Rebecca E. Nakles

Signal transducer and activator of transcription 5 as a key signaling pathway in normal mammary gland developmental biology and breast cancer

STAT5 consists of two proteins, STAT5A/B, that impact mammary cell differentiation, proliferation, and survival. In normal development, STAT5 expression and activity are regulated by prolactin signaling with JAK2/ELF5, EGF signaling networks that include c-Src, and growth hormone, insulin growth factor, estrogen, and progesterone signaling pathways. In cancer, erythropoietin signaling can also regulate STAT5. Activation levels are influenced by AKT, caveolin, PIKE-A, Pak1, c-Myb, Brk, beta-integrin, dystroglycan, other STATs, and STAT pathway molecules JAK1, Shp2, and SOCS. TGF-β and PTPN9 can downregulate prolactin- and EGF-mediated STAT5 activation, respectively. IGF, AKT, RANKL, cyclin D1, BCL6, and HSP90A lie downstream of STAT5.

BRCA1 deficient mouse models to study pathogenesis and therapy of triple negative breast cancer

Genetically engineered mice along with allograft and xenograft models can be used to effectively model triple negative breast cancer both for studies of pathophysiology as well as preclinical prevention and therapeutic drug studies. In this review eight distinct genetically engineered mouse models of BRCA1 deficiency are discussed in relationship to the generation of triple negative mammary cancer. Allograft models derived from some of these genetically engineered mice are considered and xenograft models derived from breast cancers that developed from BRCA1 mutation are presented. Examples of the use of genetically engineered, allograft and xenografts models for preventive and therapeutic studies are presented.

Human Pancreatic Cancer-Associated Stellate Cells Remain Activated after in vivo Chemoradiation

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive fibrotic reaction or desmoplasia and complex involvement of the surrounding tumor microenvironment. Pancreatic stellate cells are a key mediator of the pancreatic matrix and they promote progression and invasion of pancreatic cancer by increasing cell proliferation and offering protection against therapeutic interventions. Our study utilizes human tumor-derived pancreatic stellate cells (HTPSCs) isolated from fine needle aspirates of pancreatic cancer tissue from patients with locally advanced, unresectable pancreatic adenocarcinoma before and after treatment with full-dose gemcitabine plus concurrent hypo-fractionated stereotactic radiosurgery. We show that HTPSCs survive in vivo chemotherapy and radiotherapy treatment and display a more activated phenotype post-therapy. These data support the idea that stellate cells play an essential role in supporting and promoting pancreatic cancer and further research is needed to develop novel treatments targeting the pancreatic tumor microenvironment.

Assessing estrogen signaling aberrations in breast cancer risk using genetically engineered mouse models.

Aberrations in estrogen signaling increase breast cancer risk. Molecular mechanisms that impact breast cancer initiation, promotion, and progression can be investigated using genetically engineered mouse models. Increasing estrogen receptor alpha (ERα) expression levels twofold is sufficient to initiate and promote breast cancer progression. Initiation and promotion can be increased by p53 haploinsufficiency and by coexpressing the nuclear coactivators amplified in breast cancer 1 (AIB1) or the splice variant AIB1Δ3. Progression to invasive cancer is found with coexpression of these nuclear coactivators as well as following a single dose of 7,12‐dimethylbenz(a)anthracene. Loss of signal transducer and activator of transcription 5a reduces the prevalence of initiation and promotion but does not protect from invasive cancer development. Cyclin D1 loss completely interrupts mammary epithelial proliferation and survival when ERα is overexpressed. Loss of breast cancer gene 1 increases estrogen signaling and cooperates with ERα overexpression in initiation, promotion, and progression of mammary cancer.

Time-lapse imaging of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer.

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 μm x 700 μm fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Association of Over-Expressed Estrogen Receptor Alpha with Development of Tamoxifen Resistant Hyperplasia and Adenocarcinomas in Genetically Engineered Mice.

Background Estrogen receptor alpha (ERα) and cyclin D1 are frequently co-expressed in human breast cancer. Some, but not all, studies link tamoxifen resistance to co-expression of cyclin D1 and ERα. In mice over-expression of either cyclin D1 or ERα in mammary epithelial cells is sufficient to induce mammary hyperplasia. Cyclin D1 over-expression in mice leads to mammary adenocarcinoma associated with activated estrogen signaling pathways. ERα over-expression in mice leads to mammary hyperplasia and cancer. Significantly, disease development in these mice is abrogated by loss of cyclin D1. Methods Genetically engineered mouse models were used to determine whether or not ERα over-expression demonstrated cooperativity with cyclin D1 over-expression in cancer development, reaction to the chemical carcinogen DMBA, or tamoxifen response. Results Adding ERα over-expression to cyclin D1 over-expression increased the prevalence of hyperplasia but not cancer. Single dose DMBA exposure did not increase cancer prevalence in any of the genotypes although cyclin D1 over-expressing mice demonstrated a significant increase in hyperplasia. Tamoxifen treatment was initiated at both young and older ages to test for genotype-specific differences in response. Although normal ductal structures regressed in all genotypes at both younger and older ages, tamoxifen did not significantly reduce the prevalence of either hyperplasia or cancer in any of the genotypes. All of the cancers that developed were hormone receptor positive, including those that developed on tamoxifen, and all showed expression of nuclear-localized cyclin D1. In summary, development of tamoxifen resistant hyperplasia and cancer was associated with expression of ERα and cyclin D1. Conclusion These preclinical models will be useful to test strategies for overcoming tamoxifen resistance, perhaps by simultaneously targeting cell cycle regulatory pathways associated with cyclin D1.

Characterization of primary mammary epithelial cells with loss of BRCA1 at a single cell level

Background Loss of BRCA1 has been linked to increased cell proliferation in human mammary epithelial cells. To determine if this phenotype is mirrored in the normalappearing mammary epithelial cells from mouse models of BRCA1 deficiency, time-lapse imaging was performed on primary mammary epithelial cell (PMEC) cultures. Three distinct genetic models were tested to evaluate the role of p53 haploinsufficiency and p53 haploinsufficiency in the background of ERa over-expression to altered cell proliferation.

Abstract A26: Aromatase over‐expression results in the development of ductal growth abnormalities in the mammary gland of a conditional transgenic mouse model

Estrogens are required for the development of the normal mammary gland; however, studies suggest that estrogen and its metabolites may have mutagenic and carcinogenic potential in the mammary gland. Aromatase is key to estrogen biosynthesis, but its over‐expression may play a role in the development of breast cancer by increasing mitotic activity in breast epithelial cells. Therefore, it is highly valuable to have mouse models with increased local estrogen production to use to develop optimal therapeutic and chemopreventive strategies to treat these lesions. The purpose of this study was to evaluate if increased local estrogen production led to the development of mammary gland preneoplasia. To test our hypothesis we developed a conditional transgenic mouse model to control the spatial and temporal aromatase expression in the mammary gland. Mammary glands were collected at 4 and 12 months of age for morphological, histological, gene and protein expression studies in both MMTV‐rtTA/tet‐op‐Aromatase and wild‐type mice. RT‐PCR analysis of aromatase mRNA confirmed that the gene was conditionally expressed in the mammary gland as compared to control mice. Aromatase activity assays confirmed a significantly higher enzyme activity in the mammary glands of MMTV‐rtTA/tet‐op‐Aromatase compared to wild‐type. Mammary glands were evaluated for morphological changes using confocal microscopy, whole‐mount and hematoxylin and eosin staining. Aromatase over‐expression resulted in a higher incidence of morphological and histological abnormalities such as hyperplastic alveolar nodules, ductal dysplasia, ductal carcinoma in situ, and the unexpected persistence of terminal end buds at 12 months of age. These changes were associated with ERK1/2 and AKT activation. In summary, we generated a novel transgenic mouse system to investigate the role of aromatase over‐expression in mammary epithelial cells during mammary gland development. Increased aromatase activity resulted in the development of ductal growth abnormalities and increased ductal density. This model represents a valuable tool to investigate the effect of estrogen over‐production in the mammary gland microenvironment at specific times during development. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A26.