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的空白。长期目标是了解如何使用曲坦类衍生物通过5-HT1B/1D受体信号传导操纵eEF-2K，以达到治疗乳腺癌的目的。我们的目标是在体外建立曲坦类药物5-壬基色胺（5-NT）的作用机制，并验证其在体内是否通过相同的机制起作用。核心假设是5-NT通过乳腺癌细胞中过度表达的5-HT1B/1D受体表现出强大的抗乳腺癌活性。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致瘤信号通路施加与在体外观察到的相同的负面影响，有效地抑制体内原发性和转移性肿瘤的生长。我们的假设是基于我们的初步数据，这些数据表明，在乳腺癌细胞中，sirna介导的eEF-2K下调可诱导细胞凋亡，抑制细胞增殖和迁移。此外，包括mTOR、cMyc、IGF-1R和Src在内的关键信号蛋白下调。值得注意的是，我们发现5-NT下调eEF-2K，并且在体外和体内乳腺癌模型中表现出与通过siRNA靶向eEF-2K相似的效果。本研究的基本原理是，一旦阐明5- nt阻碍乳腺癌细胞增殖的机制，将有效制定5- HT1B/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具体目标来验证：1)描述5-NT在乳腺癌细胞中的作用机制；2)研究5-NT纳米脂质体的药代动力学和药效学特性，为优化其体内抗肿瘤活性提供指导。在第一个目标下，5-HT1B/1D水平将被调节，以确定其在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的生物标志物（如磷酸化- mtor）的水平。第二个目标是利用中性纳米脂质体的一种新的药物递送技术，将用于5-NT的递送，随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的eeff - 2k， mTOR和促瘤性信号通路。这项拟议的研究具有创新性，因为它专注于使用曲坦5-NT的一种全新的癌症治疗机制，该机制可以下调几种关键的促肿瘤激酶，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针对乳腺癌的新颖且急需的方法。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 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.