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    Research Grants Awarded

    Role of Aryl Hydrocarbon Receptor and Cross Talk with Estrogen Receptor in Response of Breast Cancer Cells to the Novel Antitumor Agent Aminoflavone

    Study Section:
    Treatment

    Scientific Abstract:
    Background: Many estrogen receptor (ER)-expressing breast cancers become refractory to ER-based therapies. Aminoflavone (AF) has been developed by NCI, and has exquisite antitumor activity in ER(+) MCF-7 and T47D cells, and in a MCF-7 nude mouse model. ER(-)breast cancer cells are less susceptible. We previously found in MCF-7 cells that AF activates the aryl hydrocarbon receptor (AhR) via translocation to the nucleus, induction of AhR?specific DNA binding activity, and expression of CYP1A1, whose transcription is governed by the AhR-ARNT basic helix-loop-helix transcription factor. CYP1A1 metabolizes AF to a species which directly or after further metabolism damages DNA. In contrast an AhR-deficient variant of MCF-7, or cells with predominantly nuclear AhR expression such as MDA-MB-231, are resistant to AF. Thus AF, unlike other antineoplastic agents, requires AhR-mediated signaling to cause DNA damage. This is a new treatment strategy for breast cancers with intact AhR signaling. Hypothesis: That AhR status and its cross talk with ER determine response to AF. Specific Aims: 1) To define the role of cytoplasmic and nuclear AhR and its partner molecules in AF sensitive and AF resistant breast cancer cell lines; 2) To define the influence of estrogen receptor, estrogen, and ER antagonists on AF-induced AhR signaling to see whether this might predict how patients? tumors will react to AF. Study Design: We will study AF driven AhR signal transduction in AF sensitive and resistant breast cancer cells in the presence and absence of estradiol and its antagonists. We will evaluate the localization of AhR expression and its relation to ER status in invasive breast cancers by using high-density tissue microarrays and immunohistochemistry. To see if AhR and ER are co-regulated, we will use chromatin immunoprecipitation (ChIP) with H2AX, ER and AhR antibodies, which allow us to identify gene regions involved in AF-mediated DNA damage, ER signaling, and AF mediated AhR activation, respectively. MCF-7 cells and subclones resistant to hormone therapies will be examined for sensitivity to AF in vitro and in vivo. We will use biopsies from xenografts to define pharmacodynamic endpoints. Potential Outcomes: We hope to establish AhR as a therapeutic target of AF in ER(+) breast cancers, and that AF will prove to be an effective treatment for patients with hormone-refractory metastatic tumors.

    Lay Abstract:
    Background: We need more treatments for breast cancer patients who have stopped responding to hormonal therapies. Aminoflavone (AF) is a novel cancer drug entering clinical trials that are sponsored by the National Cancer Institute. AF is highly active in inhibiting growth of breast cancer cells expressing the estrogen receptor (ER) in tissue culture (in vitro) and in certain animal models (in vivo). AF causes a molecule called the aryl hydrocarbon receptor (AhR) to go to the nucleus and increase the levels of an AF-metabolizing enzyme, termed cytochrome P450 1A1 (CYP1A1), which in turn can act on AF and produce a drug metabolite to damage DNA, resulting in cell kill. This is a new strategy for breast cancer treatment: to generate a DNA damaging agent selectively in the tumor cell, formed by the tumor cell from the drug AF. Hypothesis: That AhR is the molecular target of AF, and that its action is influenced by presence of the ER in breast cancer. Specific Aims: 1) To define the role of cytoplasmic and nuclear AhR and its partner molecules in AF sensitive and AF resistant breast cancer cell lines; 2) To define the influence of estrogen receptor, estrogen, and ER antagonists on AF-induced AhR signaling to see whether this might predict how patients? tumors will react to AF. Study Design: We will use an assay called chromatin immunoprecipitation (ChIP), to analyze AhR?s interaction with DNA, and determine AhR partners in AF sensitive versus resistant breast cancer cells in the presence and absence of estrogen. We will further determine the protein expression pattern of AhR and ER in a large collection of archived breast cancers and compare them to clinical outcome. Additionally we will test whether a panel of breast cancer cells that has become resistant to hormone treatment will remain sensitive to AF in vitro and in vivo. Potential Outcomes: We hope to establish AhR as a valid therapeutic target of AF, and that our results will aid the clinical development of AF by defining how to identify breast cancer patient populations who will benefit from AF treatment.