Amy A. Irving

The utility of Apc-mutant rats in modeling human colon cancer.

Prior to the advent of genetic engineering in the mouse, the rat was the model of choice for investigating the etiology of cancer. Now, recent advances in the manipulation of the rat genome, combined with a growing recognition of the physiological differences between mice and rats, have reignited interest in the rat as a model of human cancer. Two recently developed rat models, the polyposis in the rat colon (Pirc) and Kyoto Apc Delta (KAD) strains, each carry mutations in the intestinal-cancer-associated adenomatous polyposis coli (Apc) gene. In contrast to mouse models carrying Apc mutations, in which cancers develop mainly in the small intestine rather than in the colon and there is no gender bias, these rat models exhibit colonic predisposition and gender-specific susceptibility, as seen in human colon cancer. The rat also provides other experimental resources as a model organism that are not provided by the mouse: the structure of its chromosomes facilitates the analysis of genomic events, the size of its colon permits longitudinal analysis of tumor growth, and the size of biological samples from the animal facilitates multiplexed molecular analyses of the tumor and its host. Thus, the underlying biology and experimental resources of these rat models provide important avenues for investigation. We anticipate that advances in disease modeling in the rat will synergize with resources that are being developed in the mouse to provide a deeper understanding of human colon cancer.

Supplementation by vitamin D compounds does not affect colonic tumor development in vitamin D sufficient murine models.

Epidemiological studies indicate that sunlight exposure and vitamin D are each associated with a lower risk of colon cancer. The few controlled supplementation trials testing vitamin D in humans reported to date show conflicting results. We have used two genetic models of familial colon cancer, the Apc(Pirc/+) (Pirc) rat and the Apc(Min/+) (Min) mouse, to investigate the effect of 25-hydroxyvitamin D(3) [25(OH)D(3)] and two analogs of vitamin D hormone on colonic tumors. Longitudinal endoscopic monitoring allowed us to test the efficacy of these compounds in preventing newly arising colonic tumors and in affecting established colonic tumors. 25(OH)D(3) and two analogs of vitamin D hormone each failed to reduce tumor multiplicities or alter the growth patterns of colonic tumors in the Pirc rat or the Min mouse.

Candidate serum biomarkers for early intestinal cancer using 15N metabolic labeling and quantitative proteomics in the ApcMin/+ mouse.

Current screening procedures for colorectal cancer are imperfect and highly invasive and result in increased mortality rates due to low compliance. The goal of the experiments reported herein is to identify potential blood-based biomarkers indicative of early stage intestinal cancers using the ApcMin/+ mouse model of intestinal cancer as an experimental system. Serum proteins from tumor-bearing ApcMin/+ mice were quantitatively compared to tumor-free Apc+/+ wild-type mice via in anima metabolic labeling with 14N/15N-labeled Spirulina algae and an LTQ Orbitrap mass spectrometer. Out of 1116 total serum proteins quantified, this study identified 40 that were differentially expressed and correlated with the increase in intestinal neoplasms. A subset of these differentially expressed proteins underwent a secondary quantitative screen using selected reaction monitoring-mass spectrometry with stable isotope-labeled peptides. Using both quantitative techniques, we identified MGAM and COL1A1 as downregulated and ITIH3 and F5 as upregulated in serum. All but COL1A1 were similarly differentially expressed in the mRNA of neoplastic colonic tissues of ApcMin/+ mice compared to normal wild-type tissue. These differentially expressed proteins identified in the ApcMin/+ mouse model have provided a set of candidate biomarkers for future validation screens in humans.

Cyp1b1 exerts opposing effects on intestinal tumorigenesis via exogenous and endogenous substrates.

Cytochrome P450 1B1 (Cyp1b1) metabolism contributes to physiologic functions during embryogenesis but also to carcinogenic activation of polycyclic aromatic hydrocarbons (PAH). We generated Cyp1b1-deficient mice carrying the Min allele of the adenomatous polyposis coli gene. These Cyp1b1-deficient Min mice developed twice as many tumors as Min controls, which, however, remained similar in size and histology. Tumors from older (130 days) Cyp1b1-deficient Min mice selectively exhibited focal areas of nuclear atypia associated with less organized epithelia. The metabolism of endogenous substrates by Cyp1b1, therefore, suppresses tumor initiation but also affects progression. Treatment of Min mice with 7,12-dimethylbenzanthracene (DMBA) doubled both tumor multiplicity and size within 20 days but not when mice lacked Cyp1b1. This was paralleled by an abnormal staining of crypts with beta-catenin, phospho-IkappaB kinase, and RelA, which may represent an early stage of tumorigenesis similar to aberrant crypt formation. Cyp1b1 deletion did not affect circulating DMBA and metabolites. Cyp1b1 expression was higher in the tumors compared with normal small intestines. Increased tumorigenesis may, therefore, arise from generation of DMBA metabolites by Cyp1b1 in the developing tumors. Benzo(a)pyrene (BP), which is similarly activated by Cyp1b1 in vitro, did not affect tumorigenesis in Min mice. By contrast, BP and DMBA each suppressed tumor multiplicity in the absence of Cyp1b1. Cyp1b1 metabolism of DMBA and endogenous oxygenation products may each affect a tumor-promoting nuclear factor-kappaB activation, whereas Ah receptor activation by PAH affects suppression. Tumorigenesis may, therefore, depend on activation of PAH by Cyp1b1 and on offsetting suppression by Cyp1b1 of endogenous tumor-enhancing substrates.

Cholecalciferol or 25-Hydroxycholecalciferol Neither Prevents Nor Treats Adenomas in a Rat Model of Familial Colon Cancer

BACKGROUND Epidemiologic studies in humans have shown associations between greater sunlight exposure, higher serum 25-hydroxycholecalciferol [25(OH)D3] concentrations, and reduced colon cancer risk. However, results from a limited number of vitamin D supplementation trials in humans have not shown a protective effect. OBJECTIVE We sought to determine whether adding to the diet increasing amounts of either 25(OH)D3, the stable metabolite measured in serum and associated with cancer risk, or cholecalciferol (vitamin D3), the compound commonly used for supplementation in humans, could reduce emergent adenomas (chemoprevention) or decrease the growth of existing adenomas (treatment) in the colons of vitamin D-sufficient rats carrying a truncation mutation of adenomatous polyposis coli (Apc), a model of early intestinal cancer. METHODS Apc(Pirc/+) rats were supplemented with either vitamin D3 over a range of 4 doses [6-1500 μg/(kg body weight · d)] or with 25(OH)D3 over a range of 6 doses [60-4500 μg/(kg body weight · d)] beginning after weaning. Rats underwent colonoscopy every other week to assess effects on adenoma number and size. At termination (140 d of age), the number of tumors in the small intestine and colon and the size of tumors in the colon were determined, and serum calcium and 25(OH)D3 measurements were obtained. RESULTS At lower doses (those that did not affect body weight), neither of the vitamin D compounds reduced the number of existing or emergent colonic tumors (P-trend > 0.24). By contrast, supplementation at higher doses (those that caused a suppression in body weight gain) with either 25(OH)D3 or vitamin D3 caused a dose-dependent increase in colonic tumor number in both males and females (P-trend < 0.003). CONCLUSIONS No evidence for protection against colon tumor development was seen with lower dose supplementation with either cholecalciferol or 25-hydroxycholecalciferol. Thus, the association between sunlight exposure and the incidence of colon cancer may involve factors other than vitamin D concentrations. Alternative hypotheses warrant investigation. Furthermore, this study provides preliminary evidence for the need for caution regarding vitamin D supplementation of humans at higher doses, especially in individuals with sufficient serum 25(OH)D3 concentrations.

Monoallelic silencing and haploinsufficiency in early murine intestinal neoplasms

Studies of tumors from human familial adenomatous polyposis, sporadic colon cancer, and mouse and rat models of intestinal cancer indicate that the majority of early adenomas develop through loss of normal function of the Adenomatous polyposis coli (APC) gene. In murine models of familial adenomatous polyposis, specifically the multiple intestinal neoplasia mouse (Min) and the polyposis in the rat colon (Pirc) rat, most adenomas have lost their WT copy of the Apc gene through loss of heterozygosity by homologous somatic recombination. We report that large colonic adenomas in the Pirc rat have no detectable copy number losses or gains in genomic material and that most tumors lose heterozygosity only on the short arm of chromosome 18. Examination of early mouse and rat tumors indicates that a substantial subset of tumors shows maintenance of heterozygosity of Apc in genomic DNA, apparently violating Knudsons two-hit hypothesis. Sequencing of the Apc gene in a sampling of rat tumors failed to find secondary mutations in the majority of tumors that maintained heterozygosity of Apc in genomic DNA. Using quantitative allele-specific assays of Apc cDNA, we discovered two neoplastic pathways. One class of tumors maintains heterozygosity of ApcMin/+ or ApcPirc/+ RNA expression and may involve haploinsufficiency for Apc function. Another class of tumors exhibits highly biased monoallelic expression of the mutant Apc allele, providing evidence for a stochastic or random process of monoallelic epigenetic silencing of the tumor suppressor gene Apc.

The Concentrations of EGFR, LRG1, ITIH4, and F5 in Serum Correlate with the Number of Colonic Adenomas in ApcPirc/+ Rats

The development of noninvasive methods for early detection of colon cancer is critical for the successful management of this disease. Using a targeted quantitative proteomics technique, we assessed the ability of 12 serum proteins to detect the presence of colonic polyps in the ApcPirc/+ rat model of familial colon cancer. Serum protein candidates were selected from gene transcripts upregulated in colonic tumors of ApcPirc/+ rats and from a prior study of serum proteins differentially expressed in mice carrying intestinal adenomas. Proteins were quantified at early stages of polyp formation in a rat cohort monitored longitudinally by colonoscopy over a period of 75 days. Of the 12 proteins monitored at three distinct time points, seven showed differential expression in at least one time point in the serum from ApcPirc/+ rats compared with wild-type rats. Tumor multiplicity correlated with protein expression changes, and most tumors grew during the study. EGFR, LRG1, ITIH4, and F5 displayed the most robust tumor-associated protein expression changes over time. Receiver operator characteristic analysis using these four proteins resulted in a sensitivity of 100%, a specificity of 80%, and an area under the curve of 0.93 at 135 days of age, when the Pirc rats bore an average of 19 tumors in the colon and seven in the small intestine. The results of this study demonstrate that the quantitative analysis of a panel of serum proteins can detect the presence of early intestinal tumors in a rat model, and provides support for future measurements in humans. Cancer Prev Res; 7(11); 1160–9. ©2014 AACR.

The conserved protective cyclic AMP-phosphodiesterase function PDE4B is expressed in the adenoma and adjacent normal colonic epithelium of mammals and silenced in colorectal cancer

Conservation over three mammalian genera—the mouse, rat, and human—has been found for a subset of the transcripts whose level differs between the adenoma and normal epithelium of the colon. Pde4b is one of the triply conserved transcripts whose level is enhanced both in the colonic adenoma and in the normal colonic epithelium, especially adjacent to adenomas. It encodes the phosphodiesterase PDE4B, specific for cAMP. Loss of PDE4B function in the ApcMin/+ mouse leads to a significant increase in the number of colonic adenomas. Similarly, Pde4b-deficient ApcMin/+ mice are hypersensitive to treatment by the inflammatory agent DSS, becoming moribund soon after treatment. These observations imply that the PDE4B function protects against ApcMin-induced adenomagenesis and inflammatory lethality. The paradoxical enhancement of the Pde4b transcript in the adenoma versus this inferred protective function of PDE4B can be rationalized by a feedback model in which PDE4B is first activated by early oncogenic stress involving cAMP and then, as reported for frank human colon cancer, inactivated by epigenetic silencing.

Vitamin D deficiency in the Apc Pirc/+ rat does not exacerbate colonic tumorigenesis, while low dietary calcium might be protective

ABSTRACT Human studies have shown that individuals with colon cancer tend to have lower serum 25-hydroxy-vitamin D3 [25(OH)D3] levels compared with healthy controls, but whether this link is causative, a result of the disease or an indicator of another factor altogether has yet to be demonstrated. In humans, vitamin D, calcium and UV exposure are inextricably linked; therefore, understanding the individual and combined roles of each of these will require animal models specifically designed to address these questions. To begin to untangle this network, our group has employed the ApcPirc/+ rat, which contains a truncating mutation in the Apc gene, leading to the development of colonic tumors. Our group previously utilized this model to demonstrate that vitamin D supplementation above normal does not reduce colonic tumor burden and, in fact, increased tumor multiplicity in a dose-dependent manner. In the current study, we tested whether vitamin D deficiency plays a causative role in tumor development using two strains which differ in their susceptibility to intestinal tumorigenesis. In the colon, vitamin D deficiency did not increase the development of tumors in either strain, and was actually protective in one strain. Unexpectedly, low dietary calcium combined with vitamin D deficiency significantly suppressed tumor development in the small intestine and colon of both strains. The vast majority of tumors in the human intestine occur in the colon, and we find no evidence to support a direct role of vitamin D deficiency in increasing colonic tumorigenesis, and low calcium might protect against tumor development. This article has an associated First Person interview with the first author of the paper. Summary: Using rat models of familial and inflammation-induced colon cancer, this study demonstrates that vitamin D deficiency does not increase risk of developing disease, and that low dietary calcium might be protective.

Suppression of experimental autoimmune encephalomyelitis by ultraviolet light is not mediated by isomerization of urocanic acid

BackgroundUltraviolet B irradiation confers strong resistance against experimental autoimmune encephalomyelitis, a model of multiple sclerosis. This protection by ultraviolet B is independent of vitamin D production but causes isomerization of urocanic acid, a naturally occurring immunosuppressant.MethodsTo determine whether UCA isomerization from trans to cis is responsible for the protection against experimental autoimmune encephalomyelitis afforded by ultraviolet B, trans- or cis-urocanic acid was administered to animals and their disease progression was monitored.ResultsDisease incidence was reduced by 74% in animals exposed to ultraviolet B, and skin cis-urocanic acid levels increased greater than 30%. However, increasing skin cis-urocanic acid levels independent of ultraviolet B was unable to alter disease onset or progression.ConclusionsIt is unlikely that urocanic acid isomerization is responsible for the ultraviolet B-mediated suppression of experimental autoimmune encephalomyelitis. Additional work is needed to investigate alternative mechanisms by which UVB suppresses disease.