Inactivation of germline mutant APC alleles by attenuated somatic mutations: a molecular genetic mechanism for attenuated familial adenomatous polyposis

Abstract

Germline mutations of the adenomatous polyposis coli (APC) tumor-suppressor gene result in familial adenomatous polyposis (FAP). Patients with FAP typically develop hundreds to thousands of benign colorectal tumors and early-onset colorectal cancer. A subset of germline APC mutations results in an attenuated FAP (AFAP) phenotype, in which patients develop fewer tumors and develop them at an older age. Although a genotype-phenotype correlation between the locations of APC germline mutations and the development of AFAP has been well documented, the mechanism for AFAP has not been well defined. We investigated the mechanism for AFAP in patients carrying a mutant APC allele (APC(AS9)) that has a mutation in the alternatively spliced region of exon 9. APC(AS9) was found to down-regulate beta-catenin-regulated transcription, the major tumor-suppressor function of APC, as did the wild-type APC. Mutation analysis showed that both APC(AS9) and the wild-type APC alleles were somatically mutated in most colorectal tumors from these patients. Functional analysis showed that 4666insA, a common somatic mutation in APC(AS9) in these tumors, did not inactivate the wild-type APC. Our results indicate that carriers of APC(AS9) develop fewer colorectal tumors than do typical patients with FAP because somatic inactivation of both APC alleles is necessary for colorectal tumorigenesis. However, these patients develop colorectal tumors more frequently than does the general population because APC(AS9) is inactivated by mutations that do not inactivate the wild-type APC.

Figure 1

Regulation of CRT by APCdA9. SW480 cells were transfected with the combination of pCMVβgal, a reporter plasmid (pTOPFLASH, pFOPFLASH, or pcDNA3Luc), and an APC expression vector, as indicated. Results of three separate experiments using pCIN to express APC are shown. The activities of the wild-type APC and APCdA9 in the regulation of CRT are compared with that of APC1309. Similar results were obtained using pCMV-NEO-BAM to express APC (data not shown).

Figure 2

Detection of somatic APC mutation. The result of IVSP analysis of codons 686–1686 of a representative single crypt from each tumor is shown. The use of single isolated crypts in this assay allowed the easy detection of LOH in tumor 1-2. “F” indicates the position of the full-length product presented in tumors 1-1, 1-3A, 1-4, and 1-5. Dots indicate mutant products. The positions of the molecular-weight standards are shown on the left.

Figure 3

Regulation of β-catenin by APC1556. A, Expression of APC1556. MCF7 and HCT116 cell lines were transfected with pCIN alone (—) or pCIN expressing the indicated mutant APC. APC proteins were detected by immunoblotting. “wt” indicates the endogenous wild-type APC. Dots indicate mutant APC proteins. B, Regulation of CRT by APC1556. SW480, DLD-1, and HCT116 cell lines were transfected with a combination of pRL-TK, pTOPFLASH, and an APC expression vector, as indicated. The unblackened and blackened bars indicate expression of APC by pCIN and by pCMV-NEO-BAM, respectively. The mean and SD of the result of triplicate experiments are shown. The activities of the wild-type APC, APC1556, APCdA9, and APCdA,1556 in the regulation of CRT are compared with that of APC1309. (C) Reduction of β-catenin by APC1556. SW480 cells were transfected with pUHD15-1 and pTBI, expressing indicated APC. Cells were immunostained with a monoclonal antibody against β-catenin. Transfected cells were identified by their expression of green fluorescence protein (GFP), because pTBI also expressed GFP. Nuclei were revealed by staining with 4,6-diamidino-2-phenylindole (DAPI). Arrowheads indicate transfected cells.

Figure 4

Inactivation of APC_AS9, but not wild-type APC, by the 4666insA mutation. “APC_F” indicates the full-length APC mRNA. “×” indicates mutations. “Tumor Suppression” indicates whether each APC allele can function as a tumor suppressor, which is determined by the combined activities of transcripts of each allele. The mutation 4666insA slightly reduces the activity of proteins encoded by both APC_F and APCdA9 and does not abolish the tumor-suppressor function of APC. APC_AS9 does not express full-length APC protein, because its APC_F expresses a severely truncated protein owing to the mutation in the alternatively spliced region of exon 9. However, APC_AS9 expresses APCdA9 identical to that expressed by the wild-type APC and has reduced but sufficient tumor-suppressor activity. Further decrease of the reduced tumor-suppressor activity of APC_AS9 by the 4666insA mutation causes APC_AS9 to have insufficient tumor-suppressor activity.