Transgenic mouse models of hormonal mammary carcinogenesis: advantages and limitations

Mouse models of breast cancer, especially transgenic and knockout mice, have been established as valuable tools in shedding light on factors involved in preneoplastic changes, tumor development and malignant progression. The majority of mouse transgenic models develop estrogen receptor (ER) negative tumors. This is seen as a drawback because the majority of human breast cancers present an ER positive phenotype. On the other hand, several transgenic mouse models have been developed that produce ER positive mammary tumors. These include mice over-expressing aromatase, ERα, PELP-1 and AIB-1. In this review, we will discuss the value of these models as physiologically relevant in vivo systems to understand breast cancer as well as some of the pitfalls involving these models. In all, we argue that the use of transgenic models has improved our understanding of the molecular aspects and biology of breast cancer.     

The effects of aromatase overexpression on mammary growth and gene expression in the aromatase x transforming growth factor alpha double transgenic mice

The transforming growth factor alpha (TGF) and its receptor (EGFR) are expressed in many breast cancers. Typically, the progression of estrogen dependent primary breast cancers into a hormone-independent state, due to the loss of the estrogen receptor, is associated with increased levels of TGF and EGFR, leading to aggressive breast carcinomas. The relationship between breast tumorigenesis and TGF is evident in the transgenic mice overexpressing TGF in the mammary glands. In the aromatase transgenic mice, the mammary glands exhibit preneoplastic developments but do not form frank tumors. To test the interactions between growth factor overexpression with tissue estrogen, we have crossed the aromatase transgenic mice with the TGF transgenic mice to produce a double transgenic strain. The histological data for the mammary glands of aromatase x TGF double transgenic mice show that these mice develop hyperplastic changes similar to the aromatase parental strain but no tumors are formed. Consistently, the expression of cyclin D1 and PCNA is diminished in the double transgenic strain as compared to the parental strains. In addition, the expression of TGF, EGF and EGFR are also decreased in the double transgenic strain, suggesting that continuous estrogen presence in the tissue due to aromatase overexpression downregulates the expression of EGFR and its ligands.     

Use of letrozole as a chemopreventive agent in aromatase overexpressing transgenic mice

Our recent studies have shown that overexpression of aromatase results in increased tissue estrogenic activity and induction of hyperplastic and dysplastic lesions in mammary glands, and gynecomastia and testicular cancer in male aromatase transgenic mice. Our studies also have shown that aromatase overexpression-induced changes in mammary glands can be abrogated with very low concentrations of letrozole, an aromatase inhibitor without any effect on normal physiology. In the present study, we have examined the effect of prior low dose letrozole treatment on pregnancy and lactation. We have also investigated the effect of low dose letrozole treatment on subsequent mammary growth and biochemical changes in these animals. There was no change in the litter size, birth weight and no visible birth defects in letrozole-treated animals. Although, there was an insignificant increase in mammary growth in aged animals after 6 weeks of letrozole treatment, the levels of expression of estrogen receptor, progesterone receptor and genes involved in cell cycle and cell proliferation remained low compared to control untreated animals. These observations indicate that aromatase inhibitors such as letrozole can be used as chemopreventive agents without effecting normal physiology in aromatase transgenic mice.     

Characterization of six type A strains of Clostridium botulinum that contain type B toxin gene sequences

Six Clostridium botulinum isolates exhibiting type A toxicity as measured by the mouse bioassay were found to contain both type A and type B neurotoxin DNA sequences. The six strains were divided into three groups based on the DNA sequence of the type B neurotoxin gene. Members of each group exhibited 100% sequence identity over the 3876 bp type B toxin open reading frame. The type B toxin sequence of all groups differed at more than 60 positions when compared to the BGB control strain.     

Estrogen receptor alpha is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice

Aromatase transgenic mice exhibit hyperplastic and dysplastic changes, attesting to the importance of local estrogen in breast carcinogenesis. These mice also show increased levels of the estrogen receptor and β (ER, ERβ) suggesting that this receptor may play an important role in the initiation of estrogen-mediated mammary hyperplasia observed in these mice. To address the specific role of ER in the mammary development and in the induction of estrogen-mediated hyperplasia in aromatase transgenic mice, we have generated MMTV-aromatase × ER knockout cross (referred as aromatase/ERKO). Even though ERβ is expressed in aromatase/ERKO mice, lack of ER leads to impaired mammary growth in these mice. The data suggest that ER plays an important role in the mammary gland development as well as in the induction of mammary hyperplasia in aromatase transgenic mice. Lack of ER expression in the aromatase/ERKO mice resulted in a decrease in the expression of Cyclin D1, PCNA and TGFβ relative to the aromatase parental strain. The studies involving aromatase/ERKO mice show that lack of ER results in impaired mammary development even in the presence of continuous tissue estrogen, suggesting estrogen/ER-mediated actions are critical for mammary development and carcinogenesis.     

HER-2/neu x aromatase double transgenic mice model: the effects of aromatase overexpression on mammary tumorigenesis

A majority of breast cancers are hormone-responsive, and require estrogen for growth, and respond to hormonal therapy that blocks estrogen receptor action. Breast tumors with low levels of or completely lacking estrogen receptor fail to respond to antiestrogen therapy yet require estrogen for tumor initiation. To address the importance of local estrogen in oncogene-mediated breast tumorigenesis, we have crossed MMTV-aromatase with MMTV-HER2/neu and examined the incidence of breast cancer in double transgenic mice in comparison with parental strains. Double transgenic mice show normal mammary development and express both transgenes at similar levels to that of parental strains. Tumor incidence in double transgenic mice (<5%) decreased compared to HER2/neu mice (>65%). In addition to a significant decrease in tumorigenesis, these mice expressed ER as well as high levels of ERβ along with decreased levels of cyclin D1 and phosphorylated pRb among other changes. Furthermore, experiments using THC (ER-agonist and ERβ-antagonist) clearly demonstrate the critical role of ERβ in HER2/neu-mediated tumorigenesis. These studies provide the first genetic evidence that estrogen receptor, mainly ERβ than ER and its dependent changes play an important role in regulating mammary tumorigenesis. These findings provide further evidence for development and testing of novel therapeutic approaches based on selective regulation of estrogen receptors (ER and β)-dependent actions for the treatment and prevention of breast cancers.     

Modulation of in situ estrogen synthesis by proline-, glutamic acid-, and leucine-rich protein-1: potential estrogen receptor autocrine signaling loop in breast cancer cells

In situ estrogen synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms especially in postmenopausal women. Several recent studies demonstrated activity of aromatase, an enzyme that plays a critical role in estrogen synthesis in breast tumors. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1/MNAR) is an estrogen receptor (ER) coregulator, and its expression is deregulated in breast tumors. In this study, we examined whether PELP1 promotes tumor growth by promoting local estrogen synthesis using breast cancer cells (MCF7) that stably overexpress PELP1. Immunohistochemistry revealed increased aromatase expression in MCF7-PELP1-induced xenograft tumors. Real-time PCR analysis showed enhanced activation of the aromatase promoter in MCF7-PELP1 clones compared with MCF7 cells. Using a tritiated-water release assay, we demonstrated that MCF7-PELP1 clones exhibit increased aromatase activity compared with control MCF-7 cells. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II, and growth factor signaling enhanced PELP1 activation of aromatase. PELP1-mediated induction of aromatase requires functional Src and phosphatidylinositol-3-kinase pathways. Mechanistic studies revealed that PELP1 interactions with ER-related receptor-alpha and proline-rich nuclear receptor coregulatory protein 2 lead to activation of aromatase. Immunohistochemistry analysis of breast tumor array showed increased expression of aromatase in ductal carcinoma in situ and node-positive tumors compared with no or weak expression in normal breast tissue. Fifty-four percent (n = 79) of PELP1-overexpressing tumors also overexpressed aromatase compared with 36% (n = 47) in PELP1 low-expressing tumors. Our results suggest that PELP1 regulation of aromatase represents a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.     

The multifaceted role of aspartate-family amino acids in plant metabolism

Plants represent the major sources of human foods and livestock feeds, worldwide. However, the limited content of the essential amino acid lysine in cereal grains represents a major nutritional problem for human and for livestock feeding in developed countries. Optimizing the level of lysine in cereal grains requires extensive knowledge on the biological processes regulating the homeostasis of this essential amino acid as well as the biological consequences of this homeostasis. Manipulating biosynthetic and catabolic enzymes of lysine metabolism enabled an enhanced accumulation of this essential amino acid in seeds. However, this approach had a major effect on the levels of various metabolites of the tricarboxylic acid (TCA) cycle, revealing a strong interaction between lysine metabolism and cellular energy metabolism. Recent studies discussed here have shed new light on the metabolic processes responsible for the catabolism of lysine, as well as isoleucine, another amino acid of the aspartate-family pathway, into the TCA cycle. Here we discuss progress being made to understand biological processes associated with the catabolism of amino acids of the aspartate-family pathway and its importance for optimal improvement of the nutritional quality of plants.     

Aromatase overexpression transgenic mice model: cell type specific expression and use of letrozole to abrogate mammary hyperplasia without affecting normal physiology

Our recent studies have shown that overexpression of aromatase results in increased tissue estrogenic activity and induction of hyperplastic and dysplastic lesions in female mammary glands and gynecomastia and testicular cancer in male aromatase transgenic mice. Both aromatase mRNA and protein are overexpressed in transgenic mammary glands and its expression is not limited to epithelial cells. However, it is more in epithelial than in stromal cells. Our results also indicate aromatase overexpression-induced changes in mammary glands can be abrogated with very low concentrations of the aromatase inhibitor, letrozole. Low concentration of letrozole had no effect on normal physiology as indicated by no significant change in the circulating levels of estradiol and follicle stimulating hormone as well as no change in estrogen responsive genes such as the progesterone receptor and lactoferrin in the uterine tissue. These observations indicate that the expression of aromatase in both epithelial and stromal cells can influence the complex interactions of biochemical pathways leading to mammary carcinogenesis and that the aromatase inhibitor, letrozole can be used as chemopreventive agents without affecting normal physiology.     

Overexpression of aromatase in transgenic male mice results in the induction of gynecomastia and other biochemical changes in mammary glands

Our previous studies have shown that overexpression of aromatase in mammary glands results in the induction of hyperplastic and dysplastic changes in female transgenic mice. In this study we show that overexpression of aromatase in male transgenic mice results in increased mammary growth and histopathological changes similar to gynecomastia. Increased estrogenic activity also results in an increase in estrogen and progesterone receptor expression in the mammary glands of transgenic males as compared to the nontransgenic males, as well as an increase in the expression of various genes involved in cell cycle and cell proliferation. We have also observed an increase in certain growth factors, such as bFGF and TGFbeta, as a result of aromatase overexpression in the male transgenic mammary glands. In order to obtain a better understanding of the biological significance of gynecomastia, a reliable model is necessary to explain the mechanisms and correlations associated with human cancers. This model, can potentially serve as a predictable and useful tool for studying gynecomastia, hormonal carcinogenesis and action of other carcinogens on hormone induced cancers.