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
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.
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| Tavares, Clint D J; Devkota, Ashwini K; Dalby, Kevin N et al. (2016) Application of Eukaryotic Elongation Factor-2 Kinase (eEF-2K) for Cancer Therapy: Expression, Purification, and High-Throughput Inhibitor Screening. Methods Mol Biol 1360:19-33 |
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| Devkota, Ashwini K; Warthaka, Mangalika; Edupuganti, Ramakrishna et al. (2014) High-throughput screens for eEF-2 kinase. J Biomol Screen 19:445-52 |
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| Zhan, Xuanzhi; Kaoud, Tamer S; Kook, Seunghyi et al. (2013) JNK3 enzyme binding to arrestin-3 differentially affects the recruitment of upstream mitogen-activated protein (MAP) kinase kinases. J Biol Chem 288:28535-47 |
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