Positional Mapping and Candidate Gene Analysis of the Mouse Ccs3 Locus That Regulates Differential Susceptibility to Carcinogen-Induced Colorectal Cancer

Charles Meunier,Philippe Gros,Lauren Van Der Kraak,Garabet Yeretssian,Mathieu Blanchette,Normand Groulx,Ingrid Labouba,Anne-Marie Mes-Masson,Nicole Beauchemin,Maya Saleh,Pablo Cingolani,Claire Turbide,Amanda Ewart Toland

doi:10.1371/journal.pone.0058733

Abstract

The Ccs3 locus on mouse chromosome 3 regulates differential susceptibility of A/J (A, susceptible) and C57BL/6J (B6, resistant) mouse strains to chemically-induced colorectal cancer (CRC). Here, we report the high-resolution positional mapping of the gene underlying the Ccs3 effect. Using phenotype/genotype correlation in a series of 33 AcB/BcA recombinant congenic mouse strains, as well as in groups of backcross populations bearing unique recombinant chromosomes for the interval, and in subcongenic strains, we have delineated the maximum size of the Ccs3 physical interval to a ∼2.15 Mb segment. This interval contains 12 annotated transcripts. Sequencing of positional candidates in A and B6 identified many either low-priority coding changes or non-protein coding variants. We found a unique copy number variant (CNV) in intron 15 of the Nfkb1 gene. The CNV consists of two copies of a 54 bp sequence immediately adjacent to the exon 15 splice site, while only one copy is found in CRC-susceptible A. The Nfkb1 protein (p105/p50) expression is much reduced in A tumors compared to normal A colonic epithelium as analyzed by immunohistochemistry. Studies in primary macrophages from A and B6 mice demonstrate a marked differential activation of the NfκB pathway by lipopolysaccharide (kinetics of stimulation and maximum levels of phosphorylated IκBα), with a more robust activation being associated with resistance to CRC. NfκB has been previously implicated in regulating homeostasis and inflammatory response in the intestinal mucosa. The interval contains another positional candidate Slc39a8 that is differentially expressed in A vs B6 colons, and that has recently been associated in CRC tumor aggressiveness in humans.

Highlights

The pathogenesis of colorectal cancer (CRC) is associated with the sequential accumulation of mutations in specific genes, which causes stepwise progression from pre-neoplastic lesions to full blown adenocarcinoma [1]
Phenotyping a subset of 23 AcB/BcA strains for susceptibility to AOM-induced CRC initially showed that differential susceptibility of A and B6 mouse strains to CRC is regulated by a single locus designated Ccs3
Genome-wide association studies (GWAS) have pointed at an impressive plurality of genetic factors contributing to CRC susceptibility in humans [4,5]

Summary

Introduction

The pathogenesis of colorectal cancer (CRC) is associated with the sequential accumulation of mutations in specific genes, which causes stepwise progression from pre-neoplastic lesions to full blown adenocarcinoma [1]. Only in recent years and with the advent of genomewide association studies has the degree of complexity in interactions between the genetic and environmental components contributing to the etiology of human colorectal cancer been appreciated [3,4,5,6]. As many as 16–20 common low-penetrance variants have been identified in genome-wide association studies (GWAS) for human sporadic CRC [9,10]. Half of those loci are tightly linked or allelic with components of the TGFß signaling pathway: SMAD7, GREM1, BMP2, BMP4, RHPN2 and LAMA5 ([11,12], reviewed in [13]). It has been proposed that as many as 170 such loci may contribute to CRC susceptibility in humans [13]

Methods

Results

Conclusion