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    Phosphopeptide Antibodies as Prognostic Indicators for Breast Cancer

    Scientific Abstract:
    Phosphopeptide Antibodies as Prognostic Indicators for Breast Cancer (A) Background: Estrogen receptors are used as markers to predict the response of breast tumors to anti-estrogen drugs such as tamoxifen; however, approximately 30-50% of breast tumors with immunoreactive estrogen receptors do not initially respond to tamoxifen. One proposed mechanism for tamoxifen resistance is that phosphorylation of estrogen receptors by tyrosine kinases such as Her-2/neu could activate estrogen receptors in the absence of estradiol, thereby circumventing the estrogen-blocking effect of tamoxifen. Tyrosine kinases that have been implicated in estrogen receptor phosphorylation include Her-2/neu, the EGF receptor, and Src. These kinases have also been implicated as markers for aggressive tumor behavior. Breast tumors in African-American women tend to be more aggressive than those in Caucasian women. The molecular basis of this difference has not yet been determined, but may involve preferential activation of specific tyrosine kinases. We have generated polyclonal antibodies that recognize estrogen receptor alpha (ER-alpha) only when it is phosphorylated on Tyr-537, and antibodies that bind activated phosphorylated forms of Her-2/neu and Src family kinases. We will also prepare monoclonal antibodies directed against the ER-alpha Phosphotyrosine-537 epitope. (B) Objectives/Hypothesis: The long-term goal of the proposed research is to determine whether these phosphopeptide antibodies, when used in conjunction with commercially available antibodies against other estrogen receptor phosphorylation sites, can help improve predictions of prognosis and response to tamoxifen. We propose to use these antibodies to test the hypothesis that activation of Her-2/neu, EGF receptors and Src family kinases correlates with: (1) estradiol-independent phosphorylation, dimerization and activation of estrogen receptors, (2) tamoxifen resistance, and (3) poorer prognosis in African-American breast cancer patients. (C) Specific Aims: (1) To determine whether activation of Her-2/neu, EGF receptors and Src family kinases correlates with site-specific phosphorylation, dimerization, and activation of estrogen receptors. (2) To prepare monoclonal antibodies directed against Phosphotyrosine-537 of ER-alpha. (3) To use a panel of polyclonal and monoclonal phosphopeptide antibodies to determine whether expression of activated, phosphorylated forms of estrogen receptors, Her-2/neu, EGF receptors and Src family kinases in breast cancer cells correlates with tamoxifen resistance, and occurs more frequently in tumors from African-American breast cancer patients than in breast tumors from Caucasian patients. (D) Study Design: Monoclonal antibodies directed against phosphorylated Tyr-537 of ER-alpha will be produced. A panel of polyclonal and monoclonal phosphospecific antibodies will be used to assess phosphorylation of estrogen receptors at different sites in breast carcinoma cell lines, xenografts and human breast tumors by immunoprecipitation, immunoblotting and immunohistochemistry. Phosphorylation of estrogen receptors at each site will be correlated with: (1) the activation state of Her-2/neu, EGF receptors and Src family kinases; (2) the oligomerization state of ER-alpha as assessed by analytical ultracentrifugation; (3) binding of ER-alpha to estrogen response element (ERE) probes in gel shift assays; and (4) transcriptional activation of ERE reporter constructs. The frequency of phosphorylation of estrogen receptors, Her-2/neu, EGF receptors and Src family kinases will be compared in tamoxifen-responsive and tamoxifen-resistant breast tumors, and in African-American and Caucasian breast cancer patients by immunohistochemical analysis with phosphopeptide antibodies. (E) Potential Outcomes and Benefits of the Research: Correlating estrogen receptor phosphorylation with activation of specific kinases may facilitate identification of pathways that contribute to tamoxifen-resistant, phosphorylation-dependent proliferation of breast tumor cells in different ethnic groups. These studies may improve the ability to predict which estrogen receptor-positive breast cancer patients are likely to benefit from antiestrogen therapies, and may help to target tyrosine kinase inhibitor therapies such as Herceptin (trastuzumab), and Iressa (gefitinib) to the subset of tamoxifen-resistant breast tumors that are most likely to be responsive. Production of monoclonal antibodies directed against the phosphorylated ER-alpha Tyr-537 epitope will facilitate distribution of uniform reagents that detect tyrosine-phosphorylated estrogen receptors to other investigators for long-term clinical studies.

    Lay Abstract:
    Phosphopeptide Antibodies as Prognostic Indicators for Breast Cancer. The anticancer drug tamoxifen inhibits the growth of some breast cancer cells by blocking the binding of the hormone estrogen to the estrogen receptor. The presence of estrogen receptors in breast tumors is currently used to predict which patients will benefit from tamoxifen. However, some estrogen receptor-positive tumors are initially resistant to tamoxifen. One of the major goals of the proposed research is to determine whether laboratory tests using antibodies we have developed can distinguish which tumors will respond to anti-estrogen therapies like tamoxifen. The usual mechanism for estrogen receptor activation is for estrogen to bind to a particular site on the receptor, causing other changes that enable the receptor to affect the behavior of the cell. By blocking this site, tamoxifen prevents estrogen from binding. It has been proposed that addition of phosphate groups to estrogen receptors can lead to activation of the receptor. This process, known as phosphorylation, is mediated by enzymes called kinases. One proposed explanation for tamoxifen resistance is that some kinases can activate the estrogen receptor just by phosphorylating it, thereby circumventing the need for estrogen binding. Since this mechanism does not require binding of estrogen, tamoxifen would not be effective. Kinases that are believed to activate estrogen receptors by phosphorylation include Her-2/neu, EGF receptors, and Src. Activated forms of these kinases are thought to contribute to more aggressive tumor behavior, poor prognosis and to tamoxifen resistance. Our laboratory has produced antibodies that can bind to phosphate groups on active forms of Src and Her-2/neu, but not to inactive forms of these enzymes. We have also generated antibodies that bind to a phosphate group at a specific amino acid on estrogen receptor alpha (Tyrosine-537). The antibodies detect active/phosphorylated forms of Src, Her-2/neu, and estrogen receptors in breast tumor tissue samples by a staining procedure called immunohistochemistry. Using our antibodies in conjunction with commercially available antibodies, we will determine whether activation of specific kinases correlates with phosphorylation of estrogen receptors. We will identify breast tumor tissue samples where staining for active kinases matches up with staining for phosphorylated estrogen receptors. For example, a tumor that stains for active/phosphorylated Her-2/neu and phosphorylated estrogen receptors would be consistent with the possibility that active Her-2/neu leads to estrogen-independent activation of estrogen receptors. Such a tumor would be likely to be resistant to tamoxifen but may be responsive to the anti-Her-2/neu antibody Herceptin. Identifying tumors that express both phosphorylated estrogen receptors and activated kinases may improve prediction of which breast tumors will be resistant to tamoxifen, and may help target tyrosine kinase-inhibitor therapies like Herceptin and Iressa to the subset of tamoxifen-resistant breast tumors that are most likely to be responsive. Activation of these kinases may be relevant for understanding why tumors from African-American breast cancer patients tend to be more aggressive than those of Caucasian breast cancer patients. Kinases that are more frequently activated in African-American breast cancer patients may contribute to the disparity in prognosis between African-American and Caucasian breast cancer patients. Collaborating with other investigators, we will use phosphorylation-specific antibodies to stain tumors from African-American and Caucasian breast cancer patients in different age groups to determine whether differences in the frequency of activation of Her-2/neu, EGF receptors, and Src family kinases correlate with more aggressive tumor behavior and estrogen receptor phosphorylation. Although estrogen receptors are generally considered markers for good prognosis, phosphorylated estrogen receptors may identify breast tumors with poorer prognosis because of activation of kinases. Correlating estrogen receptor phosphorylation with activation of specific kinases may help identify pathways that contribute to tamoxifen-resistant, phosphorylation-dependent proliferation of breast tumor cells in different ethnic groups. Knowing which pathways are activated may provide information needed to choose drugs that more effectively treat tumors in African-American breast cancer patients.