APC and the three-hit hypothesis.

The seminal 'two-hit hypothesis' implicitly assumes that bi-allelic tumour suppressor gene (TSG) mutations cause loss of protein function. All subsequent events in that tumour therefore take place on an essentially null background for that TSG protein. We have shown that the two-hit model requires modification for the APC TSG, because mutant APC proteins probably retain some function and the two hits are co-selected to produce an optimal level of Wnt activation. We wondered whether the optimal Wnt level might change during tumour progression, leading to selection for more than two hits at the APC locus. Comprehensive screening of a panel of colorectal cancer (CRC) cell lines and primary CRCs showed that some had indeed acquired third hits at APC. These third hits were mostly copy number gains or deletions, but could be protein-truncating mutations. Third hits were significantly less common when the second hit at APC had arisen by copy-neutral loss of heterozygosity. Both polyploid and near-diploid CRCs had third hits, and the third hits did not simply arise as a result of acquiring a polyploid karyotype. The third hits affected mRNA and protein levels, with potential functional consequences for Wnt signalling and tumour growth. Although some third hits were probably secondary to genomic instability, others did appear specifically to target APC. Whilst it is generally believed that tumours develop and progress through stepwise accumulation of mutations in different functional pathways, it also seems that repeated targeting of the same pathway and/or gene is selected in some cancers.

Germline deletions of EXO1 do not cause colorectal tumors and lesions which are null for EXO1 do not have microsatellite instability.

Exonuclease 1 (EXO1) is a candidate gene for colorectal tumor susceptibility because it is believed to play a role in mismatch repair. There have been several studies investigating the role of EXO1 in mismatch repair but few investigating its role in causing clinical disease. In one recent study, germline variants of EXO1 were reported to be associated with predisposition to colorectal cancer in families with phenotypes similar to hereditary nonpolyposis colon cancer (HNPCC). We recently identified nine individuals from two British families with multiple cutaneous and uterine leiomyomatosis with independently arising heterozygous germline deletions of 1q42.3 approximately q43 encompassing not only FH, the multiple leiomyomatosis-associated gene, but also several flanking genes, including EXO1. We investigated these families for any indication of predisposition to colorectal cancer or other HNPCC spectrum cancers by means of detailed questionnaires, interviews, and examination of EXO1-null skin leiomyomata for microsatellite instability (MSI). No individual in these families had developed colorectal cancer or known colorectal adenomas, and none had any symptoms warranting gastrointestinal or other investigation. EXO1-null tumors showed no evidence of MSI. This study questions the functional significance of previously reported variants of EXO1 reported in HNPCC-like families and suggests that in humans there may be other as yet undiscovered proteins that have exonuclease function overlapping with that of EXO1 in DNA mismatch repair. Also of interest is the absence of phenotypic abnormality apart from multiple leiomyomatosis in any deletion carrier even though the adjacent genes RGS7, KMO, CHML, and OPN3 were also deleted.

Analysis of chromosomal instability in human colorectal adenomas with two mutational hits at APC.

In vitro data show that the adenomatous polyposis coli (APC) protein associates with the mitotic spindle and that mouse embryonic stem cells with biallelic Apc mutations are karyotypically unstable. These findings led to suggestions that APC acts in chromosomal segregation and that APC inactivation leads to chromosomal instability (CIN). An alternative hypothesis based on allelic loss studies in colorectal adenomas proposes that CIN precedes and contributes to genetic changes at APC. We determined whether colorectal adenomas with two mutations at APC show features consistent with these models by studying 55 lesions (average size 5 mm; range 1-13 mm) from patients with familial adenomatous polyposis. A variety of methods was used depending on available material, including flow cytometry, comparative genomic hybridization, and loss of heterozygosity (LOH) analysis. Selected adenomas were assessed for proliferative activity by Ki-67 immunocytochemistry. Seventeen of 20 (85%) tumors were diploid, two were near-diploid, and one was hypotetraploid. Just one (near-diploid) tumor showed increased proliferative activity. LOH was found occasionally on chromosome 15q (2 of 49 tumors), but not on chromosome 18q (0 of 48). In 20 adenomas, LOH at APC was associated with loss at 5q but not 5p markers, with the former encompassing a minimum of 20 Mb. However, three of these lesions analyzed by comparative genomic hybridization displayed normal profiles, suggesting, together with other data, that the mechanism of LOH at APC is probably somatic recombination. Our results therefore do not support the hypothesis that CIN precedes APC mutations in tumorigenesis. Regarding the model in which APC mutations lead directly to CIN, if APC mutations do have this effect in vivo, it must be subtle. Alternatively, CIN associated with APC mutations might be essentially an in vitro phenomenon.

SMAD4 mutations in colorectal cancer probably occur before chromosomal instability, but after divergence of the microsatellite instability pathway.

Loss of chromosome 18q21 is well documented in colorectal cancer, and it has been suggested that this loss targets the DCC, DPC4/SMAD4, and SMAD2 genes. Recently, the importance of SMAD4, a downstream regulator in the TGF-beta signaling pathway, in colorectal cancer has been highlighted, although the frequency of SMAD4 mutations appears much lower than that of 18q21 loss. We set out to investigate allele loss, mutations, protein expression, and cytogenetics of chromosome 18 copy number in a collection of 44 colorectal cancer cell lines of known status with respect to microsatellite instability (MSI). Fourteen of thirty-two MSI(-) lines showed loss of SMAD4 protein expression; usually, one allele was lost and the other was mutated in one of a number of ways, including deletions of various sizes, splice site changes, and missense and nonsense point mutations (although no frameshifts). Of the 18 MSI(-) cancers with retained SMAD4 expression, four harbored missense mutations in the 3' part of the gene and showed allele loss. The remaining 14 MSI(-) lines had no detectable SMAD4 mutation, but all showed allele loss at SMAD4 and/or DCC. SMAD4 mutations can therefore account for about 50-60% of the 18q21 allele loss in colorectal cancer. No MSI(+) cancer showed loss of SMAD4 protein or SMAD4 mutation, and very few had allelic loss at SMAD4 or DCC, although many of these MSI(+) lines did carry TGFBIIR changes. Although SMAD4 mutations have been associated with late-stage or metastatic disease, our combined molecular and cytogenetic data best fit a model in which SMAD4 mutations occur before colorectal cancers become aneuploid/polyploid, but after the MSI(+) and MSI(-) pathways diverge. Thus, MSI(+) cancers may diverge first, followed by CIN(+) (chromosomal instability) cancers, leaving other cancers to follow a CIN(-)MSI(-) pathway.