Different APC genotypes in proximal and distal sporadic colorectal cancers suggest distinct WNT/β-catenin signalling thresholds for tumourigenesis

Abstract

Biallelic protein-truncating mutations in the adenomatous polyposis coli (APC) gene are prevalent in sporadic colorectal cancer (CRC). Mutations may not be fully inactivating, instead producing WNT/β-catenin signalling levels 'just-right' for tumourigenesis. However, the spectrum of optimal APC genotypes accounting for both hits, and the influence of clinicopathological features on genotype selection remain undefined. We analysed 630 sporadic CRCs for APC mutations and loss of heterozygosity (LOH) using sequencing and single-nucleotide polymorphism microarrays, respectively. Truncating APC mutations and/or LOH were detected in 75% of CRCs. Most truncating mutations occurred within a mutation cluster region (MCR; codons 1282-1581) leaving 1-3 intact 20 amino-acid repeats (20AARs) and abolishing all Ser-Ala-Met-Pro (SAMP) repeats. Cancers commonly had one MCR mutation plus either LOH or another mutation 5' to the MCR. LOH was associated with mutations leaving 1 intact 20AAR. MCR mutations leaving 1 vs 2-3 intact 20AARs were associated with 5' mutations disrupting or leaving intact the armadillo-repeat domain, respectively. Cancers with three hits had an over-representation of mutations upstream of codon 184, in the alternatively spliced region of exon 9, and 3' to the MCR. Microsatellite unstable cancers showed hyper-mutation at MCR mono- and di-nucleotide repeats, leaving 2-3 intact 20AARs. Proximal and distal cancers exhibited different preferred APC genotypes, leaving a total of 2 or 3 and 0 to 2 intact 20AARs, respectively. In conclusion, APC genotypes in sporadic CRCs demonstrate 'fine-tuned' interdependence of hits by type and location, consistent with selection for particular residual levels of WNT/β-catenin signalling, with different 'optimal' thresholds for proximal and distal cancers.

Figure 1

Distribution of somatic APC mutations by amino acid for 630 sporadic CRCs. (a) Frequencies of protein-truncating and missense mutations are shown above and below the x axis, respectively. (b) Cumulative frequency of truncating mutations; the somatic mutation cluster region (codons 1282–1581) is highlighted. APC protein domains and exon structure are indicated.

Figure 2

Distribution of truncating APC mutations according to the number and types of somatic hits: cases with one hit (1 mutation/LOH−) are shown in blue, with two hits (1 mutation/LOH+ and 2 mutations/LOH−) in red, and with three hits (2 mutations/LOH+ and 3 mutations/LOH−) in green. The y axis shows case number. Regions with few truncating mutations are highlighted.

Figure 3

Distribution of truncating APC mutations upstream of the somatic MCR according to number of intact 20AARs left by the MCR hit for CRCs with two mutations, one within the MCR and one 5′ to the MCR (n=130). Cancers with 2–3 intact 20AARs show an overrepresentation of 5′ non-MCR mutations downstream of codon 767 leaving an intact armadillo-repeat domain, whereas cancers with 1 intact 20AAR show an overrepresentation of 5′ non-MCR mutations upstream of codon 768 disrupting or removing the armadillo-repeat domain. Cases with 3 intact 20AARs are shown in green, 2 intact 20AARs in blue, and 1 intact 20AAR in red.

Figure 4

Distribution of truncating APC mutations for proximal (red) and distal (green) CRCs. The MCR is approximately codons 1411–1581 for proximal cancers and approximately codons 1282–1494 for distal cancers. Proximal and distal cancers exhibit an overall enrichment for mutations leaving 2–3 and 0–1 intact 20AARs, respectively, as illustrated by the cumulative frequency distributions.

Figure 5

Number of intact 20AARs for truncating mutations in proximal and distal cancers by colorectal sub-region. Cancers from the hepatic flexure were grouped with the ascending colon, and those from the splenic flexure were combined with the descending colon because of small numbers of cases.

Figure 6

Frequency of APC genotypes in proximal and distal CRCs with two hits to APC. Black asterisk: >5% of proximal cancers. Grey asterisk: >5% of distal cancers. x20AAR, number of intact 20 amino-acid repeats; ARM+, intact armadillo-repeat domain; ARM−, disrupted armadillo-repeat domain; CN, copy-neutral LOH; Del, deletion LOH.