How good are rodent models of carcinogenesis in predicting efficacy in humans? A systematic review and meta-analysis of colon chemoprevention in rats, mice and men

Tumours in rodent and human colon share many histological and genetic features. To know if rodent models of colon carcinogenesis are good predictors of chemopreventive efficacy in humans, we made a meta-analysis of aspirin, beta-carotene, calcium, and wheat bran studies. Controlled intervention studies of adenoma recurrence in human volunteers were compared with chemoprevention studies of carcinogen-induced tumours in rats, and of polyps in Min (Apc(+/-)) mice: 6714 volunteers, 3911 rats and 458 mice were included in the meta-analyses. Difference between models was small since most global relative risks were between 0.76 and 1.00. A closer look showed that carcinogen-induced rat studies matched human trials for aspirin, calcium, carotene, and were compatible for wheat bran. Min mice results were compatible with human results for aspirin, but discordant for calcium and wheat bran (no carotene study). These few results suggest that rodent models roughly predict effect in humans, but the prediction is not accurate for all agents. Based on three cases only, the carcinogen-induced rat model seems better than the Min mouse model. However, rodent studies are useful to screen potential chemopreventive agents, and to study mechanisms of carcinogenesis and chemoprevention

A Multi-Scale Agent Based Model of Colon Carcinogenesis

Colorectal cancer (CRC) is a major cause of cancer mortality and there remain aspects of its formation which are not understood. The colon contains an epithelium punctuated by flask shaped invaginations called the crypts of Lieberkühn. These crypts are monoclonal in nature while adenomas are thought to be polyclonal, suggesting that multiple crypts are involved in carcinogenesis. It has been reported that fields of mutated tissue surround adenomas but the causes and growth of these fields are not well understood. There are two competing hypotheses regarding growth, the first being that mutated cells from one crypt invade neighbouring crypts, and the second that mutated crypts replicate themselves more often than wild-type crypts. To investigate these processes two agent based models were developed. The first model represents cells as agents and is similar to previous models in the field, but is novel in including the geometry of the crypt mouth. This is necessary to model multiple interacting crypts. This model is the first in the literature to be used to represent multiple crypts and is used to investigate invasion of neighbour crypts by mutated cells. The second model represents whole crypts as agents, which allows the entire colon to be simulated for multiple decades of biological time, as far as we are aware this is the first such model. The cell scale model predicts that crypt invasion does not occur, but that mutated cells can invade the flat mucosa above neighbouring crypts. Analysis of in-vivo data is consistent with this prediction. The crypt as agent model predicts fields of ~41,000 crypts, in agreement with data in the literature, this corresponds to a field ~23mm in diameter. This project models pre-cancerous fields for the first time over a variety of scales, making specific novel predictions which are in agreement with in-vivo data where such data exist. Two agent based models were created to study the development of precancerous fields, one a model with cells as agents to study cell scale phenomena and the other with crypts as agents to allow processes to be studied on larger spatial and temporal scales. These models could potentially be used to refine clinic practice by predicting the required frequency of post-intervention monitoring of patients or the necessity of further intervention

The Guanylate Cyclase C-cGMP Signaling Axis Opposes Intestinal Epithelial Injury and Neoplasia.

Guanylate cyclase C (GUCY2C) is a transmembrane receptor expressed on the luminal aspect of the intestinal epithelium. Its ligands include bacterial heat-stable enterotoxins responsible for traveler\u27s diarrhea, the endogenous peptide hormones uroguanylin and guanylin, and the synthetic agents, linaclotide, plecanatide, and dolcanatide. Ligand-activated GUCY2C catalyzes the synthesis of intracellular cyclic GMP (cGMP), initiating signaling cascades underlying homeostasis of the intestinal epithelium. Mouse models of GUCY2C ablation, and recently, human populations harboring GUCY2C mutations, have revealed the diverse contributions of this signaling axis to epithelial health, including regulating fluid secretion, microbiome composition, intestinal barrier integrity, epithelial renewal, cell cycle progression, responses to DNA damage, epithelial-mesenchymal cross-talk, cell migration, and cellular metabolic status. Because of these wide-ranging roles, dysregulation of the GUCY2C-cGMP signaling axis has been implicated in the pathogenesis of bowel transit disorders, inflammatory bowel disease, and colorectal cancer. This review explores the current understanding of cGMP signaling in the intestinal epithelium and mechanisms by which it opposes intestinal injury. Particular focus will be applied to its emerging role in tumor suppression. In colorectal tumors, endogenous GUCY2C ligand expression is lost by a yet undefined mechanism conserved in mice and humans. Further, reconstitution of GUCY2C signaling through genetic or oral ligand replacement opposes tumorigenesis in mice. Taken together, these findings suggest an intriguing hypothesis that colorectal cancer arises in a microenvironment of functional GUCY2C inactivation, which can be repaired by oral ligand replacement. Hence, the GUCY2C signaling axis represents a novel therapeutic target for preventing colorectal cancer

MicroRNA-135b promotes cancer progression by acting as a downstream effector of oncogenic pathways in colon cancer

MicroRNA deregulation is frequent in human colorectal cancers (CRCs), but little is known as to whether it represents a bystander event or actually drives tumor progression in vivo. We show that miR-135b overexpression is triggered in mice and humans by APC loss, PTEN/PI3K pathway deregulation, and SRC overexpression and promotes tumor transformation and progression. We show that miR-135b upregulation is common in sporadic and inflammatory bowel disease-associated human CRCs and correlates with tumor stage and poor clinical outcome. Inhibition of miR-135b in CRC mouse models reduces tumor growth by controlling genes involved in proliferation, invasion, and apoptosis. We identify miR-135b as a key downsteam effector of oncogenic pathways and a potential target for CRC treatment

The Tumor Suprressor APC: Nuclear Functions and Regulation by Heat Shock Response

Because mutation of the tumor suppressor APC initiates ~80% of all colorectal cancers, understanding APC function is central for better diagnostic, preventive, and therapeutic strategies for the disease. In-vitro studies have indicated that APC shuttles between the cytoplasm and nucleus, using two nuclear localization signals (NLS) and five nuclear export signals (NES). To better understand the role of nuclear APC, our lab made a mouse model with mutations in both NLS (ApcmNLS) sequences. In this dissertation, I report higher Wnt signalling and increased proliferation in intestinal epithelial cells from ApcmNLS/mNLS mice, and observe that these mice are more susceptible to colitis-induced colon tumorigenesis. Furthermore, ApcMin mice, a well-characterized Apc mouse model that carries an Apc truncation mutation, have increased intestinal polyp multiplicity, size, and proliferation index when they also carry the ApcmNLS allele. Taken together, these data support a role for nuclear Apc in cell proliferation, inhibition of Wnt signalling, and tumor suppression. ApcmNLS/Min mice also display extra-intestinal phenotypes, including enhanced mammary tumorigenicity and more severe anaemia, than in ApcMin/+ mice, suggesting a role for nuclear Apc in other tissues. My studies also identified and characterized a polymorphism in the promoter of the Pla2ga2 (Mom-1) gene that might be responsible for the attenuated phenotype observed in ApcMin/+ mice in some genetic backgrounds. I developed a simple, reliable, PCR-based test for this polymorphic allele that will allow easy screening of mouse colonies. The mechanisms by which cellular APC levels are regulated are not completely understood. In this dissertation, I show that induction of the heat-shock response increases APC levels both in colon cancer cell lines and in mouse intestinal epithelial cells. I tested two compounds to induce the heat-shock response and found altered tumor multiplicity, size, and regional distribution in two mouse models with different germline mutations in Apc. I also showed that a novel non-toxic heat-shock response inducer, KU-32, protects against colitis-mediated tumorigencity in mice. I propose that regulation of APC levels via heat-shock response contributes to many aspects of APC and intestinal tumor biology, and can serve as a novel molecular target for prevention and treatment of colorectal cancer

Methylation of NOTCH genes in normal and at-risk colorectal epithelium

PhD ThesisIntroduction Colorectal cancer (CRC) arises from genetic defects in stem cells. NOTCH signalling plays a key role in stem cell replication control. NOTCH-related genes are overexpressed in CRC. The mechanism for this is not known but could include epigenetic activation of NOTCH oncogenes via promoter hypomethylation. Methylation can be modulated by environmental stimuli including dietary factors such as butyrate, produced by bacterial fermentation of non-digestible carbohydrates in the colon. Butyrate exerts potent anti-oncogenic effects in the colorectal mucosa. Methods Participants were recruited at endoscopy and included those at normal risk of CRC (n=75), or higher risk of CRC because of previous adenomatous polyps (n=28) or ulcerative colitis (n=12). Participants provided 9 rectal biopsies. Normal risk participants were randomised to resistant starch (Hi-maize 260) or polydextrose supplementation in a 2x2 factorial placebo controlled trial for 50 days. Methylation of several CpG sites in the promoters of JAG1 (NOTCH pathway ligand) and RBP-J (NOTCH intracellular activator) was quantified using pyrosequencing. Results For JAG1 there was trend towards lower methylation at all CpG sites in those at higher CRC risk. Methylation at RBP-J CpG 11 was lower in polyp patients than in controls (18.0(1.5) vs. 23.6% (0.8), p=0.011). At JAG1 CpG 4, methylation increased following polydextrose supplementation compared to placebo (3.1(0.4) vs. 1.7%(0.4), p=0.009). A similar, but non-significant, trend was observed at other CpG sites for JAG1. Conclusions DNA methylation of NOTCH signalling genes is altered in macroscopically normal colorectal mucosa of patients at higher CRC risk. The observed changes in JAG1 methylation after polydextrose supplementation are consistent with a protective effect against carcinogenesis.Northumbria Healthcare NHS Foundation Trust: The BBSRC (grant reference BH090948)

Hydrophobic bile acids, genomic instability, Darwinian selection, and colon carcinogenesis

Sporadic colon cancer is caused predominantly by dietary factors. We have selected bile acids as a focus of this review since high levels of hydrophobic bile acids accompany a Western-style diet, and play a key role in colon carcinogenesis. We describe how bile acid-induced stresses cause cell death in susceptible cells, contribute to genomic instability in surviving cells, impose Darwinian selection on survivors and enhance initiation and progression to colon cancer. The most likely major mechanisms by which hydrophobic bile acids induce stresses on cells (DNA damage, endoplasmic reticulum stress, mitochondrial damage) are described. Persistent exposure of colon epithelial cells to hydrophobic bile acids can result in the activation of pro-survival stress-response pathways, and the modulation of numerous genes/proteins associated with chromosome maintenance and mitosis. The multiple mechanisms by which hydrophobic bile acids contribute to genomic instability are discussed, and include oxidative DNA damage, p53 and other mutations, micronuclei formation and aneuploidy. Since bile acids and oxidative stress decrease DNA repair proteins, an increase in DNA damage and increased genomic instability through this mechanism is also described. This review provides a mechanistic explanation for the important link between a Western-style diet and associated increased levels of colon cancer