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Supplement: Nutritional Genomics and Proteomics in Cancer Prevention

The Use of Genetically Altered Mice for Breast Cancer Prevention Studies¹

Claudine Kavanaugh^*, and Jeffrey E. Green^*,2

^* Laboratory of Cellular Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD and Division of Preventive Oncology, Cancer Prevention Fellowship Program, National Cancer Institute, Bethesda, MD 20892

² To whom correspondence should be addressed. E-mail: jegreen{at}nih.gov.

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

 
Chemoprevention through nutritional and dietary changes may offer an important means of inhibiting the development and progression of breast cancer, which would have a major impact on public health. Studies to assess the efficacy of potential chemopreventive compounds are difficult to perform in large human populations, whereas the use of genetically engineered mice (GEM) for preclinical testing offers several advantages. GEM models can be utilized to assess the inhibitory effects of nutritional and chemopreventive agents on well-defined oncogenic signaling pathways. Because several transgenic mouse models progress through a well-defined temporal series of stages leading to invasive carcinoma formation, they may be particularly useful for determining cancer stage-specific responses to nutritional and chemopreventive agents. The C3(1)SV40 T/t-antigen transgenic mouse mammary cancer model has been utilized for chemopreventive research in which mammary tumors develop over a well-characterized time course. Several compounds have been shown to inhibit mammary tumor development in this model, including retinoids, di-fluoromethylornithine (DFMO), dehydroepiandrosterone (DHEA), antiangiogenic compounds and nonsteroidal antiinflammatory drugs (NSAID). All of the chemopreventive agents used in the C3(1)Tag mammary mouse model appear to affect the promotion stage of tumorigenesis, suggesting that these agents may be useful in inhibiting the transition of human ductal carcinoma in situ (DCIS) to invasive carcinoma. Selective combinations of chemopreventive agents may be particularly useful for targeting multiple signaling pathways involved in cancer development and progression leading to improved clinical responses. The application of gene expression profiling to chemopreventive studies will aid in the selection of appropriate models for preclinical testing and further define mechanisms of action.

KEY WORDS: • genetically engineered mice • chemoprevention • breast cancer

Breast cancer is the second leading cause of cancer death in women in the U.S., and an estimated 192,200 new cases will be diagnosed this year ( 1). Because the development of the majority of breast cancer is primarily influenced by nongenetic factors, environmental conditions clearly play a major role in cancer development ( 2). It has been estimated that one-third of all cancers, including breast cancer, may be preventable by modifiable lifestyle factors such as nutrition, exercise and smoking ( 1).

Chemopreventive agents are given to prevent or reduce the incidence of cancer, although it is not always clear at what stage of oncogenesis these agents work. Many nutritional compounds have been associated with chemopreventive action, including soy, vitamins, minerals and fiber ( 3). Chemoprevention through nutritional and dietary changes may, therefore, offer a means of significantly reducing the incidence and progression of breast cancer. In fact, identifying women at risk for breast cancer and targeting them with proven prevention strategies from animal and human research is a major goal of the National Cancer Institute ( 3).

Directly testing prevention strategies in people at risk for breast cancer is difficult, particularly testing interventions that are focused on diet. The identification of women at higher risk for developing breast cancer is imprecise, although some predictive models such as the Gail model ( 4) have proved to be useful. Human studies are further complicated by the great genetic heterogeneity in the human population. Additionally, clinical trials require large cohorts, are quite expensive and are difficult and imprecise when controlling for actual dietary consumption and recording dietary intake ( 5). Therefore, the use of genetically engineered mouse models of mammary cancer for preclinical testing may offer a means to overcome many of the difficulties encountered in human trials. The use of such animal models provides a tool to perform studies in a controlled experimental environment in which the accurate delivery of chemopreventive agents can be measured.

Genetically altered mouse models have a defined genetic background, thereby eliminating differences in genetic heterogeneity. The responses to particular preventive or nutritional agents can be assessed according to particular oncogenic pathways that have been activated in the genetically engineered mice. Because many genetically altered mice develop tumors after a predictable time course, the stage-specific responses of nutritional and chemopreventive agents can be assessed and potentially translated into stages of human cancer progression.

Validation of animal models

Genetically engineered mouse models of mammary cancer have been important in elucidating molecular signaling pathways associated with the promotion and progression of cancer. Transgenic animal models have been generated to mimic human disease through a variety of mechanisms: overexpression or activation of genes associated with human cancer development (oncogenes), elimination of target (suppressor) genes via gene knock-out and the generation of dominant negative proteins to disrupt the function of regulatory genes. Mammary tumors arising in transgenic models are generally estrogen-receptor negative, which makes them attractive for testing agents against hormonally nonresponsive tumors. The validation of transgenic mammary cancer models as systems to help understand human breast cancer has included several parameters such as the natural history of disease progression, histopathology, gene and/or protein expression patterns, genomic alterations, biologic properties such as hormone-responsiveness and metastasis to other organs. The histopathology of mammary tumors from genetically engineered mice was recently reviewed and compared to that of human tumors. The outcome of this review found both histopathologic similarities between human and murine tumors as well as some unique phenotypic differences ( 6). However, relatively few studies have been reported that attempt to validate such models for their usefulness in the context of preclinical testing of preventive or therapeutic agents.

Targeting pathways involved in oncogenesis

The use of transgenic mammary models allows for the detailed study of stage-specific responses to chemopreventive agents, further defining the appropriate timing for intervention with these compounds. Numerous genes and proteins involved in critical cellular signaling pathways may be particularly attractive targets for the inhibition of cancer progression using potential chemopreventive agents ( Fig. 1). The inhibition of key pathways involved in oncogenesis may prevent or at least delay tumor progression. Many of these targets have been evaluated in chemically induced mammary cancer models, but have not been widely investigated in genetically engineered mice that spontaneously develop tumors through a defined oncogenic alteration.

View larger version (26K): [in this window] [in a new window]   FIGURE 1  Potential molecular targets for chemoprevention in breast cancer. AA, arachidonic acid; COX, cyclooxygenase; CDK, cyclin dependent kinases; DHEA, dehydroepiandrosterone; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ER, estrogen receptor; HDAC, histone deacetylase inhibitors; IGF, insulin growth factor; IGFR, insulin growth factor receptor; MAPK, mitogen-activated protein kinase; ODC, ornithine decarboxylase; PGE2, prostaglandin E2; RA, retinoic acid; RI, receptor 1; RII, receptor 2; RXR, retinoid X receptor; TGF, transforming growth factor.

Selenium is an essential mineral that has been associated with chemopreventive qualities in many cancer models including the breast ( 13). Rats have a significantly lower incidence of chemically induced mammary tumors if they receive selenium supplementation as compared to nonsupplemented animals ( 13). The mechanisms for selenium action are generally unknown, but may involve the inhibition of angiogenesis through decreased vascular endothelial growth factor (VEGF)³ expression ( 14), induction of apoptosis and retardation of cell cycle progression ( 15, 16).

Epidemiological data suggest that diets high in vitamin D may reduce the risk of breast cancer ( 17, 18). Laboratory studies have determined that vitamin D can reduce rates of cellular proliferation by attenuating growth factor signaling, as well as increasing the rate of apoptosis ( 19). Additionally, vitamin D analogs can induce TGF-ß expression in mammary tissue ( 20), which has a negative effect on epithelial growth.

Sodium butyarate (NaB), a dietary micronutrient that is produced by bacteria in the colon through the digestion of starch and fiber, can significantly reduce chemically induced mammary carcinogenesis in rats ( 21). The chemoprotective effects of NaB appear to be mediated through several signaling pathways. It inhibits cell proliferation by blocking the G2-M transition phase of the cell cycle, and increases cellular apoptosis in many mammary cancer cell lines ( 22). NaB also increases levels of insulin growth factor binding protein-3 (IGFBP-3) in mammary tissue, which may lower the effective activity of IGF-1, a growth factor associated with increased breast cancer risk ( 22, 23).

C3(1)SV40 T/t-antigen model of mammary cancer

Our laboratory developed and has extensively characterized the C3(1)SV40 large T/t-antigen (Tag) transgenic mouse model of mammary cancer ( 24). The overexpression of the early region of SV40 in the mammary epithelium induces mammary tumors in mice, at least in part, through the Tag inactivation of the tumor suppressors p53 and Rb ( 25). The expression of this transgene results in the progressive development of mammary lesions leading to invasive carcinoma formation ( 26). At 8 wk of age, C3(1)SV40Tag mice develop mammary epithelial atypia that progresses at 12 wk of age to mammary intraepithelial neoplasia (MIN), which is histologically similar to human ductal carcinoma in situ (DCIS). Invasive carcinomas generally appear after 16 wk of age ( 27, 28). Approximately 15% of the mice develop mammary tumor metastases to the lung in the FVB/N background ( 26), whereas 50% of mice develop metastases in an FVB/N x Sv129 hybrid background ( 29). One notable feature of this model is that the tumors that develop do not express estrogen receptor and are estrogen-independent ( 30). The expression of the C3(1)/Tag transgene does not appear to be stimulated by estrogen or pregnancy (30; Green, J. E., unpublished data).

    Chemoprevention studies in the C3(1)SV40Tag model. Retinoids and retinoid analogs can alter cell growth, differentiation and apoptosis in many types of cells ( 31). A naturally occurring ligand for the retinoid receptors is 9-cis retinoic acid (RA). RA was shown to inhibit mammary tumor progression in the C3(1)Tag transgenic mouse model ( 32) where mice were treated continuously for 6 mo with either 10 mg or 50 mg RA/kg/d starting at 5 wk of age. Tumor incidence was significantly delayed in the RA treated mice, and tumor multiplicity was decreased by 50%. However, 9-cis RA has toxic side effects that limit its usefulness. In an additional study using the retinoid X receptor (RXR)-selective retinoid analog, LGD1069, tumor incidence and multiplicity significantly decreased, but with significantly less toxicity ( 33).

DFMO is an inhibitor of ornithine decarboxylase, an essential enzyme in polyamine synthesis. Seven-week-old female C3(1)Tag mice who were administered 4,000 or 8,000 mg/kg DFMO orally for 12 wk had decreased tumor incidence and tumor multiplicity as compared to controls in a dose-dependent manner ( Fig. 2). DFMO appeared to inhibit tumor progression rather than to inhibit tumor initiation, because there were no differences in the incidence of preinvasive MIN lesions after several weeks of treatment ( 34).

View larger version (23K): [in this window] [in a new window]   FIGURE 2  The effect of DFMO on tumor incidence and multiplicity in the C3(1)SV40 large T-antigen transgenic mouse. C3(1)/Tag mice received either 4,000 or 8,000 mg of DFMO beginning at 7 wk of age. A dose-dependent inhibitory effect on tumor incidence was observed with a maximum reduction of 20% after 120 d. Tumor multiplicity was reduced by 50% in the same group of mice. [Adapted from Green, J. E., Shibata, E., Shibata, M. A., Moon, R., Anver, M., Kelloff, G. & Lubet, R. (2001) DFMO and DHEA inhibit mammary tumor progression but not initiation in C3(1)/SV40 T/t-antigen transgenic mice. Cancer Res. 61: 7449–7455.]

Antiangiogenic compounds inhibit tumor growth by preventing the generation of new blood vessels to support tumor expansion and metastases. Testing these compounds in transgenic mammary models will aid in understanding their efficacy and mechanisms of action, especially in the early premalignant stage where the "angiogenic switch" occurs ( 35). It is thought that for tumors to grow beyond 1 mm in diameter, a relative increase in proangiogenic factors must dominate as compared to antiangiogenic factors.

A study using the antiangiogenic compound, endostatin, in the C3(1)/Tag mammary cancer mouse model resulted in a significant inhibition of tumor formation ( 36, 37). A limited 3-wk course of recombinant murine endostatin administered during the preinvasive stage of tumorigenesis when the angiogenic switch appears to occur ( 37), delayed tumor development, decreased tumor burden and significantly increased animal survival. Endostatin significantly reduced VEGF mRNA levels in the mammary tumors, consistent with inhibition of the angiogenic switch.

Another study using a mutated form of human endostatin, where an alanine residue is substituted for a proline residue at position 125, demonstrated a significant inhibition of tumor formation, decreased tumor multiplicity and burden, and modestly increased survival of the C3(1)Tag animals ( 38). The mutated endostatin did not decrease preinvasive MIN lesions, but did decrease the expression of many proangiogenic factors including VEGF, VEGF receptors, angiopoietin-2, Tie-2, cadherin-5 and platelet-endothelial cell adhesion molecules. These data suggest that endostatin can inhibit angiogenesis in the C3(1)Tag mouse model ( 38). Ongoing studies in our laboratory are evaluating the effects of inhibiting VEGF signaling through the inactivation of the VEGF-RII receptor.

Nonsteroidal antiinflammatory drugs (NSAID) are a class of drugs that inhibit the production of prostaglandins (PG) by blocking the activity of cyclooxygenase enzymes (COX). COX is the rate-limiting enzyme in PG synthesis that catalyzes arachidonic acid to prostaglandin H₂ (PGH₂). There are two isoforms of COX: COX-1 is constitutively expressed and maintains cellular homeostasis, and COX-2 is expressed in inflammatory responses. PGH₂ can be converted to various prostanoids including PGE₂, a known contributor to tumor promotion and apoptosis inhibition ( 39). COX-2 is overexpressed in 56% of human breast tumors, and retrospective epidemiological data suggest a lower risk of breast cancer in women who take NSAIDs ( 40– 42). Our laboratory used celecoxib (a COX-2 specific inhibitor) and indomethacin (an inhibitor of both COX-1 and -2) to inhibit mammary tumorigenesis in the C3(1)Tag mouse model. C3(1)Tag mice overexpress COX-2 in MIN lesions as well as advanced stage tumors (Kavanaugh and Green, unpublished data). Preliminary results suggest that celecoxib significantly delays tumor incidence and multiplicity (Kavanaugh and Green, unpublished data). Although indomethacin had some protective effect, it was not as effective as celecoxib in inhibiting tumorigenesis. Determining the multiple molecular pathways through which NSAID mediate their chemopreventive effects is an active area of research in our laboratory.

All of the chemopreventive agents used in the C3(1)Tag mammary mouse model seem to affect the promotion of tumorigenesis ( Fig. 3) and therefore may be relevant to inhibiting the progression of DCIS to invasive carcinomas in human disease. Identifying chemopreventive agents that will suppress tumorigenesis at an earlier stage of tumorigenesis is actively being evaluated. Although almost all studies to date have focused on the effects of using one agent for chemoprevention, it seems likely that approaches using combinations of chemopreventive agents could target different molecular signaling pathways, thereby mediating synergistic effects to inhibit tumor progression.

View larger version (23K): [in this window] [in a new window]   FIGURE 3  Mammary tumor progression in the C3(1)/Tag transgenic mouse. At 8 wk of age, C3(1)SV40Tag mice develop mammary epithelial atypia, which progresses at 12 wk of age to mammary intraepithelial neoplasia (MIN) that is similar to human ductal carcinoma in situ (DCIS), and to invasive carcinomas generally after 16 wk of age. The chemopreventive compounds used in this model seem to delay the tumor promotion stage of tumorigenesis. DFMO di-fluoromethylornithine; DHEA, dehydroepiandrosterone.

The quest for understanding molecular mechanisms involved in oncogenesis is being rapidly advanced through the use of cDNA microarray technology, a powerful method to explore gene expression patterns of thousands of genes in a single experiment. This technology can provide a wealth of information by: 1) comparing gene expression profiles between different transgenic mammary cancer models; 2) comparing the expression profiles of mouse lesions to those of human breast tumors; and 3) determining how different treatments or therapeutic agents may modulate gene expression in vitro and in vivo, giving insights into the mechanisms of action of these agents.

A recent study in our laboratory evaluated the changes in gene expression of tumors from several different transgenic mammary models including the C3(1)Tag, MMTV-neu, MMTV-c-myc, WAP-Tag, MMTV-ras and MMTV-Polyomavirus middle T-antigen ( 42). Normal mammary gland RNA was used as the reference RNA, and five separate mammary tumors from each model were used on the cDNA arrays. Approximately 8,700 unique genes were evaluated for changes in gene expression. The mammary tumors from all the transgenic models shared many similarities, including the induction of cell cycle regulators, glycolytic pathway, metabolic regulators, zinc finger proteins and protein tyrosine phosphatases ( 43). More than 930 genes had altered expression patterns that varied significantly among the different tumor models. Hierarchical clustering using this subset of genes identified gene expression signatures, which were characteristic of particular oncogenic pathways. The gene expression patterns of various transgenic mammary models may be clustered by the function of the initiating oncogene and that signature gene expression patterns can be identified for each class of model ( 43). The T-antigen models have >100 genes that uniquely cluster together, more than any other of the transgenic mammary cancer models. T-antigen expression affected many pathways, including those involving the cell cycle, apoptosis, DNA replication, RNA metabolism, signal transduction and other genes associated with human cancer. T-antigen uniquely induced the expression of calcium-binding and calcium-regulated genes such as calcyclin, calcium calmodulin-dependent kinase II, annexin A3 and caldesmon and calumenin. These data suggest that calcium signaling may play a role in tumorigenesis in the C3(1)Tag model. Many cellular functions are modulated by calcium signaling such as cell division, differentiation and proliferation ( 44). Because several of the calcium-regulated genes have been shown to be dysregulated in human breast cancer, these data suggest that the Tag models may be useful for studying nutritional or chemopreventive agents that interfere with these signaling pathways.

Although the myc-induced mammary tumors are clustered closely with the T-antigen tumors, a unique set of genes had altered expression patterns in the myc carcinomas. C-myc affected three major signaling pathways: genes involved in the cell cycle pathways, transcription factors and ribosomal RNA genes. The three other transgenic tumor types studied, PyMT, neu and ras, had very similar clustering patterns. Based on these data sets, models may be chosen for preclinical studies based on the dysregulation of particular molecular targets involved in the formation of the tumors.

Ongoing studies in our laboratory are determining the cancer stage-specific changes in gene expression, which occur in the C3(1)Tag model. These data may provide important insights into molecular targets and signaling pathways involved in the transition of preinvasive lesions into invasive carcinomas. Inhibiting the action of such genetic changes could have significant translational impact. Finally, additional microarray studies are underway to identify molecular responses in vitro and in vivo that are induced by various nutritional and chemopreventive agents.

Genetically engineered mice can be utilized to assess the effects of nutritional and chemopreventive agents that target numerous signaling pathways to inhibit tumor progression. Because several transgenic mouse models progress through a well-defined series of stages leading to cancer, they may be particularly useful for determining cancer stage-specific responses to nutritional and chemopreventive agents. Selective combinations of chemopreventive agents could be particularly useful for targeting multiple signaling pathways involved in cancer development and progression leading to improved clinical responses. Finally, gene expression profiling of mouse models may aid in identifying appropriate models for testing particular classes of nutritional or chemopreventive agents and lead to a better understanding of their mechanisms of action.

	FOOTNOTES

 
¹ Published in a supplement to The Journal of Nutrition. Presented at the "Nutritional Genomics and Proteomics in Cancer Prevention Conference" held September 5–6, 2002, in Bethesda, MD. This meeting was sponsored by the Center for Cancer Research, National Cancer Institute; Division of Cancer Prevention, National Cancer Institute; National Center for Complementary and Alternative Medicine, National Institutes of Health; Office of Dietary Supplements, National Institutes of Health; Office of Rare Diseases, National Institutes of Health; and the American Society for Nutritional Sciences. Guest editors for the supplement were Young S. Kim and John A. Milner, Nutritional Science Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD.

³ Abbreviations used: COX, cyclooxygenase enzymes; DCIS, ductal carcinoma in situ; DFMO, di-fluoromethylornithine; DHEA, dehydroepiandrosterone; GEM, genetically engineered mice; MIN, mammary intraepithelial neoplasia; NaB, sodium butyarate; NSAIDs, nonsteroidal antiinflammatory drugs; PG, prostaglandins; PGH₂, prostaglandin H₂; RA, retinoic acid; RXR, retinoid X receptor; VEGF, vascular endothelial growth factor.

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