A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer

Abstract

Progress toward the understanding and management of human colon cancer can be significantly advanced if appropriate experimental platforms become available. We have investigated whether a rat model carrying a knockout allele in the gatekeeper gene Adenomatous polyposis coli (Apc) recapitulates familial colon cancer of the human more closely than existing murine models. We have established a mutagen-induced nonsense allele of the rat Apc gene on an inbred F344/NTac (F344) genetic background. Carriers of this mutant allele develop multiple neoplasms with a distribution between the colon and small intestine that closely simulates that found in human familial adenomatous polyposis patients. To distinguish this phenotype from the predominantly small intestinal phenotype found in most Apc-mutant mouse strains, this strain has been designated the polyposis in the rat colon (Pirc) kindred. The Pirc rat kindred provides several unique and favorable features for the study of colon cancer. Tumor-bearing Pirc rats can live at least 17 months, carrying a significant colonic tumor burden. These tumors can be imaged both by micro computed tomography scanning and by classical endoscopy, enabling longitudinal studies of tumor genotype and phenotype as a function of response to chemopreventive and therapeutic regimes. The metacentric character of the rat karyotype, like that of the human and unlike the acrocentric mouse, has enabled us to demonstrate that the loss of the wild-type Apc allele in tumors does not involve chromosome loss. We believe that the Pirc rat kindred can address many of the current gaps in the modeling of human colon cancer.

Fig. 1.

Isolation and identification of the Pirc line of rats. (A) Scheme for the colorimetric yeast assay. Two thousand five hundred and thirty bases of Apc exon 15 spanning codons 757–1,600 were amplified with primers chimeric for Apc sequence and homology to a “universal vector” that accepts any such chimeric amplicon (11). The amplicon was then gap-repaired into the universal vector and transformed into ADE2-deficient yeast. Screening of 1,360 F1 progeny yielded a single yeast plate with half-red and half-white colonies, which is the expected ratio for a heterozygous mutant. (B) Sequence trace of the founder rat showing heterozygosity for the A→T transversion at nucleotide 3409 of Apc (Upper) compared with a wild-type littermate (Lower). (C) Structure of the human Apc gene. Arrows indicate orthologous locations of mouse model and Pirc truncating mutations and the two most common FAP mutation sites. The color bar below indicates the genotype–phenotype correlation of sites of protein truncation to disease severity.

Fig. 2.

Histological and gross appearance of Pirc tumors. (A) H&E of a focal adenocarcinoma with high-grade dysplasia. (B) Enlargement of the larger rectangle in A, showing invasion into the stalk. (C and D) Enlargement of the smaller rectangle in A (C), showing high-grade dysplasia compared with normal crypts from the same section (D). (E) H&E of a peduncular colonic adenoma. (F) β-catenin (red) and DAPI (blue) immunofluorescence of the same tumor. The dashed line delineates dysplastic (above the line) and hyperplastic and normal tissue (below the line). (G) Magnification of the rectangle shown in F. (H) H&E of a colonic microadenoma (central crypt) surrounded by normal crypts, which is representative of all colonic microadenomas examined. (I) β-catenin (red). (J) β-catenin (red) merged with DAPI (blue). (Scale bars: A and E, 1 mm; H, 0.1 mm.)

Fig. 3.

LOH analysis for chromosome 18 on (F344xWF) F1 and F2 tumors. The three SNPs tested, ss48531311 (17 Mb) and Apc^am1137 (27 Mb) on the p arm and ss48531727 (43 Mb) on the q arm, were all heterozygous in the normal tissue. The centromere (open circles) lies at approximately the 38-Mb position. LOH status at each SNP was determined by using a quantitative Pyrosequencing assay. Four possible tumor genotypes are given (left to right): LOH involving only the two loci on the p arm, LOH involving only Apc^am1137, maintenance of heterozygosity (MOH) at all three loci, and LOH for all three loci. We have diagrammed homozygosity; it must be noted that these Pyrosequencing assays cannot distinguish between hemizygosity (deletion) and homozygosity (recombination).

Fig. 4.

In vivo imaging of Pirc tumors. MicroCT (A), endoscopic (B), and dissection (C) views of three colonic tumors in an 11-month-old F344 Pirc male. (Scale bar: 1 cm.)