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Novel Imaging Techniques to Compare Estrogen Receptor Structure in Tumors and Normal Tissues of Intact Living Animals
Background: Much is known of the effect of estrogens and anti-estrogens used to treat breast cancer on the structure and interactions of the estrogen receptor (ER) in vitro. However, some anti-estrogens (SERMs) behave as estrogens in some tissues. This implies that some aspects of ER structure or interactions are modified or differentially used in each tissue.
Objective/Hypothesis: Our hypothesis is that understanding breast cancer progression and identifying more effective breast cancer treatments will rely on an ability to detect ER structure and interaction at different tumor stages and in specific tissues of intact animals. This is currently not possible.
Specific Aims: Our immediate goal is to develop tools for non-invasive imaging of ER structure in whole animals.
Study Design: We will measure the structure of a dual-fluorescent protein-tagged ER in mouse models using the technique of fluorescence resonance energy transfer (FRET). Preliminary studies in breast cancer cell culture lines established principals for FRET measurement of estradiol and tamoxifen regulation of ER structure and interaction. The fluorophore-tagged ERs will be expressed in specific tissues of intact transgenic mice and in tumor cells transplanted into nude mice. We then will establish FRET collection and analysis parameters for detecting ER structure at those sites using small animal fluorescence imaging.
Potential Outcomes and Benefits of the Research: The goal of these studies is to establish FRET collection and analysis principals that overcome confounding factors in whole animal imaging. For breast cancer, the long-term aim is to establish how specific drugs, such as tamoxifen, have differing estrogenic and anti-estrogenic effects on ER structure and interactions in different tissues. These live animal molecular imaging techniques, together with knock-in or transgenic labeling of specific proteins in specific tissues of the animal, would be widely applicable for the investigation of any molecular process associated with any aspect of breast cancer initiation, progression and treatment. Indeed, such atomic-level imaging techniques would improve our biochemical and structural knowledge of any molecular target thought to be important in any disease.
The establishment and progression of many breast cancers involves changes in the shape of the protein that binds the estrogen hormones. Estrogens play a central role in the development of a large proportion of breast cancers. Indeed, estrogen depletion with arimidex (anastrozole), or treatment with the drug tamoxifen that binds to the estrogen-binding protein, prevents an estrogen-induced change in the shape of the estrogen-binding protein. However, these events only can be followed in cell culture or in the test tube. Thus, the timing and contribution of tissue and tumor-specific events to these shape changes remains unknown during tumor initiation, progression or metastases in a complex living animal.
Imaging tools detect the number and location of tumors and guide treatment selection by the patient and physician. Similar imaging techniques are used in animal models of breast cancer to establish how experimental treatments affect the course of the disease. Current imaging tools do not measure protein shape. Our hypothesis is that imaging techniques that measure the effects of estrogen and anti-estrogen treatment on the shape of the estrogen-binding protein in different organs of an animal will help establish the molecular basis of breast cancer development and treatment.
In cultured beast cancer cells, we developed an imaging technique in which a small molecular “light bulb” is attached to the estrogen-binding protein. When that light bulb is turned on, it activates another light bulb attached to a different part of the estrogen-binding protein only when the two light bulbs essentially touch each other. The amount of this “energy transfer” identifies protein shape. We have used these sophisticated techniques to measure the effects of estrogens and existing or developmental breast cancer treatments on the estrogen-binding protein, at least in cultured cells.
In the proposed studies, our goal is to establish these very powerful techniques for imaging specific actions of the estrogen-binding protein in specific tissues in whole animals. Our longer-term goal is to track changes in the estrogen-binding protein associated with tumor formation or progression, and to determine how estrogens and tamoxifen may affect those changes differently in different tissues and in tumors. Such techniques are highly relevant to establishing the molecular basis not only of breast cancer development and treatment, but also of virtually any disease.