Importance of epidermal growth factor receptor signaling in establishment of adenomas and maintenance of carcinomas during intestinal tumorigenesis

Abstract

We used the hypomorphic Egfr(wa2) allele to genetically examine the impact of impaired epidermal growth factor receptor (Egfr) signaling on the Apc(Min) mouse model of familial adenomatous polyposis. Transfer of the Apc(Min) allele onto a homozygous Egfr(wa2) background results in a 90% reduction in intestinal polyp number relative to Apc(Min) mice carrying a wild-type Egfr allele. This Egfr effect is potentially synergistic with the actions of the modifier-of-min (Mom1) locus. Surprisingly, the size, expansion, and pathological progression of the polyps appear Egfr-independent. Histological examination of the ilea of younger animals revealed no differences in the number of microadenomas, the presumptive precursor lesions to gross intestinal polyps. Pharmacological inhibition with EKI-785, an Egfr tyrosine kinase inhibitor, produced similar results in the Apc(Min) model. These data suggest that normal Egfr activity is required for establishment of intestinal tumors in the Apc(Min) model between initiation and subsequent expansion of initiated tumors. The role of Egfr signaling during later stages of tumorigenesis was examined by using nude mice xenografts of two human colorectal cancer cell lines. Treatment with EKI-785 produced a dose-dependent reduction in tumor growth, suggesting that Egfr inhibitors may be useful for advanced colorectal cancer treatment.

Figure 1

Intestinal lesion numbers in Apc^Min mice vs. Egfr status. (A) Macroadenoma analysis. Each dot represents polyp number from a single 3-mo-old mouse with horizontal lines representing means. Egfr⁺ designates mice carrying a wt Egfr allele (genotypes Egfr^+/+, Egfr^+/wa2, and Egfr^+/tm1Mag; n = 28); Egfr^wa2 designates mice with the waved-2 phenotype (genotypes Egfr^wa2/wa2 and Egfr^wa2/tm1Mag; n = 37). (B) Microadenoma analysis. Each dot represents the ileal microadenoma score of a single, 1-mo-old mouse (Egfr^+/wa2, n = 14; Egfr^wa2/wa2, n = 10).

Figure 2

Early ileal lesions in Apc^Min mice. (A and B) Adjacent sections of an Egfr⁺ cystic microadenoma. (C) Egfr⁺ crypt, containing a cluster of three nuclear β-catenin-positive cells, flanked by normal crypts. (D) Close up of boxed crypt from C. (E) Egfr^wa2 crypt with several β-catenin-positive nuclei. (A) H&E staining. (B–E) β-catenin immunohistochemistry. Arrowheads in D mark the β-catenin-positive nuclei.

Figure 3

Intestinal polyp sizes in Apc^Min mice with reduced Egfr activity. (A) Genetic analysis. Solid and dashed lines indicate best linear fit of polyp diameter vs. age for 16 Egfr⁺ and 20 Egfr^wa2 mice, respectively. Closed (Egfr⁺) and open (Egfr^wa2) circles indicate diameter means at each time point. (B) Pharmacological analysis. Dots represent individual polyp diameters from DMSO-treated (controls; n = 5) and EKI-785-treated (treatment; n = 5) mice. Means are represented by horizontal lines.

Figure 4

Growth curves of human CRC cell line xenografts in nude mice treated with EKI-785. Volumes of tumors derived from HCA-7 (A) and HCT-116 (B) cells. EKI-785 doses (mg/kg body weight) are given at the end of each growth curve.

Figure 5

Model of Egfr activity requirements during intestinal tumorigenesis. Red dots represent nuclear β-catenin-positive cells. Solid green lines indicate evidence for Egfr activity during establishment and adenocarcinoma expansion. Dashed green lines indicate that a requirement for Egfr activity during adenoma expansion, progression, and invasion has yet to be demonstrated conclusively.