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.

Promotion of mammary cancer development by tamoxifen in a mouse model of Brca1-mutation-related breast cancer.

Loss of full-length Brca1 in mammary epithelial cells of the mouse mammary tumor virus (MMTV)-Cre Brca1 conditional exon 11 deletion mouse model results in the development of mammary adenocarcinomas with similar genetic changes to those found in human BRCA1-mutation-related breast cancers. We used this experimental model to evaluate the chemopreventive effect of tamoxifen on the development of mammary preneoplasia and adenocarcinoma. No protective effects of tamoxifen administration on mammary cancer development were found. Instead, tamoxifen treatment significantly increased rates of mammary epithelial cell proliferation and the prevalence of mammary hyperplasia at 6 months of age. Tamoxifen-exposed mice developed adenocarcinomas at younger ages than control mice and a higher percentage of mice developed adenocarcinomas by 12 months of age. Both whole mouse and tissue culture cell models were used to test if loss of full-length Brca1 was associated with a relative increase in the agonist activity of tamoxifen. Tamoxifen induced increased ductal growth in MMTV-Cre Brca1 conditional mice compared to wild type. Estrogen receptor alpha (ERalpha) expression was downregulated in the tamoxifen-induced hyperplasias. Reducing BRCA1 levels in MCF-7 cells using siRNA resulted in a relative increase in the agonist activity of tamoxifen. Results suggest a model of mammary cancer progression in which loss of full-length Brca1 altered the agonist/antagonist activity of tamoxifen, resulting in tamoxifen-induced mammary epithelial cell proliferation with subsequent loss of ERalpha expression and development of ERalpha-negative hyperplasias and adenocarcinomas.

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.

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.