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Molecular Mechanisms of LRH-1 Function in Breast Tissue
Background: Breast cancer remains a major health issue for women. Treatments for early stage breast cancer after surgical removal of the tumor often involve hormone therapy. Sixty percent of all breast cancers are estrogen receptor (ER) positive and anti-estrogen therapy has been shown to be effective in breast cancer treatment. There are several ways of blocking the effects of estrogen in ER-positive breast cancers. Anti-estrogens, such as tamoxifen are commonly used to block the estrogen receptor activity. The aromatase enzyme encoded by CYP19 can convert androgens into estrogens and inhibitors of these enzymes are especially effective in treating post-menopausal breast cancer patients. Tamoxifen and aromatase inhibitors block estrogen signaling. However, these hormone treatments are not always functional for ER-positive breast cancers. In classic steroidogenic tissues such as the ovary, aromatase gene expression is regulated by two nuclear receptors of subfamily V, Steroidogenic Factor-1 (SF-1) and Liver Receptor Homologue-1 (LRH-1). However, SF-1 is not expressed in adipose stromal cells and breast cancer cell lines. Recently, Dr. Simpson’s group reported that the close homologue to SF-1, LRH-1, is expressed in preadipocytes, and in primary and in breast cancer cell lines. He also found that the CYP19 (aromatase) promoter was activated by LRH-1. However, LRH-1 function is still unclear in the mammary gland and breast adipose. In a more recent study, we reported that the crystal structure of the LRH-1 ligand binding domain. This structure suggested that LRH-1 adopts an active conformation with a large empty ligand binding pocket. Further computational docking to this pocket identified several estrogen like ligands for LRH-1.
Objective/Hypothesis: In this proposal I wish to focus on the molecular mechanisms of LRH-1 function in the normal breast development and in breast cancer.
Specific Aims: Aim 1 will determine the expression profile of LRH-1 in the mouse mammary gland, at different developmental stages and in mouse breast cancer models. I will ask if LRH-1 expression correlates with the growth rate of breast cancer cells and if LRH-1 is present in ER-positive versus ER-negative breast cancers. Aim 2 will ask if LRH-1 is phosphorylated and how phosphorylation affects cofactor recruitment. I will also ask whether the breast-cancer enriched nuclear receptor coactivator amplified in breast cancer (AIB1) interacts with LRH-1 and how phosphorylation alters recruitment of coactivators (AIBI) by LRH-1.
Study Design: In Aim 1, I will first check expression of LRH-1 in breast tissue using immunohistochemistry (IHC). Now, we are currently making an anti LRH-1 antibody for IHC, based on protocols used successfully for SF-1. Second, I check expression level of LRH-1 transcripts in breast cancer cells and alter the growth conditions using estrogen or anti-estrogen treatment to determine if the levels of LRH-1 are affected. sRNAi will be used in cultured breast cancer cell lines to eliminate LRH-1. I have established that LRH-1 expression levels are higher in normal breast versus tumor. In Aim 2, I will map the phosphorylation site in LRH-1 using in vitro kinase assays and study how phosphorylation affects recruitment of the breast-enriched coactivator AIB1 using standard GST-pull down assays and co-immunoprecipitation (Co-IP) on an endogenous DNA response element present in the aromatase promoter. Additionally, I will determine the effect of transcriptional activity of phosphorylated LRH-1 and AIB1 using cellular transfection assays and a non-phosphorylatable LRH-1 mutant.
Potential Outcome and Benefits of the Research: I wish to understand LRH-1 function in breast tissue and its potential regulation of aromatase activity. My studies might provide valuable information for developing new strategies against breast cancer.
Breast cancer is the most common type of cancer in women with approximately one in nine women developing this disease over a lifetime. Furthermore, this cancer is the second leading cause of cancer-related deaths for women in the United States for women between the ages of 35 to 54. Every year approximately 180,000 new cases of breast cancer occur, and it is estimated that 12% of women in the U.S. will at some point be diagnosed with the disease. Further insights into how different factors, such as type of cancer, age of onset and family genetic history are linked to the underlying mechanisms of breast cancers may improve the available methods of treatment. While many treatments for early breast cancer include surgery, many women also undergo additional chemotherapy or hormone therapy. Here in this application, I am focused on a potential role for a new type of hormone receptor present in breast tissue in the progression of breast cancer disease. My proposed research plan deals with novel ways to regulate levels of hormones and is therefore related directly to standard hormone therapies widely used in the treatment of breast cancer.
Normally, use of anti-estrogens in hormone therapy works by blocking the actions of the estrogen receptor to slow cell-proliferation in breast cancer. This treatment is used in preventive treatment and in advanced cancer treatment in patients with estrogen receptor-positive tumors. In the last several years, new anti-estrogen agents have become available including tamoxifen and Selective Estrogen Receptor Modulators (SERMs). Tamoxifen is an anti-estrogen, whereas SERMs chemically resemble estrogen and trick the breast cancer cells into accepting it in place of estrogen. However, unlike estrogen, they do not stimulate breast cancer cell growth. Other SERMs being studied for breast cancer include tamoxifen (which is very similar to tamoxifen), idoxifene, and droloxifene. Long term use of these drugs may lead to increased risk for blood clots and tamoxifen, and possibly toremifene also pose an increased risk for uterine (endometrial) cancer, though not enough to offset the benefits from breast cancer prevention. It is still not known if the newer SERMs will also carry some medical risks with long-term use. Probably the biggest problem associated with anti-hormone therapy is the lack of effectiveness in treating breast cancer in some women when used for long periods of time.
Another treatment aimed at reducing estrogen is the use of aromatase inhibitors. Aromatase is an enzyme that converts androgens (the male hormone) into estrogen in both pre-and post-menopausal women. Aromatase inhibitors block this enzyme, which is present in both the ovary and in other fat-containing tissues body tissues, including the breast. For post-menopausal women, estrogen is produced by the local conversion of testosterone (produced in the adrenals) by aromatase. Therefore, inhibitors offer great promise for breast cancer and do not have the potential complications of tamoxifen. Clinical studies support this strategy for breast cancer treatment. Here, we are focused on how a novel hormone receptor affects normal breast tissue and contributes to breast cancer. We wish to understand 1) how the actual gene encoding aromatase activity is regulated by this receptor, and 2) if this receptor regulates mammary gland or tumor proliferation. Our ultimate goal is to provide new molecular insights that might enable next generation drugs design in the treatment of breast cancer using molecular biology system.