The nuclear receptor coactivator amplified in breast cancer-1 is required for Neu (ErbB2/HER2) activation, signaling, and mammary tumorigenesis in mice.

Overexpression of the oncogene amplified in breast cancer 1 (AIB1)/steroid receptor coactivator-3 (SRC-3) induces mammary tumorigenesis in mice. In breast cancer, high levels of AIB1/SRC-3 and the growth factor receptor HER2/neu predict resistance to endocrine therapy and poor outcome. However, a mechanistic relationship between AIB1/SRC-3 and HER2/neu in the development of breast cancer has not been shown. Here, we show that deletion of one allele of SRC-3 significantly delays Neu-induced mammary tumor development in mice. Homozygous deletion of SRC-3 in mice completely prevents Neu-induced tumor formation. By ages 3 to 4 months, Neu/SRC-3(+/-) mice exhibit a noticeable reduction in lateral side-bud formation, accompanied by reduced cellular levels of phosphorylated Neu compared with Neu/SRC-3(wt) mice. In Neu-induced tumors, high levels of SRC-3, phosphorylated Neu, cyclin D1, cyclin E, and proliferating cell nuclear antigen expression are observed, accompanied by activation of the AKT and c-Jun NH(2) kinase (JNK) signaling pathways. In comparison, phosphorylated Neu, cyclin D1, and cyclin E are significantly decreased in Neu/SRC-3(+/-) tumors, proliferation is reduced, and AKT and JNK activation is barely detectable. Our data indicate that AIB1/SRC-3 is required for HER2/neu oncogenic activity and for the phosphorylation and activation of the HER2/neu receptor. We predict that reducing AIB1/SRC-3 levels or activity in the mammary epithelium could potentiate therapies aimed at inhibiting HER2/neu signaling in breast cancer.

Comparison of mouse mammary gland imaging techniques and applications: reflectance confocal microscopy, GFP imaging, and ultrasound.

BACKGROUND: Genetically engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue. METHODS: We evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging. RESULTS: In the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice. CONCLUSION: In conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary.

Real-time imaging and characterization of human breast tissue by reflectance confocal microscopy.

Real-time technologies can increase the efficiency of obtaining informative biopsies and accelerate interpretation of biopsy pathological review. Cellular aberrations inherent to cancer cells, including nuclear size, can currently be detected, but few technologies are available to evaluate adequacy of specimens in real time. The aims of this study are: 1. to determine if near-infrared reflectance confocal microscopy (RCM) can be used to assess epithelial/stromal content of core needle breast biopsy samples in real time, 2. to determine if epithelial cell nuclear size can be measured on RCM images, and 3. to test if RCM images can be accurately read for presence/absence of histologically relevant features of malignancy. Breast biopsies are obtained following a medically indicated breast core needle diagnostic biopsy for RCM examination. Acetic acid is used as a contrast agent to visualize structures within breast tissue. Structures are identified and optically serially sectioned, and digital images are cataloged. Relative amounts of epithelial, fatty, and collagenous tissue are determined. RCM biopsies are formalin-fixed and stained for hematoxylin and eosin (H and E) comparison with RCM images. RCM data are comparable to data from H and E sections. Epithelial cell nuclear size is measured on stored digital RCM images. We compare RCM and H and E images from 16 patients and 25 core needle biopsy samples.

Reflectance confocal microscopy for characterization of mammary ductal structures and development of neoplasia in genetically engineered mouse models of breast cancer.

The earliest steps of breast cancer begin with aberrations in mammary ductal structure. Techniques that enable an investigator to image in situ and then analyze the same tissue using biochemical tools facilitates identification of genetic networks and signaling pathways active in the imaged structure. Cellular confocal microscopy (VivaCell-TiBa, Rochester, New York) is used to image mammary ductal structures and surrounding vasculature in situ in intact wild-type and genetically engineered mice that develop ER alpha-initiated ductal carcinoma in situ (DCIS) and ER alpha-driven invasive mammary cancer. In wild-type mice, normal mammary ductal structures that appear from puberty through lactation are visualized and serially sectioned optically, and a developmental atlas is created. Altering tissue preparation enabled visualization of the vasculature surrounding the ductal structures. In the genetically engineered mice, aberrant mammary ductal structures and cancers are imaged and compared to corresponding normal structures. Different preparation techniques are able to preserve tissue for routine histological analyses and RNA isolation. Comparative studies demonstrate that reflectance confocal imaging provides more cellular detail than carmine-alum-stained mammary gland whole mounts and equivalent detail with hematoxylin and eosin stained tissue sections. In summary, reflectance confocal microscopy is a tool that can be used to rapidly and accurately analyze mammary gland structure.

Deregulated estrogen receptor alpha expression in mammary epithelial cells of transgenic mice results in the development of ductal carcinoma in situ.

A conditional tetracycline-responsive transgenic mouse model with deregulated estrogen receptor alpha expression in mammary epithelial cells developed ductal hyperplasia (DH), lobular hyperplasia, and ductal carcinoma in situ (DCIS) by 4 months of age. Higher proliferative rates were found in both normal and abnormal ductal and lobular structures. DH and DCIS but not normal ductal structures showed an increased percentage of cells with nuclear-localized cyclin D1. No differences in either the prevalence or extent of these phenotypes following exogenous 17beta-estradiol treatment were found suggesting that alteration of ERalpha expression was the rate-limiting factor in initiation of DH, lobular hyperplasia, and DCIS.

Overexpression of an N-terminally truncated isoform of the nuclear receptor coactivator amplified in breast cancer 1 leads to altered proliferation of mammary epithelial cells in transgenic mice.

Amplified in breast cancer 1 (AIB1, also known as ACTR, SRC-3, RAC-3, TRAM-1, p/CIP) is a member of the p160 nuclear receptor coactivator family involved in transcriptional regulation of genes activated through steroid receptors, such as estrogen receptor alpha (ER(alpha)). The AIB1 gene and a more active N-terminally deleted isoform (AIB1-Delta3) are overexpressed in breast cancer. To determine the role of AIB1-Delta3 in breast cancer pathogenesis, we generated transgenic mice with human cytomegalovirus immediate early gene 1 (hCMVIE1) promoter-driven over-expression of human AIB1/ACTR-Delta3 (CMVAIB1/ACTR-Delta3 mice). AIB1/ACTR-Delta3 transgene mRNA expression was confirmed in CMV-AIB1/ACTR-Delta3 mammary glands by in situ hybridization. These mice demonstrated significantly increased mammary epithelial cell proliferation (P < 0.003), cyclin D1 expression (P = 0.002), IGF-I receptor protein expression (P = 0.026), mammary gland mass (P < 0.05), and altered expression of CCAAT/enhancer binding protein isoforms (P = 0.029). At 13 months of age, mammary ductal ectasia was found in CMV-AIB1/ACTR-Delta3 mice, but secondary and tertiary branching patterns were normal. There were no changes in the expression patterns of either ER(alpha) or Stat5a, a downstream mediator of prolactin signaling. Serum IGF-I levels were not altered in the transgenic mice. These data indicate that overexpression of the AIB1/ACTR-Delta3 isoform resulted in altered mammary epithelial cell growth. The observed changes in cell proliferation and gene expression are consistent with alterations in growth factor signaling that are thought to contribute to either initiation or progression of breast cancer. These results are consistent with the hypothesis that the N-terminally deleted isoform of AIB1 can play a role in breast cancer development and/or progression.

Explant-cell culture of primary mammary tumors from MMTV-c-Myc transgenic mice.

We have established an explant-cell culture system for mammary gland tumors from c-myc oncogene-expressing transgenic mice and potentially other transgenic strains. By coating culture dish surfaces with fetal bovine serum and using culture media supplemented with low serum and growth factors, the mammary tumor specimens could be maintained in culture for over 3 mo. Throughout the culture period, the explants produced abundant outgrowths of epithelial cells. As the outgrowths of epithelial cells filled the dishes, the explants were serially transferred from one dish to another-a process that could be repeated at least six times, thus providing a continuous supply of primary tumor cells. This culture system provides a useful tool for studying the biology of mouse mammary gland tumors and possibly tumors from other organ sites.

Loss of protein phosphatase 2A expression correlates with phosphorylation of DP-1 and reversal of dysplasia through differentiation in a conditional mouse model of cancer progression.

A conditional mouse model of time-dependent dysplasia reversal demonstrated that reversal and differentiation of dysplastic salivary gland tissue at the 4-month reversible stage was characterized by the appearance of a phosphorylated slower mobility form of Differentiation Related Transcription Factor 1-polypeptide-1 that was correlated with cellular differentiation. The phosphorylated form of DP-1 was not found at the 7-month irreversible stage or in adenocarcinomas. At the 4-month reversible stage, protein phosphatase 2A expression was down-regulated coincident with loss of oncogene expression, whereas PP2A expression persisted at the 7-month irreversible stage. Results are consistent with the hypothesis that persistent PP2A expression prevented the appearance of the phosphorylated form of DP-1 required for cellular differentiation and reversal of dysplasia after loss of oncogene expression.

Introduction of estrogen receptor-alpha into the tTA/TAg conditional mouse model precipitates the development of estrogen-responsive mammary adenocarcinoma.

Conditional expression of estrogen receptor (ER)-alpha) was introduced into tetracycline-responsive MMTV-tTA/tetop-TAg mice to develop a mouse model of estrogen-responsive ER-alpha-positive mammary adenocarcinoma. Mammary adenocarcinomas developed in the mice with a mean latency of 11 months. Precursor lesions including ductal hyperplasia and hyperplastic alveolar nodules were present by the age of 4 months. The mammary adenocarcinomas exhibited histological features similar to human breast cancers. ER steroid-binding studies conducted on adenocarcinoma lysates demonstrated binding to estradiol. Tumor explant studies in the presence and absence of estradiol in ovariectomized athymic nude mice revealed that growth of mammary tumors was stimulated by estrogen. In addition, the presence of ER-alpha altered the tumor spectrum in other MMTV-targeted tissues in the tTA/TAg female mice. Lymphomas, which develop in 40% of tTA/TAg female mice, were found in only 4% of tTA/TAg/ER-alpha mice (P = 0.014, chi-square test). These experiments demonstrate that the introduction of an ER-alpha transgene targeted to mammary epithelial cells can be used to develop mouse models of ER-alpha-responsive mammary cancer.

Conditional mouse models demonstrate oncogene-dependent differences in tumor maintenance and recurrence.

Diversity in the pathophysiology of breast cancer frustrates therapeutic progress. We need to understand how mechanisms activated by specific combinations of oncogenes, tumor suppressors, and hormonal signaling pathways govern response to therapy and prognosis. A recent series of investigations conducted by Chodosh and colleagues offers new insights into the similarities and differences between specific oncogenic pathways. Expression of three oncogenes relevant to pathways activated in human breast cancers (c-myc, activated neu and Wnt1) were targeted to murine mammary epithelial cells using the same transgenic tetracycline-responsive conditional gene expression system. While the individual transgenic lines demonstrate similarly high rates of tumor penetrance, rates of oncogene-independent tumor maintenance and recurrence following initial regression are significantly different, and are modifiable by mutations in specific cooperating oncogenes or loss of tumor suppressor gene expression. The experiments make three notable contributions. First, they illustrate that rates of tumor regression and recurrence following initial regression are dependent upon the pathways activated by the initiating oncogene. The experiments also demonstrate that altered expression or mutation of specific cooperating oncogenes or tumor suppressor genes results in different rates of tumor regression and recurrence. Finally, they exemplify the power of conditional mouse models for elucidating how specific molecular mechanisms give rise to the complexity of human cancer.

Chemoprevention of mammary carcinogenesis in a transgenic mouse model by alpha-difluoromethylornithine (DFMO) in the diet is associated with decreased cyclin D1 activity.

Mechanisms underlying the chemopreventive effect of difluoromethylornithine (DFMO) on the development of mammary cancer were investigated utilizing the whey acidic protein promoter-T antigen transgenic mouse model of breast cancer progression. Mice were exposed to four different doses of DFMO in the diet (3.5, 4.9, 7.0 and 10 g/kg diet). Tumor latency was increased in a dose-dependent manner. DFMO at the highest dose significantly delayed tumor onset (131 days as compared to 109 days in control unexposed mice, P=0.018). Analyses of preneoplastic mammary tissue collected 1 month after DFMO treatment demonstrated that DFMO (10 g/kg diet) significantly increased the ratio of apoptotic to proliferative indices (P=0.013) and significantly reduced the percentage of cells demonstrating nuclear localized cyclin D1 (P=0.013). Nuclear localizations of p27, p21 and Stat5a were not affected. Inhibitory effects of DFMO on cell growth and survival were lost as the cells progressed to cancer. In conclusion, the chemopreventive effects of DFMO on mammary cancer progression were mediated by changes in both apoptosis and cell proliferation in preneoplastic cells. Alterations in cyclin D1 activity in preneoplastic cells could represent an early biomarker of chemopreventive action and are consistent with a mechanistic role for cyclin D1 in progression of mammary cancer.

Conditional over-expression of estrogen receptor alpha in a transgenic mouse model.

Attempts to delineate the mechanisms of estrogen action have promoted the creation of several estrogen receptor alpha (ERalpha) mouse models in the past decade. These traditional models are limited by the fact that the receptors are either absent or present throughout all stages of development. The purpose of this work was to develop a conditional transgenic model that would provide an in vivo method of controlling the spatial and temporal regulation of ERalpha expression. The tetracycline responsive system was utilized. Three lines of transgenic mice carrying a transgene composed of the coding sequence for murine ERalpha placed under the regulatory control of a tet operator promoter (tet-op) were generated. These three lines of tet-op-mERa mice were each mated to an established line of transgenic mice expressing a tetracycline-dependent transactivator protein (tTA) from the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Double transgenic MMTV-tTA/tet-op-mERalpha mice were produced. All three lines demonstrated dominant gain of ERalpha shown by RT-PCR, immunoprecipitation, and immunohistochemistry. Transgene-specific ERalpha was expressed in numerous tissues including the mammary gland, salivary gland, testis, seminal vesicle, and epididymis. Expression was silenced by administration of doxycycline in the drinking water. This model can be utilized to evaluate the consequences of ERalpha dominant gain in targeted tissues at specific times during development. In this study dominant gain of ERalpha was associated with a reduction in epididymal/vas deferens and seminal vesicle weights consistent with the proposed action of ERalpha on fluid transport in the male reproductive tract. Combining this model with other dominant gain and gene knockout mouse models will be useful for testing effects of ERalpha action in combination with specific gene products and to evaluate if developmental and stage-specific expression of ERalpha can rescue identified phenotypes in gene knockout mice.