Animal models for breast cancer

ELSEVIER Mutation Research 333 (1995) 37-44

Fundamental and Molecular Mechanisms of Mutagenesis

Animal models for breast cancer

Saraswati Sukumar * , Katherine McKenzie, Ying Chen

&folecular Biology of Breast Carzcer Laboratop, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd.. La Jolla, CA 92037. USA

Abstract

Rodent mammary tumors induced by chemical carcinogens have proven to be very useful in the genetic analysis of initiation, promotion and progression of mammary carcinogenesis. We are studying rat mammary carcinomas induced by the chemical carcinogen, N-nitroso-N-methylurea. The earliest genetic event observed in the mammary gland is the activation of Ha-ras oncogenes, which is followed by promotion of the initiated cells by hormones involved in puberty. Preferential amplification of the mutated Ha-ras allele, of PRAD-I and IGF2, loss of expression of the mitogenic growth factor gene, MK, and mutation in the tumor suppressor gene, ~53, are seen in the mammary tumors during tumor progression.

Keywords: Breast cancer: Animal model; N-Nitrosa-N-methylurea

1. Introduction

Animal models systems provide an invaluable tool to understand the complexity of multistep carcinogenesis. Carcinogen-induced mammary tumors in rats and mice have been particularly useful in furthering our understanding of mammary gland tumorigenesis.

The three broadly defined events in tumor formation are initiation, promotion and progression (Yuspa and Poirier, 1988). During initiation, carcinogens form adducts with DNA and cause mutations in oncogenes and/or tumor suppressor genes that be- come fixed upon mitosis. During promotion the initiated cells are stimulated to divide by mechanical, hormonal or genetic factors and form preneoplasias and benign tumors. This stage can occur long after

* Corresponding author.

the cells have been initiated and can be reversed if the promotional factors are removed. Progression to a malignant phenotype with acquisition of metastatic capabilities is the final stage of tumorigenesis. This stage is believed to be brought on by the increasing genetic instability of the tumor, leading to mutations at key genetic sites.

Rat models of mammary cancer present identifi- able states of preneoplasia and neoplasia of the mammary gland. Therefore genetic events can be analyzed in terms of when they occur during the tumorigenic process and whether their appearance is sufficient for the maintenance of the phenotype. The suitability of the rat as a model for human breast tumorigenesis also lies in the ease with which hormone-dependent tumors can be generated by carcinogens. In their advanced stages, the tumors metas- tasize to the lung. It is therefore possible to predict, in this model, the progression of events during the tumorigenic process. By examining preneoplastic,

0027.5107/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDl 0027-5 107(95)00 129-8

38 S. Sukurnar et al. /Mututiorz Rrseorch 333 f 1995) 37-41

neoplastic and metastatic tissues, researchers can delineate the genetic alterations occurring during tumorigenesis.

In this paper, we describe the ways in wh.ich rat models have given us insight into the specific molecular alterations driving tumor initiation, promotion and progression of breast cancer.

2. Initiation of carcinogenesis by activation of ras oncogenes and other factors contributing to initiation of mammary tumorigenesis

Some of the highly potent carcinogens for elicit- ing mammary cancer are methylating agents such as N-nitroso-N-methylurea (NMUI and polycyclic hy- drocarbons such as 7,12-dimethylbenz( alanthracene (DMBA). The mechanism of action of NMU and DMBA adds to the utility of the model. NMU is a direct alkylating carcinogen that is active at physiological pH for about half an hour (Gullino et al., 197.5) and as short as 8 min in cell culture (Jensen et al., 1977). Therefore, any consequences of NMU action will occur very early in the tumorigenic process. DMBA is an indirect acting carcinogen, but mammary epithelial cells are capable of metaboliz- ing it to the active proximal carcinogens (Guzman et al.. 1988). Thus, mammary epithelial cells efficiently activate both of these carcinogens. They can be used both in vivo and in vitro to isolate and study differ- ent stages of tumorigenesis.

When carcinogens are administered to mammary cells, specific genetic targets for tumor initiation can be identified. Mutational activation of rus oncogenes is closely linked to mammary carcinogenesis in animal models. A single administration of NMU or DMBA to a susceptible rodent can cause a point mutation in the 12th. 13th or 61 st MS codon which is sufficient for activation of the oncogene (Zarbl et

al.. 1985). The rus oncogene codes for p2 1. p2 1 is a plasma

membrane associated protein that binds GTP and GDP with high affinity and possesses a GTPase activity. The G to A transition in Ha-r-as changes the amino acid from a glycine to a glutamic acid residue. Substitution of glycine by other amino acids with the exception of proline gives rise to an activated ~21. Mutation in the rus gene result in two kinds of alterations, mutations that reduce the affinity of GDP and GTP and those that abolish the intrinsic GTPase activity. Loss of the intrinsic hydrolytic activity leads to loss of self-regulation by hydrolysis of GTP, resulting in a t-us protein that is constitutively activated (Haubruck and McCormick, 1991).

Initiation of mammary carcinogenesis occurs at the genetic level. The type of carcinogen used dic- tates the mutation that appears in the lesion. For example, NMU mutates DNA by methylating guanine residues at the Oh and N’ positions. Although most of these lesions are repaired by 06-methylguanine DNA methyltransferase, some mutations es- cape repair resulting in 06-methylguanine adducts. The cell misreads the unrepaired guanine, leading to G to A transitions (Loechler et al., 1984). G to A transitions have been established as the major muta- genic lesion caused by NMU (Singer and Kusmierek. 1982; Eadie et al., 1984).

Approximately 90% of the NMU-induced rat mammary tumors is a single G to A point mutation in the 12th codon of the Ha-rus oncogene’(Sukumar et al., 1983). The G to A transition in the rus oncogene supports the idea that NMU is directly responsible for the mutation because it is characteris- tic of the genetic change observed in DNA exposure to NMU (Zarbl et al., 1985; Sukumar et al., 1988). The mutation detectable in the DMBA-induced tumors derived from rats (Zarbl et al., 198.5) or 70-80% of those derived from the mouse hyperplastic out- growth line UCD/Dl (Dandekar et al., 1986) was an A to T transition in the 61st codon of Ha-rus. The A to T transition conforms to the expected chemical reactivity of DMBA on DNA.

DMBA, on the other hand, forms adducts with Ha-rus is not the only oncogene found activated both dG and dA nucleotides (Balmain and Brown, in NMU-induced mammary tumors from rats and 1988). The mutations generated by the formation of mice. Ki-rus carries a G to A mutation in the 12th

these adducts predict the transition of dG or dA residues to alternative nucleotides. Several studies have identified DNA adduct formation in mammary epithelial cells when animals are administered DMBA (Singletary et al., 1990; Liu and Milner. 1992) indi- cating that this mechanism is active in mammary

cells.

S. S&mar et al. /Mutation Research 333 11995137-44 39

codon of about 50% of rat mammary tumors (Sukumar, 1989; Kumar et al., 1990) and the 12th codon of about 17% of pituitary isografted mice (Guzman et al., 1992). No investigations have un- covered mutations in N-rus in carcinogen treated mammary glands.

Because of its commonality, several investigators have attempted to establish the role of point mutated rus genes as an initiator of tumorigenesis. One av- enue of investigation to achieve this end is the assessment of the number of cells carrying the activating rus mutation in the rat mammary gland shortly after exposure to NMU. Researchers have developed a clonogenic assay for determining the number of cells initiated during tumorigenesis (Gould et al., 1991). Estimations were made by exposing the mammary gland in situ to NMU or DMBA, maintaining the glands in culture and using a limiting dilution transplantation. They find that one in 43,000 cells are mutated 3 weeks after exposure to NMU and that one in 5 of these events consists of a point mutation in the ras oncogene. Mutations can be detected as early as 2 weeks after the NMU exposure as deter- mined by PCR amplification of the mutated Ha-rus (Kumar et al., 1990) and limiting dilution transplantation assay (Zhang et al., 1990). However other researchers using mismatched PCR technology con- tend that there are pre-existing G3’ to A35 ras

mutations in the breast cells of normal rats and that carcinogen exposure merely selects for the cells with mutation (Cha et al., 1994). Based on their data that an estimated one in IO5 cells of the rat mammary gland carry transforming Ha-rus mutations, it is surprising that the spontaneous mammary tumor incidence is only about 3%. and none of nearly 100

tumors examined carry mutated rus oncogenes (Marshal1 Anderson, personal communication). While the direct role of NMU on Ha-rus is open to question. clearly Ha-rus confers a selective advantage of tumorigenesis on cells carrying the mutation.

Perhaps the best evidence of rus involvement in the initiation of mammary tumors is that when activated rus genes are transfected into mammary cells and transplanted to mice. the cells give rise to preneoplastic lesions (Miyamoto et al.. 1990). However this evidence also indicates that rus activation is not sufficient for complete tumorigenesis.

The hormonal status of the animal at the time of

carcinogen exposure has a profound effect on both the type of lesions generated and the genetic alterations they harbor. The stage of the rat’s estrous cycle at the time of carcinogen exposure can dictate the type and the number of lesions (Haslam and Bern, 1977; Lindsey et al., 198 I ; Braun et al.. 1989). Late proestrus and estrus are times when circulating progesterone (Pg> and prolactin (Prl) levels are highest. Rats administered NMU during this period develop a greater number of terminal endbuds which are the precursors to intraductal hyperplasia and HANS. Intraductal hyperplasias harbor the majority of Ha-rus mutations (Sakai and Ogawa, 1991) and are precursors to adenocarcinoma. However, the percentage of tumors with activating Ha-rus mutations is lowest (8%) when the carcinogen is administered during estrus (S. Nandi, personal communication). The percentage of tumors carrying the Ha-rus mutation is highest (85%) when NMU is administered during metestrus. These observations argue that the administration of carcinogen when the mammary gland is under the influence of increased numbers of mitogenic signals leads to an increased complexity in the initiating events at the genetic level and the number of cell types affected.

Animal models have demonstrated that the activation of specific oncogenes leads to transformed phenotypes. Ha-rus and Ki-rus are specifically impli- cated in this process in several mammary mode1 systems. There is no doubt that activation of the rus

genes is one of the first genetic lesions associated with the majority of carcinogen induced mammary cancer.

3. Hormones, diet and genetic factors contribute to promotion of carcinogenesis

Molecular events occurring at the promotional stage of carcinogenesis are less well characterized in the mammary gland. This is due, in part, to the definition of promotion. The promotional stage of tumorigenesis relies on the hormonal state of the animal and the proliferative ability of the initiated cells. Therefore factors operating at this stage of tumorigenesis are generally characterized as epigenetic. However, they must ultimately act at the genetic level to generate changes leading to malignant

40 S. Sukumur et al. /Mutution Research 333 (19%~ 37-44

tumors. In fact, many of the mediators of tumor types operating at the initiation stage are also func- tional at the promotional stage of tumorigenesis.

Mutational activation of rus genes is not sufficient for tumorigenesis. Transfection studies have demonstrated that genes such as my and ~53 can cooperate with r-us in tumor formation (Slingerland and Benchimol, 1991; Taylor et al., 1992). When it is transfected into mammary epithelial cells. Ha-rus results in the formation of lobular alveolar nodules which are mortal and do not form tumors (Miyamoto et al.. 1990). Therefore additional changes are required for complete tumorigenesis.

Studies in rat hybrids have revealed that the mutation of rus alone is insufficient for tumorigenesis. The Copenhagen and Fischer F344 rats when ex- posed to the same NMU carcinogenesis protocol as the Buf/N. or Sprague-Dawley rats do not develop mammary tumors (Isaacs, 1986) although the fraction of cells containing the Ha-rus mutation in- creases by lo- to lOO-fold (Lu and Archer, 1992). This resistance to transformation is believed to be due to an inability of the cells carrying the mutated Ha-rus gene to undergo clonal expansion due to the inheritance of an autosomally dominant gene called mammary carcinoma suppressor gene (mcs). The tumorigenicity of v-Ha-rus when it was transfected into mammary cells of Copenhagen rats can over- come the suppressor effect of mcs. However, the tumors that appear are less aggressive than those arising in Sprague-Dawley rats under the same protocol (Wang et al., 19911.

Our studies examining the role of hormones in the carcinogenesis of the mammary gland have demonstrated that initiated cells require the presence of estrogen for tumor formation. Ovariectomy prior to, or following NMU administration lowers tumor incidence to negligible levels. Subsequent replacement with estrogen returns the tumor incidence to former levels with 50% of the tumors harboring activated rus genes (Kumar et al.. 19901. These results illustrate the requirement of estrogen-induced differentiation of mammary epithelial cells to trigger neoplastic development. Moreover, it emphasizes the point that the initiation events involving Ha-rus oncogene activation are not able to exert their tumorigenic proper- ties until the harboring cells became engaged in hormone-mediated differentiation.

There are at least two ways in which estrogen could be performing its role in promotion. The generally accepted function of estrogen is to expand the population of activated cells. Additionally, some studies have shown that the metabolites of estrogen can cause genotoxic damage to DMBA-treated mammary epithelial cells (Telang et al.. 1992) thereby causing further mutations in the susceptible cells.

Hormones other than estrogen play a role in tumor progression. When tumor cells that have been initiated in culture or in syngeneic animals are grown in animals carrying pituitary implants, the number of tumors and the molecular characteristics are distinct from the tumor grown in intact animals (Takata et al., 1990; Guzman et al., 1992). When rats are given pituitary implants the tumor number per animal in- creases, but the percentage of tumors with Ha-rus activation decreases from 30 to 15%. This data indicates that overproduction of prolactin can select for cells that were not necessarily initiated by rus

activation. Jahn et al. (1991) detect elevated levels of prolactin receptor and insulin-like growth factor receptor in DMBA-induced tumors in Sprague-Daw- ley rats. They hypothesize that the elevated levels of receptor help to increase cellular proliferation in the later stages of tumorigenesis (Jahn et al.. 1991).

In summary, animal models are necessary in order to define the factors involved in tumor promotion. They provide a physiological endpoint for analysis. However, these factors have a relatively small effect

at the molecular level except for the induction of genes required for cellular expansion and some mod- ulation of Ha-rus gene expression.

4. Amplification of Ha-ras and PRAD-1 oncogene, inactivation of ~53 tumor suppressor genes contribute to tumor progression and metastasis

The study of progression in the rodent mammary model relies heavily on detecting the accumulation of changes in the genetic profile of the tumors. Animal models are particularly suited to progression analysis because alterations in the same tumor can be simultaneously followed and analyzed (Dulbecco and Armstrong, 1988; Aldaz et al., 1992; Aldaz et al., 1993). The endpoints for analysis of tumor progres-

S. Sukumar et al. /Mutation Research 333 (1995137-44 41

sion the estrogen of the and degree metastatic potential.

tumors lacking or progesterone are classified more aggressive are

indicative a greater for metastases and Page, Koenders et 1991). Estrogen

also provides useful parameter the analysis tumor progression rodent mod-

The presence a single Ha-rus onco- in the of its counterpart is

for yielding hormone-independent phenotype et al., This indicates further mutations required before attain hormone

Two mechanisms cancer progression changes in dosage and mutations in

alternate genes. information indi- that both operate in tumor

promotion progression. The that genetic are responsible tumor progression

supported by observation that metastatic phe- of rat epithelial cells be in-

by transfecting cell lines DNA from cell lines et al.,

The specific responsible for increase the metastatic of the lines were identified. The that remains be answered which, if of the or tumor

genes known date are and/or sufficient tumor progression? in rats

mice indicate this question put much simply. It be that genes are the same so that is no critical gene, an array putative targets.

extension of carcinogen induced model can used to understanding of molecular mechanisms tumor progression. progression can monitored in system by

transplanting tumors syngeneic animals et al., Dulbecco and 1988).

Each of tumor be assayed histological as as genetic

Allelic deletion Ha-rus with subsequent duplication the mutant has been as a of activation other tumor such as skin (Quintanilla al., 1986; and Brown, The allelic of Ha-rus be due linkage with nearby tumor gene

(Aldaz al., 1992; et al., In fact, et al. have performed stud-

ies several hormone-dependent hormone-independent lines and that there a bias

duplication of chromosome 1, harbors the gene, as progress. There a

specific of the Ha-ras with concurrent loss the wild-type with increas-

tumor aggressiveness two separate progression (Aldaz et 1992; Aldaz al.. 1993);

unpublished observations). observations suggest the mutated is functioning tumor progression,

of non-metastatic rat mammary with v-Ha-rus increased metastatic to its This phenotype

associated with increase in expression (Kyprianou Isaacs, 1990) in genetic ity (Ichikawa al., 1990). cells with metastatic phenotype fused with parental non-metastasizing the metastatic is lost et al.. although the expression is The fusion indicate that elevated level Ha-rus expression not sufficient conferring the phenotype suggesting the critical involves the of a suppressor gene.

Ha-r-us status advanced tumors by no the only of genetic Several

putative for progression breast cancer been recognized later established

the use animal models. examples of genes are which has expression metastatic, but non-metastatic, NMU-in-

rat tumors et al., PRAD-1, which is over-expressed in 20% of breast tumors

(Gullick, 1990; Rosenberg et al., 1991); ~53. a tumor-suppressor gene that is altered in 40% of breast cancers (Donehower and Bradley, 1993); and HER2/neu. which is amplified in up to 40% of human tumors (McGuire et al., 1990; McKenzie, 1991). PRAD-I and ~53 are two genes that may have prognostic value in human disease.

We have screened 18 lines of invasive and hormone dependent tumors for alterations in a number of oncogenes such as PRAD-I and IGF2 and tumor suppressor genes such as ~53, and MK (Tomomura et al., 1990). We have detected PRAD-I and IGF2

41 S. Sukunmr et ul. /Mutation Research 333 f 19951 37-11

gene amplification in later passages of hormone independent and metastatic tumors by differential PCR

and Southern blot analysis. In the ~53 tumor suppressor gene, a G to A transition was found in the second passage of one out of 18 tumor lines and was maintained in subsequent passages. This shows that while ~53 mutations occur during the progression of NMU-induced rat mammary tumors, they are essential to the progression of only a small fraction of the tumors. Another gene, MK. encoding a growth-re- lated heparin-binding mitogenic protein. is expressed at high levels in the primary rat mammary tumors, but not in the metastatic ones, suggesting that loss of expression of this gene may aid the metastatic pro-

cess (our unpublished observations). These studies illustrate that the role of some of the putative mark- ers for advanced disease in humans can be elucidated through the use of animal models.

Animal models are instrumental in studying mammary molecular carcinogenesis. They provide manip- ulable systems that render physiological morpholo- gies for each phase in the tumorigenic process. In addition to identifying genes that play pivotal roles in tumorigenesis, researchers use these models to isolate the mechanisms through which tumorigenesis occurs. The complexity of the genetic events involved in the tumorigenic process is becoming in- creasingly apparent as the roster of oncogenes and tumor suppressor genes that participate in tumorigenesis grows. Eventually these investigations will be significantly enhanced by newly developed tech- niques such as fluorescent in situ hybridization

(FISH) and competitive genomic hybridization (CGH) which allow for examination of the alterations in an entire genome. However, animal studies also show that understanding the role of epigenetic factors in tumorigenesis is just as essential to solving the puzzle of mammary tumorigenesis as is understanding the genetic events. Epigenetic factors mod- erate not only the type of lesions generated by carcinogens. but also the genetic changes that occur during mammary tumorigenesis.

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Animal models for breast cancer