Novel Therapeutics for Translation Control in Breast Cancer

描述（由申请人提供）：在理解真核延伸因子2激酶（eEF-2K）如何增强乳腺肿瘤细胞存活和增殖方面存在根本性差距。长期目标是了解如何使用曲坦衍生物通过5-HT 1B/1D受体信号传导来操纵eEF-2K，以治疗乳腺癌。这里的目标，这是追求这一目标的下一步，是建立5-壬基色胺（5-NT），曲坦，在体外的作用机制，并验证它通过相同的机制在体内发挥作用。中心假设是5-NT通过5-HT 1B/1D受体（其在乳腺癌细胞中过表达）的信号传导而表现出有效的抗乳腺肿瘤活性。这导致eEF-2K的下调，这反过来又负调节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发生信号通路施加与体外观察到的相同的负面影响，有效地抑制了体内原发性和转移性肿瘤的生长。该假设是基于我们的初步数据，这表明在乳腺癌细胞中，siRNA介导的下调eEF-2K诱导凋亡，抑制细胞增殖和迁移。此外，包括mTOR cMyc、IGF-1 R和Src在内的关键信号蛋白被下调。值得注意的是，我们发现5-NT下调eEF-2K，并在体外和体内乳腺癌模型中表现出与siRNA靶向eEF-2K相似的效果。这项研究的基本原理是，一旦阐明了5-NT阻碍乳腺癌细胞增殖的机制，将有效地制定5-HT 1B/1D的分子靶向策略。在强有力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进行测试：1）描述5-NT在乳腺癌细胞中的作用机制; 2）研究药代动力学和药效学特性，作为优化携带5-NT的纳米脂质体体内抗肿瘤活性的指导。在第一个目标下，将调节5-HT 1B/1D水平以确定它们在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志物（如磷酸化mTOR）的水平。在第二个目标下，一种利用中性纳米脂质体的新型但已证实的药物递送技术将用于5-NT递送，然后在裸鼠体内原位异种移植乳腺癌模型中分析转移性人乳腺肿瘤中的eEF-2K，mTOR和促肿瘤发生信号通路。这项研究具有创新性，因为它专注于使用曲坦5-NT的全新癌症治疗机制，该机制可以下调几种关键的促肿瘤发生激酶，包括eEF-2K，C-Src，IGF-1 R和mTOR。这项研究意义重大，因为它将首次允许开发针对乳腺癌的新的和急需的方法。1 公共卫生相关性：我们最近的工作表明，曲坦类药物可以促进乳腺癌细胞的反应，导致其死亡。本提案中的工作旨在了解这种反应，并开发这种药物作为乳腺癌治疗的用途。拟议的研究将具有重要意义，并将有益于公众健康。

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 PUBLIC HEALTH RELEVANCE: Our recent work indicates that a triptan drug can promote responses in breast cancer cells that lead to their death. The work in this proposal aims to understand this response and to develop the use of this drug as a breast cancer therapy. The studies proposed will be of high significance and will benefit public health.