Prevention of Mammary Cancer in Her-2neu Transgenic Mice

描述（由申请人提供）：抗癌药物的作用机制的详细知识是根据其功能设计临床试验的必要条件。在我们过去3年的工作中，我们专注于了解四硫钼酸盐（一种降铜剂）如何抑制肿瘤生长和血管生成。我们一开始就推测，由于铜参与了几种血管生成因子的分泌和功能，铜缺乏可能会对抑制肿瘤血管生成产生相当全面的、可能是普遍的影响。我们在乳腺癌、前列腺癌、肺癌和鳞状细胞癌方面的工作为支持铜缺乏的全球性和普遍性影响提供了重要证据。具体来说，我们之前在体外和体内乳腺癌模型上的研究表明，铜缺乏对NFkappaB激活的抑制是一个关键的致病事件。最近，许多实验室对NFkappaB系统的各种组成部分的功能有了大量的了解，在这里，我们建议详细定义铜缺乏如何抑制癌细胞和正常及肿瘤相关内皮细胞中NFkappaB的激活。我们的目标是将铜缺乏对NFkappaB关键激活因子（如IL-1）分泌的抑制作用与NFkappaB激活及其随后靶基因（TNF, IL-6, IL-8, lAP, MMPs）合成的内在抑制分离开来。我们的总体经验假设是，铜缺乏通过改变因子定位于细胞核后的转录激活来影响NFkappaB的激活。在NFkappaB的激活过程中，从组分的合成到DNA结合和转录激活，铜都可以发挥作用。我们还假设，由于大体积肿瘤中铜缺乏引起的生长停滞，NFkappaB活性的抑制也延伸到肿瘤细胞侵袭的关键介质，如膜1型基质金属蛋白酶（MT1-MMPs）。此外，在这篇修订后的申请中，我们考虑了另一种假设，即铜缺乏可能通过erk信号的改变介导AP-1和SP-1转录因子的影响。为了验证这些机制假设并继续详细描述铜缺乏的作用，我们提出以下具体目标：1)了解铜缺乏干扰乳腺癌和其他癌症体外模型中TNFalpha和IL-1诱导的NFkappaB激活的信号水平以及铜缺乏对AP-1和SP-1转录的影响。2)描述NFkappaB激活与肿瘤条件内皮细胞微管生长之间的相互作用。A)研究暴露于肿瘤条件下的内皮细胞中肿瘤细胞诱导原始血管生长的过程中铜的依赖性；B)将缺铜环境中IL-1分泌减少的影响与缺铜对NFkappaB激活的潜在内在抑制分离开来。3)研究缺铜对Her2/neu转基因小鼠（高NFkappaB活性）和wnt转基因小鼠（正常NFkappaB活性）早期肿瘤和导管增生及早期肿瘤病变的影响。确定在肿瘤病变、内皮和基质成分中铜缺乏作用的关键介质的表达变化。了解TM是否在her2和wnt转基因肿瘤中调控uPAR表达和erk信号。

DESCRIPTION (provided by applicant): Detailed knowledge of the mechanism of action of anti-cancer drugs is a requirement for the design of clinical trials tailored to their function. In our work conducted over the past 3 years, we have focused on understanding how tetrathiomolybdate, a copper lowering agent, inhibits tumor growth and angiogenesis. We surmised at the outset that since copper is involved in the secretion and function of several angiogenic factors, copper deficiency would have a fairly global, possibly general effect of inhibiting angiogenesis in tumors. Our work in breast cancer, prostate cancer, lung cancer, and squamous cell cancer provide important evidence in support of the global and generalizable effects of copper deficiency. Specifically, our previous work under this grant on in vitro and in vivo models of breast cancer has strongly implicated inhibition of NFkappaB activation by copper deficiency as a key causative event. Armed with a recent explosion of knowledge from many laboratories on the function of various components of the NFkappaB system, here we propose to define in detail how copper deficiency inhibits NFkappaB activation in cancer cells and in normal and tumor associated endothelial cells. We aim to separate the inhibitory effects of copper deficiency on the secretion of key activators of NFkappaB, such as IL-1 from the intrinsic inhibition of NFkappaB activation and subsequent synthesis if target genes (TNF, IL-6, IL-8, lAP, MMPs). Our overarching, empirical hypothesis is that copper deficiency affects NFkappaB activation by altering the activation of transcription after the factors are localized to the nucleus. There are many steps in the process of activation of NFkappaB, from synthesis of the components to DNA binding and transcription activation where Cu could play a role. We also postulate that due to the growth arrest elicited by copper deficiency in bulky tumors, the inhibition of NFkappaB activity also extends to key mediators of tumor cell invasion, such as membrane type 1 matrix metalloproteases (MT1-MMPs). In this revised application, in addition, we consider the alternative hypotheses that Cu deficiency has effects on the AP-1 and SP-1 transcription factors, possibly mediated by alterations of erk signaling. In order to test these mechanistic hypotheses and continue to delineate the action of copper deficiency in detail, we propose the following specific aims: 1) Understand the signaling level at which copper deficiency interferes with TNFalpha and IL-1- induced NFkappaB activation in in vitro models of breast and other cancers and the effects of Cu deficiency on AP-1 and SP-1 transcription. 2) Delineate the interaction between NFkappaB activation and microtubular outgrowth in tumor conditioned endothelial cells. A) Investigate the copper dependence of the process whereby tumor cells elicit outgrowth of primordial vessels in endothelial cells exposed to a tumor-conditioned milieu; B) Separate the effects of decreased IL-1 secretion in a copper poor environment from the potential intrinsic inhibition of NFkappaB activation by copper deficiency. 3) Investigate the molecular surrogates of copper deficiency effects in incipient tumor and ductal hyperplastic and early tumor lesions in Her2/neu transgenic mice (high NFkappaB activity), wnt transgenic mice (normal NFkappaB activity). Define changes in expression of key mediators of the action of copper deficiency in the tumor lesions, the endothelium, and the stromal components. Understand whether TM regulates uPAR expression and erk signaling in her2 and wnt transgenic tumors.