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 locus (Ccs3) on mouse chromosome 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 Â 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 genes 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) gene 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 chromosomes 3 (CBA Â C57BL/6 cross) (Angel et al., When colons were examined 19 weeks following 2000), 4 (B10.O20 Â O20 cross) (Fijneman and Demant, initiation of treatment, two types of macroscopic