Amy Greene

Molecular characterizations of Nop16 in murine mammary tumors with varying levels of c-Myc

NOP16, also known as HSPC111, has been identified as a MYC and estrogen regulated gene in in vitro studies, hence coexpression levels were strongly correlated. Importantly, high expression of NOP16 was associated with poor clinical outcome in breast cancer patients. However, coexpression of NOP16, MYC and estrogen receptor (ESR1) varied widely in tumors and cell lines suggesting that transcriptional regulation differed according to pathological environments. The goal of this study was to determine the expression patterns of Nop16, Myc and Esr1 in murine mammary tumors with disparate histopathological and molecular features. We hypothesized that tumor environments with relatively high Myc levels would have different coexpression patterns than tumor environments with relatively low Myc levels. We measured levels of Myc and Nop16 mRNA and protein in tumors from WAP-c-myc mice that were of high grade and metastasized frequently. In contrast, Myc and Nop16 mRNA and proteins levels were significantly lower in the less aggressive tumors that developed in NRL-TGFα mice. Tumors from both mouse lines express ESR1 protein and we found that Esr1 mRNA levels correlated positively with Myc levels in both models. However, Myc and Nop16 transcript levels correlated positively only in tumors from NRL-TGFα mice. We identified prominent NOP16 protein in nuclei and less prominent staining in the cytoplasm of luminal cells of ducts and lobules from normal mammary glands as well as in hyperplasias and tumors obtained from NRL-TGFα mice. This staining pattern was reversed in tumors from WAP-c-Myc mice as nuclear staining was faint or absent and cytoplasmic staining more pronounced. In summary, the regulation of expression and localization of NOP16 varies in tumor environments with high versus low MYC levels and demonstrate the importance of stratifying clinical breast cancers based on MYC levels.

Short-term Prophylactic Tamoxifen Reduces the Incidence of Antiestrogen-Resistant/Estrogen Receptor-Positive/Progesterone Receptor-Negative Mammary Tumors

Although many estrogen receptor–positive (ER+) breast cancers are effectively treated with selective estrogen receptor modulators and down-regulators (SERM/SERD), some are highly resistant. Resistance is more likely if primary cancers are devoid of progesterone receptors (PR−) or have high levels of growth factor activity. In this study, a transgenic mouse line that expresses transforming growth factor-α (NRL-TGFα mice) and that develops ER+/PR− mammary tumors was used to assess the possible effects of (a) therapeutic delivery of the SERM, tamoxifen, or SERD, ICI I82,780 (ICI), on the growth of established tumors and (b) short-term prophylactic tamoxifen administration on the initial development of new mammary tumors. To determine the therapeutic effects of tamoxifen and ICI on the growth of established tumors, mice were exposed to 3 weeks of drug treatment. Neither drug influenced tumor growth or glandular pathology. To determine if early prophylactic tamoxifen could alter tumorigenesis, a 60-day tamoxifen treatment was initiated in 8-week-old mice. Compared with placebo-treated mice, tamoxifen reduced tumor incidence by 50% and significantly decreased the degree of mammary hyperplasia. Prophylactic tamoxifen also significantly extended the life span of tumor-free mice. These data show that in this mouse model, established ER+/PR− mammary tumors are resistant to SERM/SERD treatment but the development of new mammary tumors can be prevented by an early course of tamoxifen. This study validates the utility of NRL-TGFα mice for (a) identifying candidate biomarkers of efficacious tamoxifen chemoprevention and (b) modeling the evolution of tamoxifen resistance.

Mechanisms of occupational asthma: Not all allergens are equal

Asthma is a heterogeneous lung disorder characterized by airway obstruction, inflammation and eosinophil infiltration into the lung. Both genetics and environmental factors influence the expression of asthma, and not all asthma is the result of a specific immune response to allergen. Numerous asthma phenotypes have been described, including occupational asthma, and therapeutic strategies for asthma control are similar regardless of phenotype. We hypothesized that mechanistic pathways leading to asthma symptoms in the effector phase of the disorder differ with the inciting allergen. Since route of allergen exposure can influence mechanistic pathways, mice were sensitized by identical routes with a high molecular weight occupational allergen ovalbumin and a low molecular weight occupational allergen trimellitic anhydride (TMA). Different statistical methods with varying selection criteria resulted in identification of similar candidate genes. Array data are intended to provide candidate genes for hypothesis generation and further experimentation. Continued studies focused on genes showing minimal changes in the TMA-induced model but with clear up-regulation in the ovalbumin model. Two of these genes, arginase 1 and eotaxin 1 are the focus of continuing investigations in mouse models of asthma regarding differences in mechanistic pathways depending on the allergen. Microarray data from the ovalbumin and TMA model of asthma were also compared to previous data usingAspergillus as allergen to identify putative asthma ‘signature genes’, i.e. genes up-regulated with all 3 allergens. Array studies provide candidate genes to identify common mechanistic pathways in the effector phase, as well as mechanistic pathways unique to individual allergens.

Downstream bioengineering of ACE chromosomes for incorporation of site-specific recombination cassettes.

Advances in mammalian artificial chromosome technology have made chromosome-based vector technology amenable to a variety of biotechnology applications including cellular protein production, genomics, and animal transgenesis. A pivotal aspect of this technology is the ability to generate artificial chromosomes de novo, transfer them to a variety of cells, and perform downstream engineering of artificial chromosomes in a tractable and rational manner. Previously, we have described an alternative artificial chromosome technology termed the ACE chromosome system, where the ACE platform chromosome contains a multitude of site-specific, recombination sites incorporated during the creation of the ACE platform chromosome. In this chapter we review a variant of the ACE chromosome technology whereby site-specific, recombination sites can be integrated into the ACE chromosome following its de novo synthesis. This variation allows insertion of user-defined, site-specific, recombination systems into an existing ACE platform chromosome. These bioengineered ACE platform chromosomes, containing user-defined recombination sites, represent an ideal circuit board to which an array of genetic factors can be plugged-in and expressed for various research and therapeutic applications.

Abstract B88: Local delivery of breast cancer chemopreventives using mesenchymal stem cells

The overall goal of this study is to evaluate the ability of bone marrow mesenchymal stem cells (MSCs) to act as vehicles for local delivery of chemopreventives in mice that develop mammary cancer. Recently, efforts to use adult‐derived stem cells to deliver therapeutic agents to tumor sites have shown great promise and we currently are extending this concept to include the delivery of gene therapy in the form of chemopreventives. Our goals in this initial project were to (1) isolate and study the behavior of transplanted MSCs in mammary glands of mice and (2) develop a gene expression system which robustly produces gene therapeutics. For gene therapy we aimed to target epidermal growth factor (EGFR/ErbB1/HER1) because activation of this growth factor pathway is a frequent component driving breast cancer proliferation and survival and recent evidence demonstrated that the EGFR pathway is more active in hyperplastic enlarged lobules compared with normal terminal ductal lobular units (AJP 2007 171, 253–262), indicating that EGFR activation is a very early event that precedes cancer development and therefore inhibiting EGFR may abrogate cancer development. To carry out this goal we developed an expression system that produces the natural inhibitory ligand of EGFR, decorin. Decorin is a small leucine rich proteoglycan with known effects in extracellular matrix assembly as well as growth inhibition via reduction in levels and activity of EGFR along with other signaling molecules. Thus far we have demonstrated the ability to isolate, culture and maintain murine MSCs from the FVB/N strain in an undifferentiated state. In addition, these MSCS do not form tumors upon transplantation into mammary glands of syngeneic mice. Furthermore, we have produced secreted decorin in Chinese hamster ovarian cells and MSCs. Finally, we demonstrate the ability of conditioned media containing decorin to inhibit mammary tumor cell proliferation in vitro. Our long-term goal is to test the efficacy of decorin‐engineered MSCs as a breast cancer prevention modality in mouse models. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B88.

Abstract A197: Development of an immunocompetent, triple negative, breast cancer model for stem cell based delivery of therapeutics

Breast cancer represents a broad‐spectrum cancer with diverse morphological and immunohistochemical features and varied clinical outcomes. Affected breast cancer patients fall into one of three classifications: 1) hormone (estrogen receptor (ER), progesterone receptor (PR))‐positive tumors, 2) tumors with demonstrated ERBB2 (HER2 + ) amplification and, 3) tumors that are ER − , PR − and ERBB2 lo . For hormone receptor‐positive (ER) tumors, a variety of ER‐targeted therapy regimens with and without chemotherapy are available. Patients whose tumors are classified as ER − , PR − and ERBB2 lo (triple negative breast cancer; TNBC) are largely limited to chemotherapy as the only therapeutic modality. We have developed an autologous, immunocompetent triple negative breast cancer (TNBC) model whose features may overcome deficiencies of xenograft models for development of TNBC therapeutics. The model consists of: FVB/N immunocompetent mice; bone derived mesenchymal stem cells (MSCs) from FVB/N mice; and a triple negative cell line (UMD‐227), which is derived from a mammary tumor that developed in an NRL‐TGFα FVB/N mouse. Our hypothesis is that expression of antitumor factors (interferon beta (IFNβ), TNF‐related apoptosis‐inducing ligand (TRAIL), and/or the epidermal growth factor receptor (EGFR) inhibitor decorin (DCN)) from MSCs, either singly or in combination, will efficiently kill triple negative breast cancers. To test this hypothesis in vitro , we have applied each therapeutic individually, as conditioned medium produced from MSCs, on our novel, triple negative, mouse mammary cancer line, UMD‐227, and measured cell proliferation by a sulforhodamine B assay. In addition, we have tested for an additive effect with IFNβ and TRAIL using MSC/IFNβ conditioned media and commercial TRAIL. The addition of MSC/IFN conditioned medium to UMD‐227 cells significantly inhibits their growth in a proliferation assay similar to that shown with the TNBC line MDA‐MB‐231. The addition of commercial TRAIL 24 hours after placing UMD‐227 cells in MSC/IFNβ conditioned medium, produces a significant decrease in cell proliferation as compared to MSC/IFN conditioned medium alone or TRAIL alone. This is consistent with the knowledge that IFNβ upregulates TRAIL receptors, making the cell more susceptible to the addition of TRAIL at the 24 hour timepoint. These results suggest that MSCs engineered to express combinations of anticancer molecules may be a more efficacious therapeutic modality for TNBC. The next step in the development of our autologous immunocompetent triple breast cancer model is the insertion of multiple factors into the MSCs. We will then test the efficacy of the combinatorial therapy delivered from MSCs on the proliferation and motility of UMD‐227s in vitro. Our final goal is to move the entire system into the autologous mouse model (NRL‐TGFα) and test the efficacy of the combinatorial therapy in vivo. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A197.