The long-range objective of this proposal is to develop non-invasive methods for predicting and evaluating the response of breast cancer to hormonal therapy, by means of magnetic resonance imaging and spectroscopy. Based on the results obtained in the last supported period, we propose to continue investigating the molecular basis for estrogen-induced changes in angiogenesis and glycolysis. Specifically, we would like to test the hypothesis that estrogen interaction with the estrogen receptor alpha (ERalpha) activates the oncogenic nuclear transcription factor c-myc, which in turn down-regulates the expression of vascular endothelial growth factor (VEGF) and up-regulates the expression of glucose transporter 1 (GLUT1), thereby affecting tumor perfusion and glucose transport and metabolism through glycolysis, Furthermore, in this proposal we describe for the first time a plan to develop a new, quantitative and noninvasive approach to evaluate the level of estrogen receptors in breast cancer by means of molecular imaging. Accordingly, we intend to synthesize and test tamoxifen-derived and estrogen-derived steroidal metal-complexes that fluoresce or modify MR parameters of exchanging water, as well as peptides conjugated to fluorescent or MRI probes that bind specifically to the ligand bound ERalpha. The experimental integration of molecular Biology and MRI will involve studies of human breast cancer cells (ERalpha+-positive and ERalpha-negative for comparison), including a newly developed ERalpha+-positive clone of HCF7 cells, 35im, that is harboring a stably transfected human c-myc gene, whose expression is stringently controlled by the bacterial reverse tetracycline transcription activator protein. We will proceed to investigate the orthotopic tumors of these cells implanted in mice (immunodeficient or sever compromised immunodeficient mice), under varying hormonal manipulation by estrogen and antiestrogens.
The specific aims are designed to test the above hypothesis at the molecular, cellular and whole tumor levels and to develop and test targeted probes for molecular imaging of the estrogen receptor. The experimental protocols include utilizing molecular and immunohistochemical methods that will characterize c-myc expression in conjunction with VEGF and GLUT1 expression, under varying hormonal treatments of cells and tumors. In addition, efforts will be devoted to develop new, non invasive MRI and MRS methods that would enable monitoring hormonal induced changes in the vasculature properties as well as glucose transport and metabolism. The synthesis of the estrogen receptor targeted probes was already initiated. The new probes will tested in vitro and in vivo for their binding specificity to ERalpha, the transport parameters into the cells and the pharmacological and toxicological effects on cells and whole animals. Prediction based on the presence of about 1000 fmol/mg protein of the receptor (the level in MCF7 cells) indicated a small but measurable change in T1 and T2*. This work will add to the basic understanding of the hormonal regulation of breast cancer and may also help design and improve new targets for anti-estrogenic and anti-angiogenic therapy of breast cancer. The methods of imaging functional estrogen activities may serve as a basis for measuring the efficacy of new selective estrogen receptor modulators in animal models, The current proposal will also bring us closer to the capacity of imaging, non-invasively, both the estrogen receptor level and the parameters demonstrating its functional activity. This, in turn, may improve significantly the assessment of prognosis and the management of the approximate 75% of the breast cancer patients with ER-positive tumors.
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