Chemical induction of colonic tumor formation

Among several naturally mutant or genetically modified rodent models, which are predominantly used to study hereditary CRC and which will be discussed later (see chapter 4.5.3), inducible tumor models to study non-hereditary tumor development exist (Neufert et al. 2007). Azoxymethane (AOM) is a specific colon carcinogen that is used to mimic sporadic CRC (Bissahoyo 2005; Rosenberg et al. 2008). Of note, AOM itself is not the final carcinogenic metabolite, it is rather a pro-carcinogen that requires metabolic activation (Neufert et al. 2007; Rosenberg et al. 2008). The first step of metabolism is the conversion of AOM to methylazoxymethanol (MAM) by hydroxylation catalyzed by CYP2E1 in the liver, which is an alcohol-inducible cytochrome P-450 isoform (Sohn et al. 2001; Rosenberg et al. 2008). MAM, which is an unstable compound that spontaneously decomposes to formaldehyde and methyldiazonium, is transported to the colon via the bloodstream (Rosenberg et al. 2008). Methyldiazonium is responsible for the alkylation of guanine and thymine and thereby facilitates base mispairings, which, if not repaired, result in initiation of tumorigenesis (Sohn et al. 2001;

Neufert et al. 2007). Bissahoyo (2005) performed mouse experiments to standardize AOM treatment. They revealed significant dose- and strain-dependent susceptibility to

AOM, and predicted 10 mg AOM per kg body weight as the standard dose that is maximal tolerated for tumor induction. They showed four weekly intraperitoneally treatments as the best route of administration, and did not observe any sex-dependent differences. They revealed that AOM is not only a tumor initiator, but also a tumor promoter. Interestingly, AOM-induced tumors were predominantly found in the distal colon, which is also the predominant localization in humans. Furthermore, the tumors were pathologically similar to sporadic CRC in humans (Bissahoyo 2005; Neufert et al.

2007). In addition, AOM-induced tumors also mirrored human CRC at the molecular level. As already described, mutations in the tumor suppressor gene APC occur early during colorectal tumorigenesis (see chapter 1.2.1 and Powell et al. 1992) and were predicted to be the initiating events in the development of CRC (Maltzman et al. 1997).

APC is known to be mutated in more than 70% of sporadic cancers (Kheirelseid et al.

2013). Maltzman et al. (1997) investigated the role of Apc in murine colorectal tumors induced by AOM by immunofluorescence staining of Apc. They observed Apc staining in the small intestine and in the colon. Interestingly, Apc staining was absent in 96.7%

of the analyzed adenomas and carcinomas induced by AOM, indicating that wild type Apc is lost in AOM-induced colonic tumors, which mirrors the situation in human CRCs (Maltzman et al. 1997).

Fifty percent of human colonic tumors that carry an intact APC gene reveal mutations of the β-catenin gene at codons 33, 41 and 45 leading to constitutive transcriptional activation (Takahashi et al. 2000b). Analyzing ten murine colonic tumors induced by AOM revealed that all tumors had mutations in the β-catenin gene at codons 33, 34, 37 and 41 (Takahashi et al. 2000b).

Cyclin D1 is a positive regulator of cell cycle progression. Cyclin D1 forms a complex

with Cdk4 and Cdk6, which regulate the progression of the G1 phase and the G1 / S phase transition. Overexpression of cyclin D1 was found in human colon cancer

and concurrent overexpression of cyclin D1 and Cdk4 was reported in adenomatous

polyps of the human colon (Wang et al. 1998). Interestingly, examination of AOM-induced murine tumors revealed a significant increase in cyclin D1 and Cdk4 mRNA levels compared to control colonic tissue (Wang et al. 1998).

The K-RAS oncogene was also proven to play a role during the development of human CRC as described earlier (see chapter 1.2.1). In humans, mutations of K-RAS have been reported in 50% of CRCs (Luceri 2000). Takahashi et al. (2000a) investigated K-ras gene mutations in tumors induced by AOM in F344 rats and found that K-ras

gene mutations were as frequent in rat colonic carcinogenesis induced by AOM as in human CRCs.

In addition, Takahashi et al. (2000a) observed elevated levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in rat colon adenocarcinomas induced by AOM. Interestingly, expression of iNOS and COX-2 are known to be often increased in human colon tumors.

Thus, the AOM-based model is a suitable tool to analyze sporadic CRC and advantages of AOM-induced carcinogenesis are for example high potency, simple route of administration, low price and the comparability to human colonic tumors at the molecular level. However, one major disadvantage is the lack of invasiveness and the rareness of metastases (Neufert et al. 2007).

In addition to the AOM-based tumor model, which mimics sporadic human CRC, a colitis model based on dextran sodium sulfate (DSS) exists (Tanaka et al. 2003). DSS is a pro-inflammatory reagent with highly variable molecular weight that is extremely toxic to the epithelial lining of the colon, leading to severe colitis with bloody diarrhea (Neufert et al. 2007; Perše and Cerar 2012). Since the DSS model is known to reflect clinical and histopathological features seen in human chronic colitis (Perše and Cerar 2012), this model is particularly used to study tumor progression driven by chronic colitis, which is known to be the case in ulcerative colitis and Crohn’s disease (Neufert et al. 2007). In rodents, DSS is given over the drinking water (Neufert et al. 2007). The response of mice to DSS highly varies and was predicted to be dependent on e.g. the molecular weight of DSS, DSS concentration and the duration of DSS administration, but also on the strain and the sex of the mice (Perše and Cerar 2012). Analyses of uptake and tissue distribution revealed that DSS penetrates the mucosal membrane in the intestine, and that DSS was mainly detected in the liver Kupffer cells and in macrophages in the mesenteric lymph node and colon (Kitajima et al. 1999). A disadvantage of this colitis model based on DSS is the fact that a long period or repeated administration of DSS is needed to induce colitis or colitis-associated CRC, and that the incidence of induced tumors is relatively low (Tanaka et al. 2003). Tanaka et al. (2003) developed a new mouse model based on the combination of AOM and DSS. This model for colitis-associated cancer consists of a single intraperitoneal administration of 10 mg AOM per kg body weight and a one-week administration of 2% DSS over the drinking water. In week 20, colonic adenocarcinomas with 100% incidence and adenomas with 38% incidence with the presence of dysplasia and

colitis with mucosal ulceration have developed. Examination of the developed tumors by immunohistochemistry and immunofluorescence stainings revealed that all of the induced colonic adenocarcinomas were positive for β-catenin, COS-2 and iNOS, but were negative for p53 (Tanaka et al. 2003).

4.4.2 The IGF1R promotes progression of colonic tumors induced by AOM and