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The Role of BRCA1 in Development and Maintenance of Genomic Integrity in the Terminal End Bud
Title: The role of BRCA1 in development and maintenance of genomic integrity in the terminal end bud
Background: Regulation of mammary gland development is controlled by stimulation from multiple hormone and growth factor pathways, including estrogen. When these signals for normal mammary gland development go awry, epithelial cells grow in a disorderly and uncontrolled manner that could ultimately lead to breast cancer. Understanding the role of breast cancer associated genes during breast development may be key to designing more effective interventions and preventive strategies for women in all risk groups. BRCA1 is a breast cancer susceptibility gene the loss of which leads to defects in several cellular processes, including gene transcription and ability of cells to repair DNA damage. BRCA1 cellular activity is likely to be particularly critical in the very early stages of normal breast glandular development.
Objective/Hypothesis: Loss of full-length Brca1 can alter the genetic control of estrogen signaling pathways in the mammary gland and thus exert a gross effect on terminal end bud (TEB) development and increase the probability of DNA mutations.
1. Determine whether or not loss of full-length Brca1 increases the activity of estrogen signaling pathways through pubertal development.
2. Test if full-length Brca1 is required for the maintenance of genomic integrity during terminal end bud and ductal development.
Specific Aim #1 –Mammary glands from a conditional Brca1 mutant mouse model will be evaluated in whole mammary gland organ culture to determine whether loss of full-length Brca1 increases rates of cell proliferation, TEB number, ductal growth or PR expression over that measured in wild-type control glands using molecular, histological, and organ culture techniques that already have been established by Dr. Jones and her mentor’s lab. Cell proliferation rates will be measured by BrdU labeling. The number of TEBs and extent of ductal development will be determined by analysis of mammary gland whole mounts. Progesterone receptor expression will be evaluated by RT-PCR and immunohistochemistry.
Specific Aim #2 – The Brca1 mutant mouse model will be combined with the l-based Big Blue mouse model to test if full-length Brca1 is required for the maintenance of genomic integrity during TEB development. The Big Blue mouse model harbors a recoverable transgene in a lambda/LIZ shuttle vector that serves as a target to detect spontaneous or chemically induced point mutations, small insertions and deletions that arise in this transgene. Mammary glands from a conditional Brca1 mutant/Big Blue mouse model will be evaluated in whole mammary gland organ culture to determine whether or not loss in full-length Brca1 alone results in a increase in the rate of mutation, if estrogen signaling pathways increase the mutation frequency and if the combination of loss of full-length Brca1 with activation of estrogen signaling further increases rates of mutation either with or without an exogenous mutagen.
Potential Outcomes and Benefits of Research:
The overall aim of the proposed study is to establish the utility and exploit the use of whole mammary gland organ culture to investigate the role of Brca1 in the early stages of mammary gland development and maintenance of genomic integrity. It is our expectation that the proposed research will lead to a better understanding of the complex interactions involved in the early stages of breast cancer and provide insight towards the development of new prevention strategies that target the evolutionary process in tumors before it reaches the late stage usually seen in clinical cancer.
At birth, the human mammary gland consists of an undeveloped bud-like structure. It is not until the onset of female puberty, when high levels of estrogen are produced, that the mammary gland begins to grow rapidly. Normal development inside of rapidly growing cells, depends on the accuracy and coordination of several complex hormone signaling pathways and the ability of certain proteins to protect the deoxyribonucleic acid (DNA) from damage (i.e. mutation). DNA is a long molecular strand of genetic instructions stored in the nucleus of every cell. Our DNA is susceptible to many types of damage throughout mammary gland development and whether or not this DNA damage is repaired properly is a primary determinant in the development of cancer. BRCA1 (breast cancer susceptibility) is the name of a gene that is altered in certain families with an inherited susceptibility to breast cancer. Several studies, including those in our lab, have shown that BRCA1 is a DNA damage repair protein. Therefore, in cells that have lost BRCA1 function, there is a greater susceptibility to breast cancer.
My proposed project seeks to understand the role of BRCA1 in mammary gland development. One way to determine the normal function of a protein in the body is to generate a mouse model that does not express the protein or expresses a modified form of the protein that does not function properly. Then, one could look at whether any of the normal physiological processes in the body are altered or abolished. In these studies we will utilize a mouse model of BRCA1-assocatiated tumorigenesis that does not express the full-length Brca1 protein. Since there are many complex hormonal signaling events that occur in the whole animal during mammary gland development that we are unable to control, we will remove the mammary gland from the mouse and place it in a tissue culture dish so that we can control the level and types of hormone stimulation as well as monitor its growth in response to various hormonal conditions. We will use this mammary gland whole organ culture model to determine whether or not loss of Brca1 effect estrogen-signaling pathways during the early stages of mammary gland development. In addition, we will explore whether Brca1 is required for the maintenance of genomic integrity during early mammary gland development
We suggest that the results may shed light on both the normal function of BRCA1 in mammary epithelial cells as well as defining whether or not alterations in hormonal exposure can influence progression of BRCA1 mutation related breast cancer. It is our expectation that the proposed research will lead to a better understanding of the complex interactions involved in the early stages of breast cancer and provide insight towards the development of new prevention strategies that target the evolutionary process in tumors before it reaches the late stage usually seen in clinical cancer.