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Role of Progestins and Progesterone Receptors in Hormone-Dependent Human Breast Cancers
“Hormone-dependent” human breast cancers stain positive for estrogen receptors (ER) and progesterone receptors (PR) and require estrogens (E) to grow. These “E-dependent” tumors will initially respond well to endocrine therapies targeted at ER. To date, PRs have essentially been used as markers of functional ER. However, considerable attention is now being given to both PRs and their ligands. PRs: PRs exist as two functionally distinct forms termed PR-A and PR-B. The ratio of the two PRs varies considerably in tumors, contrary to normal tissues, and A>B may be associated with a poorer prognosis. Additionally, PRs negatively modulate ER function - PR-A are more potent in this regard. Progestins: Early full-term pregnancies are protective against breast cancer. This is hypothesized to be due in part to high serum progesterone levels. Contrarily, synthetic progestins taken in conjunction with E as part of hormone replacement therapy (HRT) increase the risk of breast cancers. Taken together, the role of progestins and PRs in human breast cancers remains an enigma. We believe that PRs are more than mere markers of ER function – and that it is imperative to study the effects of ERs and PRs in relation to one another, and not in isolation. The objective of this proposal is to evaluate systematically how progestins and PRs affect the growth, treatment, and metastatic potential of E-dependent human breast tumors.
Aim 1. What is the role of progestins on E-dependent human breast tumor growth? These studies will determine how two progestins, the natural hormone progesterone and the synthetic progestin MPA (HRT), affect E-dependent tumor growth and gene expression. These experiments will be done in the context of the two different PRs. A. Growth of E-dependent tumors in the presence of progestins. We have established a model in which E-dependent T47D human breast cancer cells with different PR status (no PR, PR-A only, PR-B only, or both PRs) are grown into solid tumors in vivo in ovariectomized female nude mice. Tumors will be established with E, than additionally exposed to placebo, progesterone, or MPA and growth monitored. B. Gene expression profiles in progestin treated tumors. Affymetrix human gene chip technology will be used to collect data from tumors generated in A. Genes will be identified that change in accordance with PR expression and progestin treatment in the context of E-treated tumors. This aim will determine how PRs liganded by progesterone or the synthetic drug MPA affect E-dependent breast tumor growth - and will profile genetic changes that occur in these tumors.
Aim 2. How do PR expression and progestins affect aromatase inhibitor (AI) treatment of E-dependent breast cancers? Our T47D xenografts respond poorly to tamoxifen – analogous to many advanced ER+PR+ tumors in the clinical setting. Ongoing clinical trials are testing AI as a new, potentially more effective treatment option for E-dependent tumors. How PRs translate as markers for AI therapy is unknown. We will develop a model to assay the efficacy of AIs on in our T47D xenografts (with different PR expression): the aromatase (estrogen synthase) gene will be stably introduced into our T47D cell lines, tumors grown using the E precursor androstenedione, and established tumors treated with AI. We will test two types of AIs: letrozole (nonsteroidal) and exemestane (steroidal). Completion of this aim will define how PRs correlate as markers for AI therapy.
Aim 3. Do PR-A or PR-B affect the metastatic potential of E-dependent breast tumors? Breast cancer cells expressing predominantly PR-A are described as more “migratory” and “invasive” than cells expressing predominantly PR-B. We will test this hypothesis using T47D breast cancer cells expressing either PR-A or PR-B linked to the enhanced green fluorescent protein (EGFP). We will use two experimental metastasis approaches. A. Direct intravenous or intracardial injection of cells B. Orthotopic establishment of primary tumors in the mammary fat pad. For both approaches breast cancer cells will be tracked by serial sectioning major target tissues (i.e. lymph node, lung) at different time points and using fluorescent microscopy to detect EGFP expression. These experiments will test the hypothesis that tumors expressing predominantly PR-A are more aggressive.
Completion of this proposal will begin to address how PRs and progestins impact the phenotype of E-dependent human breast cancers. This is important since ERs coexist with PRs in the majority of primary human breast cancers – and the auxiliary role of PRs and progestins in these cancers remains unknown.
The two most important markers of human breast cancers are estrogen receptors (ER) and progesterone receptors (PR). Tumors positive for these receptors (ER+PR+) are considered “hormone-dependent” based on their requirement for the female hormone estradiol (E) to grow. Approximately 70% of these “E”-dependent tumors respond well initially to endocrine treatments, most notably to the antiestrogen tamoxifen. However, tamoxifen resistance is a recurrent problem in all advanced tumors. Clinical trials are now testing new therapeutic drugs called aromatase inhibitors, which target the local production of estrogens in the breast. Whereas considerable attention has been given to the study of E and ERs in human breast cancers, less attention has been afforded to PRs. This grant focuses on PRs and progestins (which bind to PRs). We have several reasons for studying PRs and progestins: 1) We believe that PRs as markers can tell use much more about a tumor’s prognosis than is currently known 2) PR are expressed as two forms termed PR-A and PR-B. Several studies have found that PR-A and PR-B are expressed unequally in primary tumors (i.e. A>B or B>A). Tumors with A>B have been preliminarily correlated with poorer prognosis. 3) PRs modulate the actions of ERs. Since ERs are the main target of endocrine therapies, PR expression may dictate outcomes. 4) The use of synthetic progestins may increase the risk of breast cancer. This stems from the Women’s Health Initiative trial in which the progestin component of hormone replacement therapy (HRT), medroxyprogesterone acetate (MPA, commonly known as Depo-Provera), was found to increase the incidence of breast cancer. Many women have ceased to use progestins as a result. Their use, however, has many additional health benefits so an understanding of the risk involved is crucial.
We propose to investigate how both progestins (P) and PRs affect the growth, treatment, and metastasis of E-dependent breast cancers. We employ the female “nude” mouse, which lacks a complete immune system, as a model to study human breast tumor growth and response to treatment. These mice are ovariectomized, have circulating levels of hormones similar to postmenopausal women, and serve as good hosts for growth of implanted human tumor cells. We can manipulate both the PR expression in the tumors, and the hormone treatment of the animals (E and P). The specific goals of this study are summarized.
Aim 1A. We will investigate how progestins and PRs affect the growth of E-dependent human breast cancers. This will be done using human breast tumors cells that contain no PR, PR-A only, PR-B only, or both PR-A and PR-B. E-dependent growth will be monitored in the presence of the natural hormone progesterone or the synthetic hormone MPA (HRT).
Aim 1B. We will determine gene expression profiles in progestin treated tumors. Using advanced “human gene chip” technology, we can determine changes that occur in progestin-treated tumors compared to tumors treated with E alone. We can distinguish changes due to both PR form expression (A vs. B) and the type of progestin used (progesterone vs. MPA). The latter is crucial since the clinical drug MPA may elicit different changes than progesterone itself.
Aim 2. We will define how PRs and progestins affect the outcome of treatment with aromatase inhibitors (AI). Our experimental breast tumors show resistance to the antiestrogen tamoxifen, similar to many advanced clinical cases. AI are currently used in clinical trials and are, preliminarily, more effective than tamoxifen. It is unknown how PRs serve as markers for AI therapy. We will test the effectiveness of AI in our tumor model in relation to PRs (A and B) and progestin treatment.
Aim 3. We will determine how PRs affect the metastatic potential of tumors. Data at the cell level have shown that PR-A containing breast cancer cells are more “migratory and invasive” and hence metastatic than PR-B containing cells. We will address this in a mouse model using fluorescently labeled cells, which can be tracked in vivo. We will determine to which tissues PR-A and PR-B containing breast cancer cells localize (i.e. in the lymph nodes or lung).
This proposal addresses how PRs and progestins affect the physiology of E-dependent breast cancer cells. The precise impact of PRs on ER function in malignant breast cells remains a mystery – our studies will address this. Many women continue to take progestins for a variety of health reasons and how this impacts the growth and treatment of breast cancers is of fundamental importance.