Chemically induced mammary tumours

These models have been very important models for hormone sensitive breast cancer.

2.2.4.2.1 7,12 Dimethylbenz(a)anthracene (DMBA)

The development of chemically induced rat models of breast cancer was pioneered by C.B.Huggins et al. (1961) and Gullino et al. (1975), who used 7,12 dimethylbezanthracene and N-methyl-nitrosamine respectively. The models were reproducible and compared to former methods relatively easy to use. The resulting tumours are generally hormone sensitive adenocarcinomas. These models reflect the dependence on ovarian and pituitary hormones for development of about half of the human cases.

The chemicals mainly used today to induce mammary tumours are 7, 12 dimethylbenzanthracene, N- methylnitrosurea and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The naturally occurring food derived PhIP is thought to have more relevance as a cause for breast cancer in women than DMBA or MNU (Ip, 1996).

7,12 dimethylbezanthracene is a polycyclic aromatic hydrocarbon which can induce hormone sensitive mammary adenocarcinomas in a frequency up to

100%, when applied by gastric gavage to virgin rats. The susceptibility is greatest at an age between 30 and 55 days, when terminal end buds are proliferating most actively (Russo et al. 1978). Histologic types of mammary tumours encountered after DMBA treatment are papillary or cribriform adenocarcinomas.

A series of experiments in the laboratory of Kerdelhue et al (1981) investigated hormonal alterations after DMBA administration. It was shown that cycles were associated with blunted preovulatory surges of LH and FSH and increased surges of 17beta-estradiol (el-Abed et al. 1987). GnRH Rc content in the anterior pituitary was lower in DMBA treated rats versus controls, especially on prooestrus which was supposed to be a possible mechanism of the formerly observed lower LH peak (Jakubowski et al. 2002). Jahn et al. (1991) reported an up-regulation of prolactin receptor and insulin-like growth factor receptor in DMBA induced tumours (Jahn et al. 1991). The developing tumours are dependent on pituitary and ovarian hormones, especially prolactin, progesterone and oestrogen. Administration of a combination of oestrogen and progesterone has a tumour inhibiting effect (Welsch, 1985). Androgens have a tumour inhibiting function in DMBA induced tumours (Teller et al. 1966). Melatonin is thought to inhibit mammary cancer growth acting as an antagonist of gonadal steroids especially oestrogen (Sanchez-Barcelo et al. 2003).

Down-regulation of melatonin was proposed as a mechanism of carcinogenesis in the DMBA-model (de Jonage Canonico et al. 2003). Russo et al. (1996) observed a marked stromal reaction consisting of collagen deposition and infiltration by mast cells and lymphocytes around intraductal proliferations which eventually progress to carcinoma. They concluded that the host response and especially the accumulation of mast cell play a role in tumour progression (Russo et al. 1996).

DMBA is a mutagenic agent, which forms DNA adducts after metabolic activation. Mutations caused are transitions of dG or dA to alternative nucleotides. An A to T transition resulted in an activating mutation in the 61^st codon of the H-ras oncogene in rat mammary tumours induced by DMBA. This could be an important factor in the pathogenesis of DMBA-induced tumours

(Stanley, 1994). Other changes of the genetic regulation of the cells which have been associated with DMBA treatment are up-regulation of NF-κB (Kim et al.

2000), VEGF (Heffelfinger et al. 2000) and alterations in the AhR pathway (Trombino et al. 2000), which is involved in the metabolism and activation of DMBA to form an ultimate carcinogen (DMBA-3,4-diol-1,2-epoxide) via the Cytochrome P450 system. Up-regulation of AhR could explain the high levels of NF-κB by induction of oxidative stress (Dalton et al. 2002; Nebert et al. 2000).

AhR and NF-κB have been shown to interact and to regulate c-myc expression and a cyclin dependent kinase (Tian et al. 2002; Puga et al. 2002).

Two studies examining DMBA and PhIP (Kuramoto et al. 2002, Shan et al.

2002) using microarray technology have identified an up-regulation of cyclin D1, one of them (Shan et al. 2002) reporting an up - regulation of CDK4, PDGF-A and STAT5A as well. It was speculated that PDGF exerts its effect via up - regulation of STAT transcription factors which in turn may account for the over expression of cyclinD1. CyclinD1 up-regulation was reported already six weeks after DMBA-treatment (Papaconstantinou et al. 2005). Among the genes up-regulated at this early time-point were Lgals7, Il-18, Igfbp2 and Pdgfa. Casein beta and thyroid hormone responsive Spot14 and Scd1 were down-regulated (Papaconstantinou et al. 2005).

2.2.4.2.2 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

The mechanism of action of PhIP involves activation by Phase II esterification resulting in the guanine adduct forming N-acetoxy-PhIP. In addition to the mutating activity of PhIP, it seems to retard the differentiation of TEBs of adolescent rats thus facilitating carcinogenesis by conserving the more susceptible TEBs. Inhibition of apoptosis and a prolactin-like effect may contribute to the carcinogenic effect of PhIP (Snyderwine et al. 2002;

Snyderwine, 1999). The de-regulation of the cyclin D1, CDK4, Rb pathway was confirmed for the PhIP model by Qiu et al. (2003). Ras is mutated in PhIP- induced tumours like in DMBA-induced tumours (Yu et al. 2002). Interestingly, PhIP is also capable of inducing elevated levels of Neu - the rat homologue of

ErbB2. (Davis and Snyderwine, 1995). In a study of Vuorio et al. (1988) no alteration of ErbB2 was detected in the DMBA-induced model. The amplification of the Neu gene - as occurs in 30% of human breast cancers - has not been observed in any rat model except in a transgenic approach of Watson et al.

(2002), which resulted in breast cancer of male rats.

2.2.4.2.3 N-methylnitrosurea (MNU)

MNU is a directly acting carcinogen. It causes activating mutations in codon 12 of the Ha-ras oncogene (Stanley, 1994).

There are two protocols for the induction of mammary tumours. One applying MNU to animals of about 55 days of age (Gullino et al. 1975) and another one treating animals at 21 days of age (Thompson et al. 1995).

The histological lesions occurring after treatment of animals at the age of 21 days are ductal hyperplasia, atypical hyperplasia and ductal carcinoma in situ (cribriform, comedo, papilliform in descending order of frequency). Some of the DCIS are surrounded by a desmoplastic stroma infiltrated by mast cells. The adenocarcinomas induced in this system resembled those induced by treatment of 55-day-old rats and show cribriform, papillary, and comedo subtypes. Invasion of the regional lymph node and metastasis to the lung has been observed in animals treated at 21 days of age. In this model, ovarian dependent and independent tumours are formed (<60% ovarian steroid dependent tumours arise in this model while >75% arise in rats treated at 50 days; Thompson et al. 2000).

In contrast to Thompson et al., Singh et al. (2000) state, that metastases are not found in the short term model except for an occasional focus in lymph nodes.

Elastosis and microcalcification, which occur in humans are not observed.

Histologic lesions such as tubular, mucinous, adenoid cystic, medullary and lobular carcinoma are not found. The range of benign lesions and hyperplasias is more limited in the rat model (Singh et al. 2000).

Genetic alterations in the MNU model are changes in Ha-ras (mutation of codon 12) mutations in p53 and mdm2 have not been found in original and passaged tumours in normal and ovariectomized rats treated with MNU (McKenzie et al.

1997). In contrast to these observations, Sukumar et al. (1995) observed a p53 mutation in one passaged MNU-induced tumour, PRAD-1 and IGF-2 amplifications and a loss of the mitogenic growth factor gene MK in later passages of MNU-induced tumours. In an investigation of MNU- and DMBA- induced tumours the following alterations in gene expression were observed. Up- regulated genes in MNU-induced tumours include rat homologues of galectin-7, the human mouse melanoma inhibitory activity/ bovine chondrocyte – derived retinoic acid sensitive protein gene (MIA/CD-RAP), the mouse stearoyl CoA desaturase-2 gene, and the mouse endoB cytokeratin/ human cytokeratin gene 18, galectin 7, MIA/CD-RAP and cytokeratin gene 18 were over expressed in DMBA-induced tumours as well (Lu et al. 1997).

Rat mammary tumour gene 1 is up-regulated in mammary tumours induced by MNU. (Chiou et al. 2001). The centrosome associated regulatory molecule and oncogene AurA (Aurora A) was reported to be over-expressed in MNU tumours, which showed centromere amplification (Goepfert et al, 2002).

Cyclin D1 is up-regulated in MNU tumours (Thompson 2000, Wang 2001).

The most prominent oncogenes or tumour suppressor genes involved in human breast cancer (BRCA1/2, p53, ATM) are either not studied or have been shown to play different roles in the two species. For BRCA1 no different expression was observed in normal and diseased tissues of the rat (Chen et al. 1996), p53 is not mutated as in humans, ATM and BRCA2 have not yet been studied. The only oncogene found with a potentially activating mutation is Ha-ras. The involvement of this oncogene plays a role in about 5% of the human cases. Its role in the development of mammary tumours in the rat is shown to be causative in some experiments (Thompson et al. 1998). The activation of ras is more frequent than mutations, though, as ras is a downstream effector of growth factor pathways, which are frequently activated in human breast cancer e.g. the erbB2 or EGF pathway (von Lintig et al. 2000).