A novel mouse model of sporadic colon cancer induced by combination of conditional Apc genes and chemical carcinogen in the absence of Cre recombinase

Abstract

Although valuable insights into colon cancer biology have been garnered from human colon cancer cell lines and primary colonic tissues, and animal studies using human colon cancer xenografts, immunocompetent mouse models of spontaneous or chemically induced colon cancer better phenocopy human disease. As most sporadic human colon tumors present adenomatous polyposis coli (APC) gene mutations, considerable effort has gone into developing mice that express mutant Apc alleles that mimic human colon cancer pathogenesis. A serious limitation of many of these Apc-mutant murine models, however, is that these mice develop numerous tumors in the small intestine but few, if any, in the colon. In this work, we examined three spontaneous mouse models of colon tumorigenesis based upon the widely used multiple intestinal neoplasia (Min) mouse: mice with either constitutive or conditional Apc mutations alone or in combination with caudal-related homeobox transcription factor CDX2P-Cre transgene - either with or without exposure to the potent colon carcinogen azoxymethane. Using the CDX2 promoter to drive Cre recombinase transgene expression effectively inactivated Apc in colonocytes, creating a model with earlier tumor onset and increased tumor incidence/burden, but without the Min mouse model's small intestine tumorigenesis and susceptibility to intestinal perforation/ulceration/hemorrhage. Most significantly, azoxymethane-treated mice with conditional Apc expression, but absent the Cre recombinase gene, demonstrated nearly 50% tumor incidence with two or more large colon tumors per mouse of human-like histology, but no small intestine tumors - unlike the azoxymethane-resistant C57BL/6J-background Min mouse model. As such this model provides a robust platform for chemoprevention studies.

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CDX2 promoter-driven, Cre-targeted Apc inactivation leads to earlier tumor onset. (A) Representative colonscopic images of control and tumor-bearing murine colons in which C57BL/6J mice served as controls for Apc^min/+ animals, whereas wild-type littermates, determined by genotyping, served as the controls for the other respective groups. (B) Kaplan–Meier curve showing onset of detectable tumors of Apc^min/+ + AOM (red), CDX2P-Cre-Apc^+/LoxP (green) and CDX2P-Cre-Apc^+/LoxP + AOM (blue) mice. Log-rank analyses revealed that CDX2 promoter-driven, Cre-targeted Apc inactivation enhances early tumor development in colon, with additional acceleration afforded by AOM treatment (P < 0.05). (C) Mean animal age of tumor onset for CDX2P-Cre-Apc^+/LoxP + AOM mice, CDX2P-Cre-Apc^+/LoxP mice and Apc^min/+ + AOM mice showing earlier onset with conditional Apc inactivation, accelerated by AOM administration — though only differences in tumor onset age between CDX2P-Cre-Apc^+/LoxP + AOM and Apc^min/+ + AOM age groups were statistically significant (*P < 0.05). (D) Similar trend mirrored in the median tumor onset ages of the three respective cohorts studied.

Figure 2.

CDX2 promoter-driven, Cre-targeted Apc inactivation leads to increased colon tumor incidence. (A) Harvested colons of experimental and control groups, longitudinally opened and reflected to reveal their lumenal surfaces. (B) Percentage of Apc^min/+ + AOM, CDX2P-Cre-Apc^+/LoxP and CDX2P-Cre-Apc^+/LoxP + AOM mice with visually detectable colon tumors revealing higher incidence rates than their respective controls (*P < 0.05, **P < 0.01, ****P < 0.0001). (C) Comparison of tumor incidence between mice with CDX2 promoter-driven, Cre-targeted Apc inactivation and Apc^min/+ controls revealed a significant increase in tumorigenesis in CDX2P-Cre-Apc^+/LoxP mice (***P < 0.001). (D) Especially noteworthy is the effect that AOM had on conditional Apc^+/LoxP animals in the absence of Cre recombinase, with no detectable tumors occurring in the absence of AOM (****P < 0.0001).

Figure 3.

CDX2 promoter-driven, Cre-targeted Apc inactivation enhances tumor multiplicity and total tumor burden (volume). (A) Average number of tumors was significantly higher in germline Apc^min/+ or conditional Apc^+/LoxP mice treated with or without AOM, compared to their corresponding controls (*P < 0.05, ***P < 0.001). Apc^min/+ mice not given AOM presented with 1.25 tumors per mouse (data not shown). (B) Older animals (>15 weeks) developed significantly more tumors without AOM treatment whereas AOM treatment accelerated early tumor development (*P < 0.05). (C) Older animals (>20 weeks) also developed significantly more tumors that were >10 mm³, when compared to younger cohorts (*P < 0.05). (D) Average tumor volume was significantly greater in AOM treated older animals (>20 weeks) with CDX2 promoter-driven, Cre-targeted Apc inactivation than in younger counterparts (*P < 0.05).

Figure 4.

Histological and immunohistochemical analyses of observed colon carcinogenesis. (A) Representative images of hematoxylin and eosin (H&E)–stained tumors showing tumorigenesis (A: left to right) showing normal colon tissue, adenoma and adenocarcinoma, respectively. (B: left to right) Corresponding representative immunohistochemical stainings of the proliferation biomarker Ki67. (C: left to right) Corresponding representative immunohistochemical stainings of the tumor biomarker β-catenin. All images were acquired at ×200 magnification.

Figure 5.

MUC1 is notably upregulated in colon tumors of all three murine models examined. (A–C) Compared to genotype-matched control mucosa, the colon tumors of Apc^min/+ + AOM mice (A), CDX2P-Cre-Apc^+/LoxP mice (B) and CDX2P-Cre-Apc^+/LoxP + AOM mice (C) each revealed substantial upregulation of MUC1 mRNA expression, as determined by qRT-PCR (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). AOM substantially increased MUC1 mRNA expression in the normal-appearing mucosa in Apc^min/+ mice compared to normal colonic mucosa. Although MUC1 mRNA levels were similar between control mucosa in Apc^+/LoxP mice and normal-appearing mucosa in CDX2P-Cre-Apc^+/LoxP mice, AOM administration to CDX2P-Cre-Apc^+/LoxP mice appeared to increase MUC1 mRNA expression in grossly normal tissues when compared to similar tissues harvested from AOM treated Apc^+/LoxP controls, but differences were not statistically significant (C). (D–H) Immunohistochemical stainings confirmed MUC1 protein upregulation, and AOM-induced potentiation of MUC1 protein upregulation, in colon tumors and normal-appearing mucosa in both Apc^+/LoxP and CDX2P-Cre-Apc^+/LoxP mice. All images were taken at ×200 magnification.