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    Awarded Grants
    EphA2, a Novel Target for Breast Cancer Therapy

    Scientific Abstract:
    Background: EphA2 is a receptor tyrosine kinase found at low levels in adult breast epithelium. In non-malignant cells, EphA2 is localized to sites of cell-cell contact facilitating interaction with its membrane-bound ligands; ligand binding causes EphA2 to negatively regulate cell growth and motility. In contrast, EphA2 is overexpressed in many breast cancer cells, where it is diffusely localized over the cell surface. Thus, EphA2 fails to properly interact with its ligands and instead promotes uncontrolled tumor cell growth, angiogenesis and invasion. EphA2 is overexpressed in ~50% of human breast cancers. ER+ MCF-7 breast cancer cells transfected with EphA2 grow in culture and form tumors in athymic mice without supplemental estrogen and are less sensitive to tamoxifen in vitro and in vivo. We have produced a monoclonal antibody (EA5) that binds to EphA2 and mimics ligand binding, inducing phosphorylation and degradation of the EphA2 receptor. EA5 restores normal EphA2 function, inhibiting breast cancer growth in vivo and restoring hormone dependence and tamoxifen sensitivity in vitro. Objective/Hypothesis: Our central hypothesis is that EphA2 plays a critical role in breast cancer progression. The guiding rationale for this proposal is that overexpression and altered function of EphA2 provides a novel target for breast cancer therapy. Specific Aims: Aim 1 will determine the efficacy of EA5 on in vivo tumor growth in both ER+ and ER- models. Aim 2 will determine ER-dependent mechanisms by which EphA2 overexpression controls breast cancer growth, hormone dependence and tamoxifen resistance. Study Design: Using an engineered ER+, EphA2 overexpressing xenograft model we will further explore the in vivo dose response, then test EA5 in combination with tamoxifen. Finally we will measure the ability of EA5, alone and in combination with bevacizumab, to inhibit growth and metastasis of ER-, naturally EphA2 overexpressing xenografts. Using an ERE-luciferase construct, we will verify that EphA2 overexpression consistently decreases ER activation. Next we will quantify the effect of EphA2 on expression of ER target gene mRNA and protein expression. Finally using GST pulldown and co-immunoprecipitation, we will determine if EphA2 exerts a direct effect by binding to ER. Potential Outcomes and Benefits: The results of studies will further solidify the foundation for clinical development of EphA2 targeted therapies. In addition the studies in aim 2 will shed further light on the mechanisms by which EphA2 promotes cell growth, metastasis and hormone dependence.

    Lay Abstract:
    Background: The need for better, and less toxic, breast cancer therapies is real and pressing. We have focused on EphA2, a protein that functions as a key ‘molecular switch’ to either stimulate or inhibit cell growth and metastasis. In normal cells, EphA2 is localized to sites of cell-cell contact where it interacts with its partners (called ephrins), which are bound to the adjacent cell. Interaction of EphA2 with the ephrins decreases cell growth (switch off). In contrast, EphA2 is overexpressed and diffusely localized over the entire cell surface in many breast cancers. Thus, EphA2 is not in a proper position to interact with the ephrins. When EphA2 does not interact with the ephrins, it promotes uncontrolled tumor cell growth, (switch on). EphA2 overexpression and altered function are found in nearly half of human breast cancers. We have produced a monoclonal antibody (named EA5) that binds to EphA2 and restores normal EphA2 function, in essence flipping the switch back to the ‘off’ position. EA5 inhibits growth of ER+ and ER- breast cancers in mice and restores estrogen dependence and tamoxifen sensitivity of cells in culture. Objective/Hypothesis: The guiding hypothesis of this proposal is that overexpression and altered function of EphA2 provides a novel target for breast cancer therapy. We propose that restoring normal EphA2 function with EA5 will be an effective therapy for breast cancer. Specific Aims: Our first studies will test the impact of EA5 on the growth of ER+ and ER- tumors in mice. Our second series of experiments will investigate the molecular mechanisms by which EphA2 controls cell growth, hormone dependence and tamoxifen resistance. Study Design: Aim 1 will utilize a well-established mouse model system to study EA5 therapy. First we will use different doses and schedules of EA2, either alone or in combination with tamoxifen to treat ER+ tumors that have been artificially made to overexpress EphA2. Then we will test the ability of EA5, alone or in combination with bevacizaumab, to decrease primary tumor growth and metastases in ER- tumors that naturally overexpress EphA2. Aim 2 will study how EphA2 and ER interact to control cell growth. Potential Outcomes and Benefits: This proposal brings us closer to our long-term goal of bringing EA5 to the clinic. We expect that EphA2 antibodies will have limited effect on normal cells both because of the low EphA2 expression and because EphA2 in normal cells is already bound to its ligand and is therefore unavailable to the antibody.