains (Moen (p105; 135.6 Mb) is a transcription factor that plays an et al., 1996). Second, the genetic control of AOM- important role in innate immunity and inflammatory induced hyperplastic lesions is likely to be complex and response by regulating production of key cytokines and multigenic, with the transfer of 12.5% of the genome chemokines. NF-kB has anti-apoptotic activities and from either A/J or B6 in BcA and AcB strains, overexpression of NF-kB has been associated with respectively, having no influence on this trait. Third, bowel inflammatory conditions (ulcerative colitis, the genetic control of AOM-induced tumors behaves as Crohn’s disease) and with colorectal tumors (Greten a simple trait, and haplotype analysis shows a major et al., 2004; Pacifico and Leonardi, 2006). More gene effect on the distal portion of chromosome 3 that specifically, mutations in the NF-kB-activating kinase we designate Ccs3. Fourth, the presence of multiple Ikkb cause a reduction in total tumor burden in mice tumors in BcA71, BcA72 and BcA87 that have low treated with a combination of AOM (i.p., single dose) numbers of hyperplastic lesions, and conversely, low and the irritant dextran sulfate (p.o., 3 weekly doses) tumor multiplicity in AcB52 despite high numbers of (Karin, 2008). These results suggest that NF-kBisa hyperplastic lesions, suggests that one is not a pre- strong candidate for Ccs3. The Rap1gds (138.7 Mb) gene requisite for the other. Finally, the intermediate number (Rap1 GTP/GDP dissociation stimulator) is another of tumors detected in AcB58 suggests the presence of interesting positional candidate. Rap1 has a role in cell additional genetic modifiers in that strain. differentiation, cell proliferation and apoptosis; Rap1 A locus (Ccs2) influencing DMH-induced colon interacts with two effectors of the Mek/Erk mitogen- tumor multiplicity in (C57BL/6J Â CBA/J) backcross activated protein kinase pathway, namely Raf-1 and B- and F2 mice has been regionally assigned to mouse Raf (Stork, 2003; Stork and Dillon, 2005). As an chromosome 3 (D3Mit147, D3Mit19; cumulative inhibitor of the Raf1 pathway, Rap-GTPase is an LOD4.6) (Angel et al., 2000). It is possible that the antagonist of Ras-GTPase, a group of proteins known chromosome 3 effect (Ccs3) detected here in AOM- to be critical in the progression of colorectal tumors. treated AcB/BcA strains is identical to Ccs2. However, Finally, there are two additional guanylate binding several lines of evidence suggest that they may be proteins with GTPase activity that map within the Ccs3 different, and this is why we have retained the interval, Gbp2 (142.2 Mb) and Gbp3 (142.3 Mb). temporary designation Ccs3. Ccs2 was detected using a The retroviral tagged cancer gene database (RTCGD; different treatment (DMH vs AOM), and a different Akagi et al., 2004; http://rtcgd.ncifcrf.gov) compiles all mouse strain combination (B6 and CBA/J). In addition known retroviral and transposon integration sites and and as opposed to Ccs3, the genetic control did not corresponding genes that are associated with the appear to segregate as a monogenic trait in appearance of hematological tumors (T-cell lymphomas, (B6 Â CBA)F2 and backcross mice, and the linkage myeloid leukemias) in mouse experimental models (from peak for Ccs2 maps distally to Ccs3 on chromosome 3 17 different laboratories). A search of this database has (Angel et al., 2000). Finally, haplotype mapping with revealed four common insertion sites in the Ccs3 region multiple single nucleotide polymorphisms from this that alter expression of four corresponding genes, region failed to identify a conserved haplotype on that namely NF-kb1, Rg9mtd2, Rap1gds1 and Bcl10. segment of chromosome 3 between A/J, CBA/J and Together with their known biological function (dis- C57BL/6J, a situation that might be expected for a cussed above), these observations further strengthen the conserved interstrain genetic control such as that candidacy of these genes as being responsible for the detected for the Char9 locus in malaria susceptibility Ccs3 effect. (Min-Oo et al., 2007) and the Nramp1 locus and Positional cloning with further reduction of the susceptibility to intracellular infections (Malo et al., genetic interval and systematic sequencing of all genes 1994). Further experimentation will be required to in the interval is currently being undertaken, and will determine if Ccs2- and Ccs3-inherited phenotype are ultimately be required to identify the causative genetic caused by the same gene. lesion at Ccs3. In addition to the 4–6 obvious candidates There are several obvious positional candidates within reviewed above, additional prioritization for character- the maximum 14 Mb Ccs3 interval that contains at least ization and validation will take into account 94 annotated genes, with 25 of them showing robust the transcript and exon profiling data presented in mRNA expression in normal colon. Bcl10 (145.6 Mb) is Figures 7 and 8. Priority will be given to genes in the a signaling molecule and caspase regulator that has Ccs3 interval that are (a) differentially expressed in proapoptotic activity important for the development normal A/J vs B6 colon, and/or (b) differentially and activation of B and T lymphocytes (Thome, 2004). expressed in normal colon vs colon tumors, and/or (c) Null Bcl10 mouse mutants show severe immunodefi- which show evidence of interstrain difference in alter- ciency (Xue et al., 2003). Bcl10 is disrupted in the nate splicing in normal colon or in colon tumors. t(1;14)(p22;q32) translocation found in humans with Finally, validation of possible positional candidates will MALT B-cell lymphoma, and is also mutated in a be obtained by functional complementation in vivo, number of other lymphoid and solid tumors, including using transgenic mice carrying bacterial artificial chro- colonic carcinoma cell lines Lovo and LS513 (Willis mosome (BAC) clones from the region. In these et al., 1999). Interestingly, it was recently reported that experiments, BAC clones overlapping the minimal

Oncogene Colon cancer susceptibility locus Ccs3 C Meunier et al 659 genetic/physical interval for Ccs3 will be transferred to 8 weeks. The animals were weighed and examined twice per the germ line of A/J (either directly or after back- week for appearance of clinical symptoms. Animals showing crossing), followed by testing for correction of the discomfort were immediately killed. End points were defined tumor susceptibility phenotype in A/J transgenic ani- as 19–20 weeks after the first injection. At least 10 mice of each mals expressing the B6 parental copy of the positional genotype were tested per experiment. After killing the mice, the candidates. entire colon was removed, flushed with cold PBS, sectioned longitudinally and fixed flat on 10% paraformaldehyde-soaked The Ccs3 interval on mouse chromosome 3 shares Whatman filter paper. The same person scored either tumors synteny relationship with interspersed portions of hu- (protruding masses with thick/opaque interior) or hyperplastic man 4q21–25 on the proximal side and human 1p22–31 lesions (flat structures showing hollow interior). The approx- on the distal side. Interestingly, human 4q and human imate volume of each tumor was calculated by the formula 1q regions have been associated with inflammatory ((diameter/2)3  p  2/3) and tabulated for each mouse and for bowel disease and CRCs. Human chromosome 4q gave each mouse strain. positive association in a genome-wide screen of 297 individuals with Crohn’s disease and ulcerative colitis Preparation of tissues, histological analyses (in a familial setting) (Cho et al., 1998). Genome The mice were killed and intestines were immediately removed, instability or loss of heterozygosity at human 4q or washed in PBS and fixed in 10% phosphate-buffered formalin both have been detected in a number of solid tumors, and processed for immunohistochemistry. Fixed tissues were including esophageal adenocarcinoma (Nancarrow dehydrated in ethanol and embedded with paraffin. Tissue et al., 2008), hepatocellular carcinoma (Herath et al., sections, 6 mm in thickness, were deparaffinized according to 2006) and CRC (Kurashina et al., 2008).Conversely, standard protocols, mounted onto slides, stained with eosin recent genome-wide association studies in several and counterstained with hematoxylin, following standard histological procedures. Grading of tumors was performed thousand patients with Crohn’s disease have identified by a pathologist (SJ). Images were acquired using a Zeiss the 1p31 region as a very strong genetic contributor Axiovert instrument (Zeiss Canada, Toronto, ON, Canada), (Cho et al., 1998; Barrett et al., 2008). Finally, a large and processed using Adobe Photoshop (www.adobe.com). body of published data has associated human 1p with colon cancer. For example, allelic imbalance at 1p has Transcript profiling with microarrays been noticed in up to 10% of small adenomas (Shih Colons were dissected, rinsed with PBS and snap-frozen in et al., 2001). Other studies have detected 1p deletions in liquid nitrogen (three mice per experimental group). Tissue colorectal polyps (Lothe et al., 1995), and 1p alterations was disrupted using a Polytron homogenizer (Brinkmann (LOH, allelic imbalance, deletions) have been detected Instruments, Mississawga, ON, Canada), and total RNA was in up to 30–80% of colorectal tumors in humans isolated using TRIzol reagent following the manufacturer’s (Couturier-Turpin et al., 2001; Cianciulli et al., 2004). instructions (Invitrogen, Burlington, Ontario, Canada). The It is tempting to speculate that the same gene or set of RNA quality was assessed using RNA 6000 NanoChips with genes may contribute to inflammatory bowel diseases the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). and colorectal tumors in humans and may underlie Biotin-labeled target for the microarray experiment were the Ccs3 effect mapped in our study. Therefore, prepared using 1 mg total RNA. The RNA was subjected to an rRNA removal procedure with the RiboMinus Human/ identifying the gene responsible for the Ccs3 phenotype Mouse Transcriptome Isolation Kit (Invitrogen) and cDNA may help further understand pathogenesis of inflamma- was synthesized using the GeneChip WT (Whole Transcript) tion and/or CRC in humans. Sense Target Labeling and Control Reagents kit as described by the manufacturer (Affymetrix). The sense cDNA was then fragmented by UDG (uracil DNA glycosylase) and APE 1 (apurinic/apyrimidinic endonuclease 1) and biotin-labeled Materials and methods with terminal deoxynucleotidyl transferase using the Gene- Chip WT Terminal labeling kit (Affymetrix). Selected RNAs Mice were used for transcriptional profiling with Affymetrix Inbred A/J and C57BL/6J mice were purchased from the oligonucleotides chips (Mouse Genome 430 2.0 array), Jackson Laboratory (Bar Harbor, ME, USA). The AcB/BcA according to the manufacturer’s recommendations. Raw data set of RCS were derived from a double backcross (N3) generated by GeneChip Operating Software were normalized between A/J and C57BL/6J parents at McGill University. The using GeneSifter microarray data analysis system (VizX Labs, breeding, genetic characteristics and genotype of these animals Seattle, WA, USA; www.genesifter.net), using RMA (Robust et al. for 625 markers have been previously described (Fortin , Multi-Array) normalization. This analysis revealed that 78 of 2001). The 35 AcB/BcA strains were maintained at the Animal the 94 annotated genes in the Ccs3 interval are expressed Care Facility of McGill University according to the guidelines in colon. of the Canadian Council on Animal Care. They were fed For exon arrays, hybridization was performed using 5 mgof ad libitum regular chow and water . biotinylated target, which was incubated with the GeneChip Mouse Exon 1.0 ST array (Affymetrix) at 45 1C for 16–20 h. Carcinogen treatment and colon tumor preparations Following hybridization, nonspecifically bound material was Azoxymethane was purchased as a single lot from Sigma (St removed by washing, and detection of specifically bound target Louis, MO, USA), and stored as 25 mg aliquots at À20 1C. For was performed using the GeneChip Hybridization, Wash and each injection, a fresh aliquot was thawed and appropriately Stain kit, and the GeneChip Fluidics Station 450 (Affymetrix). diluted. Age-matched male and female mice (12-week old) The arrays were scanned using the GeneChip Scanner 3000 7G were injected intraperitoneally with AOM (Sigma) dissolved in (Affymetrix) and raw data were extracted from the scanned saline at a dose of 10 mg/kg of body weight, once per week, for images and analysed with the Affymetrix Power Tools

Oncogene Colon cancer susceptibility locus Ccs3 C Meunier et al 660 software package (Affymetrix). The Affymetrix Power Tools tracking of haplotypes in contributing and non-contributing software package (Affymetrix) was used to quantile normalize strains. In a second analysis, analysis of variance was used to the probe fluorescence intensities and to summarize the probe first compare the number of tumors displayed in each RCS set (representing exon expression) and meta-probe set (repre- strain to the one of its corresponding parental strains. Using senting gene expression) intensities using a probe logarithmic this simple test, we qualitatively scored each strain as A/J-like intensity error model (see http://www.affymetrix.com/support/ or B6-like (binary phenotype). The obtained SDP was then technical/technotes/plier_technote.pdf). A variance stabiliza- compared to the genetic map of the RCS panel using a simple tion constant of 16 was added to all PLIER scores before log computer program scoring percentage of similarity between transformation. Only the high-confidence set of core probe sets the loci and the phenotypes. and core meta-probe sets were used in the analysis.

Linkage analysis in AcB/BcA recombinant congenic strains Conflict of interest The published genetic map of the AcB/BcA RCS set was used to identify congenic segments associated with tumor suscept- The authors declare no conflict of interest. ibility following AOM treatment (Fortin et al., 2001). Quantitative trait loci were first determined by computing single marker regressions with MapManager QTX software, using average number of tumors as a phenotypic marker Acknowledgements (Manly et al., 2001). A self-RI model, which implicitly considers each background (A/J or B6) as an independent RI We thank George Chountalos for his work in the haplotype set, was used for the analysis. The markers with highest linkage mapping of the genetic interval of Ccs3 in common mouse values were subsequently positioned on the July 2007 mouse strains. This work was supported by research grants to PG and (Mus musculus) genome data obtained from the Build 37 NB from the National Cancer Institute of Canada and the assembly by NCBI to attribute physical genomic positions. Canderel Initiative Program of the Goodman Cancer Centre. Fine-mapping of the locus interval was appraised by visual PG is a James McGill professor of biochemistry.

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