Research Grants Awarded
Refined Prediction of Hormone Response
Background. Resistance to hormonal therapy is a major clinical problem, and we do not currently have accurate predictive assays to reliably identify those women who might benefit from combined endocrine and non-endocrine or gene-targeted therapies. We have discovered that several biomarkers of oxidative stress, which are modulated by reactive oxygen species (ROS), are up-regulated in metastatic tumors that arose while the patients were taking the antiestrogen tamoxifen (Tam). Objective/Hypothesis. We hypothesize that the generation of ROS is an underlying determinant of clinical resistance. We will examine the role of four markers of ROS?MKP3, c-fos, topoisomerase 2B, and p27 in tamoxifen resistance (TR) using three cell line models of resistance. We will also test whether the measurement of these markers are useful to refine and improve decision-making for some ER-positive breast cancer patients. Specific Aims. In Aim 1 we will determine the contribution of MKP3, c-fos, Top2B, and p27 to the resistant phenotype. Overerxpression and siRNA experiments in three models of resistance, and growth as xenografts in athymic nude will be examined. The effects of antioxidant blockage with the antioxidants N-acetylcysteine (NAC) or glutathione (GSH) on resistance and signal transduction will also be examined. In Aim 2 we will determine whether the measurement of c-fos, Top2B, p27, and MKP3 RNA levels enhance the prediction of resistance in a cohort of tumors from treated patients using quantitative (q)RT/PCR multiplex assays. Study Design. Aim 1) Tam-resistant MCF-7 and T47D cells will be stably transfected with siRNAs to MKP3, c-fos, Top2B, and p27. MCF-7 and T47D cells will also be stably transfected with expression vectors to these markers as well, and these engineered lines, and MCF-7-MKP3-overexpressing cells will be tested for anchorage-independent growth and cell cycle parameters. Signal transduction and effects on expression of downstream effectors such as ER, progesterone receptor, cyclin D1, and bcl-2 will be determined using immunoblot analysis. Specific signal transduction and antioxidants will be used to examine effects on growth, cell survival, and response to antiestrogens. Aim 2) will employ multiplex qRT/PCR of these selected markers of ROS in a cohort of women treated with adjuvant Tam. We will evaluate the usefulness of ROS biomarkers to predict outcomes in this cohort. Potential Outcomes and Benefits of the Research. This is a completely novel approach to improve prediction of response, and from which to attack the problem of Tam-resistant metastatic lesions. Outcomes will be new gene targets and new predictive markers for hormonal therapy.
Tamoxifen (Tam) and aromatase inhibitors (AI) are the most frequently prescribed hormonal agents for treatment of primary breast cancer after its diagnosis. AIs and Tam will continue to be offered to patients at some time during their treatment plan as part of a hormonal therapy sequencing strategy. But unfortunately these agents fail in the treatment of many women, and resistance is manifest with the appearance of metastatic lesions. There is now evidence that overexpression of growth factors, their receptors, and downstream effector molecules, such as the Mitogen Activated Protein Kinases (MAPK)?also known as ERK-1 and 2, are involved in the development of Tam resistance (TR). We propose to first study the resistance mechanisms associated with modulation of MAPK ERK1,2 by its regulatory molecule, MAPK phospatase 3 (MKP3) in ER-positive breast cancer cells. But we have also observed that Tam-induction of reactive oxygen species (ROS), a measurement of the level of oxidative stress in cells, is critical for MKP3-mediated resistance in breast cancer cells. Therefore, we hypothesize that the generation of ROS may be an underlying denominator of clinical resistance in women treated with Tam. Thus, we will also study the role of three other selected biomarkers of ROS that we discovered in studying the gene expression profiles of metastatic tumors from women who had been treated with Tam. We will examine if these proteins that are modulated by ROS can indeed predict hormonal resistance, and we will then examine whether determination of their RNA levels can help us refine the clinical prediction of hormone response. It must be stressed that one of the most important questions remaining in hormone therapy of breast cancer today is to establish which ER-positive patients require only endocrine therapy, and then identify those women who might benefit from additional non-endocrine therapies. This proposal is highly relevant to the decision-making problems that women with breast cancer and their physicians face today, and is a high priority issue in breast cancer treatment.