Research Grants Awarded
Role Of Coup-Tfii In Endocrine-Responsive And Resistant Breast Cancer
Investigator Initiated Research
Ablation of estrogen receptor (ER) signaling by aromatase inhibitors to block estrogen production and by ER antagonists, e.g., tamoxifen (TAM), Raloxifene (Ral), and Faslodex, to block ER?s transcriptional activity are used successfully in the treatment and prevention of breast cancer. However, ~40% of patients initially antiestrogen-responsive develop tumors that are endocrine-resistant and the disease progresses to metastatic disease with poor prognosis. The mechanisms for the development of endocrine-resistance in ERalpha-positive tumors is complex and involves multiple pathways. We recently reported that the protein and mRNA expression of COUP-TFII, an orphan nuclear receptor, is reduced in antiestrogen-resistant breast cancer cell lines. Further, re-expression of COUP-TFII in resistant cells increased TAM inhibition of cell proliferation, decreased motility, and increased apoptosis. Conversely, knockdown of COUP-TFII expression in MCF-7 antiestrogen-responsive breast cancer cells decreased the ability of 4-OHT to inhibit cell proliferation. These support the overall hypothesis that COUP-TFII plays a role in antiestrogen responses in sensitive breast cancer cells and that reduced COUP-TFII expression plays a role in acquired endocrine-resistance in human breast cancer. Thus, this study directly addresses the Komen Grants Program?s Annual Research Focus, i.e., ?breast cancer biology; biomarkers of risk, disease burden, and/or treatment response or resistance?. The proposed project follows up these novel observations with 4 Specific Aims. AIM 1 is to determine the expression of COUP-TFII in breast tumors and normal breast tissue. This is a translational study to move from basic science into hopeful clinical utility. This Aim will be performed in collaboration with Dr. Peter Kulesza who will perform immunohistochemistry to examine COUP-TFII in 100 archival tumors in a retrospective study using samples with known TAM resistance/sensitivity. We will correlate COUP-TFII expression with other markers with the goal of determining if COUP-TFII will serve as a useful prognostic marker for tumors sensitive versus resistant to endocrine therapy. Since re-expression of COUP-TFII to ?normal? levels in endocrine resistant breast cancer cells re-sensitizes them to TAM inhibition, we need to understand the mechanism by which COUP-TFII supports TAM?s inhibitory activity. AIM 2 is to determine the identity of proteins that interact with COUP-TFII and to examine roles and expression in endocrine-sensitive versus resistant breast cancer cells. This Aim tests the hypothesis that COUP-TFII is a member of a network of proteins that are involved in endocrine responsiveness in breast cancer. The experimental approach uses state-of-art proteomic approaches. FLAG-tagged COUP-TFII will be expressed in MCF-7 endocrine-sensitive and LCC9 endocrine-resistant breast cancer cells. COUP-TFII-interacting proteins will be purified by two parallel immunoaffinity methods: 1) anti-FLAG affinity resin and 2) DSS-crosslinking of our own COUP-TFII antibody to Protein G agarose beads. Eluted COUP-TFII interacting proteins will be digested with tyrpsin and peptides will be sequenced and proteins identified using an approach that combines two-dimensional capillary-scale liquid chromatography (2D-LC) peptide separation with tandem mass spectrometry (MS/MS). This 2D-LC-MS/MS methodology allows a direct, unbiased and high-sensitive identification of hundreds to thousands of individual proteins from virtually any type of complex biological sample. COUP-TFII interactions in endocrine-sensitive and endocrine-resistant breast cancer cells will be systematically compared using an in-house web-based program (http://plato.kdp.louisville.edu/BIGCAT/index.php). This method has been optimized and preliminary data from 2D-LC-MS/MS and confirmed by co-immunoprecipitation indicate COUP-TFII interacts with nucleolin and Poly(ADP-ribose) polymerase (PARP) in endocrine/antiestrogen-sensitive MCF-7 breast cancer cells. AIM 3 is to validate the detected COUP-TFII-protein interactions in breast cancer cell lines and in clinical samples. These experiments will employ coimmunoprecipitation, western blot analysis, and colocalization by immunohistochemical staining. AIM 4 is to determine the role of candidate interactors in relevant in vitro functional assays. Our long term hope is that COUP-TFII may be a biomarker for early identification of breast tumors that are likely to develop endocrine resistance and thus could be used prognostically to identify patients for more aggressive therapies to prevent recurrent disease.
We are all aware of the importance of estrogen signaling and estrogen receptor (ER, particularly ERalpha) expression in breast cancer initiation and progression. Indeed, ablation of estrogen action, both by blocking its interaction with ERalpha by antiestrogens and by inhibiting conversion of peripheral androgens to estrogens, is the key treatment for early breast cancer. However, a great concern for women with breast cancer is that ~ 40% of initially ERalpha positive tumors become resistant to, or are stimulated to grow by, tamoxifen, raloxifene, and other antiestrogens. Although much studied, the mechanisms for endocrine resistance in human breast cancer remain unknown. In fact, breast cancer cells appear to have multiple mechanisms that are involved in endocrine resistance and that this is a complex problem needing focused effort. The research described in this application continues work previously funded by the Komen Grant Program in which we discovered that the levels of a protein called COUP-TFII were decreased in breast cancer cells that are antiestrogen-resistant. Further, we reported that if we re-introduced COUP-TFII into endocrine-resistant cells and treated them with tamoxifen, the cells were again inhibited by tamoxifen! Funding of this grant will allow us to confirm these observations at the protein level in human breast tumors. Thus, Aim 1 is a translational aim to move our observation on the decrease in COUP-TFII from basic science in breast cancer cells into hopeful clinical utility in breast tissue samples. This Aim will be performed in collaboration with Peter Kulesza, M.D./Ph.D., a pathologist, who will perform immunohistochemistry to examine COUP-TFII in 100 archival tumors in a retrospective study using samples with known TAM resistance/sensitivity. These experiments will be the first to examine COUP-TFII expression in correlation with other markers of clinical responses, e.g., ERalpha, PR, HER2, lymph node status, EIN, with the goal of determining if COUP-TFII will serve as a useful prognostic marker for tumors sensitive versus resistant to endocrine therapy. We need to know how COUP-TFII works to make breast tumor cells responsive to antiestrogens because if we can keep patients from developing endocrine resistance, we would prevent relapse. The experiments in Aims 2-4 will reveal new mechanistic insights into HOW COUP-TFII acts in breast cancer cells to allow them to be growth inhibited and driven to apoptosis when treated with tamoxifen. In addition, we will determine if the proteins that we identify in a cell model system that mimics the development of antiestrogen resistance also interact in human breast tumor samples. These experiments use state-of-the art and novel proteomic approaches. It is now widely appreciated that proteomics has the potential to revolutionize cancer diagnosis and management. The studies described in our application will move research into the ?post-genomic? era of investigation in breast cancer research. Our prediction is that COUP-TFII will be useful for early identification of breast tumors that are likely to develop endocrine resistance. This would have a major impact on prognosis, and more importantly on therapeutics in the most immediate sense. We hope that the basic scientific progress outlined on our proposal will enable biomarker-based use of anti-estrogens in the clinic. We believe that this is especially necessary to fully utilize the curative potential of these drugs. Further, understanding how COUP-TFII functions in concert with other proteins in an interactive pathway is important in understanding clinical responses to antiestrogens and may provide new targets for therapeutic intervention, specifically at early stages of disease.