Novel Therapeutics for Translation Control in Breast Cancer

描述(由申请人提供)：在理解真核延长因子2激酶(EEF-2K)如何促进乳腺肿瘤细胞存活和增殖方面存在着根本的空白。长期目标是了解如何使用Triptan衍生物通过5-HT1B/1D受体信号来操纵EEF-2K，从而达到治疗乳腺癌的目的。这里的目标是建立雷公藤多苷的体外作用机制，并验证其在体内是否通过相同的机制发挥作用。中心假设是，5-NT通过在乳腺癌细胞中过度表达的5-HT1B/1D受体发出信号，从而显示出强大的抗乳腺肿瘤活性。这导致EEF-2K的下调，进而负向调节mTOR。我们还假设，携带5-NT的中性电荷纳米脂质体通过对关键的促肿瘤信号通路产生与体外观察相同的负面影响，有效地抑制体内原发和转移肿瘤的生长。这一假说是基于我们的初步数据提出的，这些数据表明，在乳腺癌细胞中，siRNA介导的EEF-2K下调诱导了细胞凋亡，并抑制了细胞的增殖和迁移。此外，关键信号蛋白包括mTOR cMyc、IGF-1R和Src下调。值得注意的是，我们发现，在体外和体内乳腺癌模型中，5-NT下调EEF-2K，并显示出与siRNA靶向EEF-2K相似的效果。这项研究的理论基础是，一旦阐明了5-NT阻碍乳腺癌细胞增殖的机制，就可以有效地制定5-HT1B/1D的分子靶向策略。在强大的初步数据的指导下，我们的假设将通过追求两个特定的目标来验证：1)描述5-NT在乳腺癌细胞中的作用机制；2)研究5-NT的药代动力学和药效学性质，以指导优化携带5-NT的纳米脂质体的体内抗肿瘤活性。在第一个目标下，5-HT1B/1D水平将被调节，以确定它们在5-NT抗肿瘤活性和EEF-2K稳定性中的作用。将分析蛋白质翻译的调节以及潜在生物标记物的水平，如磷酸化mTOR。在第二个目标下，一种利用中性纳米脂质体的新型药物传递技术将用于5-NT的传递，随后将在裸鼠原位移植乳腺癌模型中分析转移性人乳腺肿瘤的EEF-2K、mTOR和促肿瘤信号通路。这项拟议的研究具有创新性，因为它专注于一种全新的使用Triptan 5-NT治疗癌症的机制，该机制发出信号下调几个关键的促肿瘤激酶，包括EEF-2K、C-Src、IGF-1R和mTOR。这项研究意义重大，因为它将首次允许开发新的和迫切需要的方法来靶向乳腺癌。1

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how eukaryotic elongation factor 2 kinase (eEF-2K) enhances breast tumor cell survival and proliferation. The long-term goal is to understand how to manipulate eEF-2K through 5-HT1B/1D receptor signaling using triptan derivatives with the therapeutic purpose of treating breast cancer. The objective here, which is the next step in pursuit of that goal, is to establish the mechanism of action of 5-nonylytryptamine (5-NT), a triptan, in vitro, and to validate that it functions via the same mechanism in vivo. The central hypothesis is that 5-NT exhibits potent anti- breast tumor activity by signaling through the 5-HT1B/1D receptors, which are over-expressed in breast cancer cells. This leads to the down-regulation of eEF-2K, which in turn negatively modulates mTOR. We also hypothesize that neutrally charged nanoliposomes carrying 5-NT effectively curb the growth of primary and metastatic tumors in vivo by exerting the same negative effects on critical pro-tumorigenic signaling pathways as those observed in vitro. The hypothesis was formulated based on our preliminary data, which indicate that in breast cancer cells, the siRNA-mediated down-regulation of eEF-2K induces apoptosis, and inhibits cell proliferation and migration. Additionally, critical signaling proteins including mTOR cMyc, IGF-1R and Src are down-regulated. Significantly, we found that 5-NT down-regulates eEF-2K and exhibits similar effects to targeting eEF-2K by siRNA in both in vitro and in vivo breast cancer models. The rationale for the proposed research is that once the mechanism by which 5-NT hinders breast cancer cell proliferation has been elucidated, strategies for the molecular targeting of 5- HT1B/1D will be effectively formulated. Guided by strong preliminary data, our hypothesis will be tested by pursuing two specific aims: 1) Delineate the mechanism of action of 5-NT in breast cancer cells; and 2) Investigate the pharmacokinetics and pharmacodynamics properties as a guide to optimizing the in vivo antitumor activity of nanoliposomes carrying 5-NT. Under the first aim, 5-HT1B/1D levels will be modulated to ascertain their role in 5-NT antitumor activity and eEF-2K stability. Modulation of protein translation will be analyzed as well as levels of potential biomarkers such as phospho-mTOR. Under the second aim, a novel yet proven drug delivery technique utilizing neutral nanoliposomes, will be used for 5-NT delivery, followed by analysis of eEF-2K, mTOR and pro-tumorigenic signaling pathways in metastatic human breast tumors in an in vivo orthotopic xenograft breast cancer model in nude mice. The proposed research is innovative because it focuses on an entirely novel mechanism of cancer therapy using the triptan 5-NT, which signals the down-regulation of several key pro-tumorigenic kinases including eEF-2K, C-Src, IGF-1R and mTOR. This research is significant because it would allow, for the first time, the development of novel and much needed approaches to target breast cancer. 1