MRI and MRS of Hormonal Induced Changes in Breast Cancer

描述(由申请人提供)： 这项建议的长期目标是开发非侵入性方法，通过磁共振成像和光谱分析来预测和评估乳腺癌对激素治疗的反应。基于上一支持阶段的结果，我们建议继续研究雌激素诱导的血管生成和糖酵解变化的分子基础。具体地说，我们想测试一种假设，即雌激素与雌激素受体α(ERpha)的相互作用激活致癌的核转录因子c-myc，进而下调血管内皮生长因子(VEGF)的表达，上调葡萄糖转运体1(GLUT1)的表达，从而通过糖酵解影响肿瘤的灌注和葡萄糖的运输和代谢。此外，在这项建议中，我们首次描述了一项计划，即开发一种新的、定量的、非侵入性的方法，通过分子成像的方法来评估乳腺癌中雌激素受体的水平。因此，我们打算合成和测试他莫昔芬衍生的和雌激素衍生的类固醇金属络合物，它们荧光或改变交换水的MR参数，以及连接到荧光或MRI探针的多肽，这些多肽与配体结合的ERpha特异结合。分子生物学和核磁共振的实验性整合将涉及对人类乳腺癌细胞的研究(对比ERpha+和ERpha阴性)，包括新开发的ERpha+阳性的HCF7细胞克隆35im，该克隆携带稳定转基因的人c-myc基因，其表达受到细菌反向四环素转录激活蛋白的严格控制。我们将继续研究这些细胞在雌激素和抗雌激素的不同激素作用下移植到小鼠(免疫缺陷或严重免疫缺陷小鼠)中的原位肿瘤。具体目的是在分子、细胞和整个肿瘤水平上验证上述假说，并开发和测试雌激素受体分子成像的靶向探针。实验方案包括利用分子和免疫组织化学方法，在细胞和肿瘤的不同激素治疗下，结合血管内皮生长因子和GLUT1的表达来表征c-myc的表达。此外，还将致力于开发新的、非侵入性的MRI和MRS方法，使其能够监测荷尔蒙诱导的血管特性以及葡萄糖运输和代谢的变化。雌激素受体靶向探针的合成已经启动。新的探针将在体外和体内测试它们与ERpha的结合特异性、进入细胞的转运参数以及对细胞和整个动物的药理学和毒理学效应。根据受体约1000fmol/mg蛋白的存在(在MCF7细胞中的水平)的预测表明，T1和T2*有微小但可测量的变化。这项工作将增加对乳腺癌激素调节的基本了解，并可能有助于设计和改进乳腺癌抗雌激素和抗血管生成治疗的新靶点。功能性雌激素活性的成像方法可以作为在动物模型中测量新的选择性雌激素受体调节剂的有效性的基础，目前的提议也将使我们更接近于无创地成像雌激素受体水平和显示其功能活性的参数。反过来，这可能会显著改善大约75%的ER阳性乳腺癌患者的预后评估和治疗。

DESCRIPTION (provided by applicant): 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.