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Mapping The Mammary Epithelial Cell Transformation In BRCA1 Carriers
Tumor Cell Biology V
Background : Identifying early steps in the cancerous transformation of primary mammary epithelial cells (PMECs) may help guide treatment and prevention strategies. Female BRCA1 carriers are highly predisposed to developing breast cancer, and their breast epithelium has a high prevalence of pre-malignant lesions. We have modified a three-dimensional (3D) culture technique to grow and analyze murine as well as human PMECs in a matrigel-based colony formation assay. In this culture PMECs will grow, differentiate and form characteristic structures. The structures can be classified into (1) “round, acinus-like” with an intact lumen, (2) “irregular”, i.e. growing in a disorganized way to large structures with filled lumina or (3) “cancer-like” with features of invasion. In this system tumor-like structures can be generated ex vivo from PMECs that in vivo had not yet formed tumors. It may therefore allow us to study very early oncogenic events. Hypothesis : The breast epithelium at risk for breast cancer formation of BRCA1 carriers harbors a heterogenous population of tumor progenitor cells at various stages of transformation that will grow in ex vivo cultures. Specific Aims: 1. Characterize ex vivo colony formation of PMECs that have been isolated from prophylactic mastectomies of BRCA1 carriers. 2. Analyze genetic changes in PMEC colonies with neoplastic features. Study Design : 1. Fresh tissues from women with the BRCA1 mutation who have opted for mastectomy or from women who chose reduction mammoplasties will be collected. Mammary epithelial cell and stromal cell fractions will be isolated and analyzed in the 3D assay. The phenotypes of the different colony types will be analyzed histologically and functionally through xeno-transplantation into nude mice. 2. In the different colony subtypes we will analyze the genetic changes that occur during clonal evolution on the pre-malignant level. Based on our preliminary data, we anticipate that PMECs from 6 to 10 BRCA1 carriers and age-matched controls will be sufficient to yield significant results. Potential Outcomes and Benefits : I hope to map the cancerous transformation of breast epithelial cells in these patients and to identify molecular targets for tumor treatment and prevention. This ex vivo model may also resemble in vivo conditions closely enough to allow for the experimental recapitulation of breast cancer formation and the testing of inhibitors of this process.
Background: Many different oncogenic events have been identified over the last several decades. However, we still lack a comprehensive model of what defines a breast cancer cell in a given patient. Recently developed culture models may provide us with a window into the initial stages of breast cancer formation, when some molecular changes have already occurred but the typical malignant growth is not yet present. We have called these cells that have already taken one or more “hits”, but do not yet form cancer, tumor progenitor cells. As a sample for the examination of such tumor progenitor cells I have chosen BRCA1 carriers because women who carry this mutation have a very high likelihood of develping breast cancer, and their breast tissue typically contains a number of pre-cancerous lesions. Hypothesis : The breast tissue of BRCA1 carriers harbors a population of cancer progenitor cells that can be identified using a combination of culture and genetic techniques. Specific Aims : 1. Use cell cultures to identify tumor progenitor cells in the healthy tissues of BRCA1 carriers who have undergone prophylactic mastectomy 2. Identify genetic changes that are characteristic for tumor progenitor cells. Study Design : Breast epithelial cells will be isolated from women with the BRCA1 mutation who have opted to have a prophylactic mastectomy. Reduction mammoplasty specimen will serve as controls. The cells will be cultured in an assay that allows for the growth of these cells in three dimensions, where their growth pattern most closely resembles the mammary gland environment. Molecular biology and statistical tools will be employed to dissect the steps of cancerous transformation in these colonies. Potential Outcomes and Benefits : The model system itself is valuable as it provides the research community with a tool to recapitulate the cancer formation process and to test inhibitory drugs. Moreover, this study aims at identifying early, crucial steps of carcinogenesis, and I hope to identify molecules that could be used in the early detection of breast cancer, or that could be targets for breast cancer prevention.