S9, a Novel Anticancer Agent, Exerts Its Anti-Proliferative Activity by Interfering with Both PI3K-Akt-mTOR Signaling and Microtubule Cytoskeleton Chao Zhang1., Na Yang1., Chun-hao Yang 2 , Hua-sheng Ding 2 , Cheng Luo 3 , Yu Zhang 3 , Mao-jiang Wu 2 , Xiong-wen Zhang 1 , Xu Shen3,4, Hua-liang Jiang3,4, Ling-hua Meng1*, Jian Ding 1 * 1 Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China, 2 Division of Organic Synthesis, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China, 3 Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China, 4 School of Pharmacy, East China University of Science and Technology, Shanghai, China Abstract Background: Deregulation of the phosphatidylinositol 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a central role in tumor formation and progression, providing validated targets for cancer therapy. S9, a hybrid of a-methylene-c-lactone and 2-phenyl indole compound, possessed potent activity against this pathway. Methodology/Principal Findings: Effects of S9 on PI3K-Akt-mTOR pathway were determined by Western blot, immunofluorescence staining and in vitro kinas assay. The interactions between tubulin and S9 were investigated by polymerization assay, CD, and SPR assay. The potential binding modes between S9 and PI3K, mTOR or tubulin were analyzed by molecular modeling. Anti-tumor activity of S9 was evaluated in tumor cells and in nude mice bearing human cancer xenografts. S9 abrogated EGF-activated PI3K-Akt-mTOR signaling cascade and Akt translocation to cellular membrane in human tumor cells. S9 possessed inhibitory activity against both PI3K and mTOR with little effect on other tested 30 kinases. S9 also completely impeded hyper-phosphorylation of Akt as a feedback of inhibition of mTOR by rapamycin. S9 unexpectedly arrested cells in M phase other than G1 phase, which was distinct from compounds targeting PI3K-Akt-mTOR pathway. Further study revealed that S9 inhibited tubulin polymerization via binding to colchicine-binding site of tubulin and resulted in microtubule disturbance. Molecular modeling indicated that S9 could potentially bind to the kinase domains of PI3K p110a subunit and mTOR, and shared similar hydrophobic interactions with colchicines in the complex with tubulin. Moreover, S9 induced rapid apoptosis in tumor cell, which might reflect a synergistic cooperation between blockade of both PI3-Akt-mTOR signaling and tubulin cytoskeleton. Finally, S9 displayed potent antiproliferative activity in a panel of tumor cells originated from different tissue types including drug-resistant cells and in nude mice bearing human tumor xenografts. Conclusions/Significance: Taken together, S9 targets both PI3K-Akt-mTOR signaling and microtubule cytoskeleton, which combinatorially contributes its antitumor activity and provides new clues for anticancer drug design and development. Citation: Zhang C, Yang N, Yang C-h, Ding H-s, Luo C, et al. (2009) S9, a Novel Anticancer Agent, Exerts Its Anti-Proliferative Activity by Interfering with Both PI3K- Akt-mTOR Signaling and Microtubule Cytoskeleton. PLoS ONE 4(3): e4881. doi:10.1371/journal.pone.0004881 Editor: Xiaoping Pan, Western Illinois University, United States of America Received October 30, 2008; Accepted January 22, 2009; Published March 18, 2009 Copyright: 2009 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Knowledge Innovation Program of Chinese Academy of Sciences (KSCX2-YW-R-25), National Natural Science Foundation of China (30721005, 90713034), Science and Technology Commission of Shanghai Municipality Pujiang Talent Program (08PJ14114), Grant 07dz05906. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lhmeng@mail.shcnc.ac.cn (L-hM); jding@mail.shcnc.ac.cn (JD) . These authors contributed equally to this work. Introduction Phosphatidylinositol 3-kinase (PI3K) plays a central role in a complex, multi-armed signaling network that orchestrates cell response including cell survival, growth, proliferation, angiogen- esis, migration and glucose metabolism [1,2]. PI3K is presumed to activate most of its downstream targets via the Akt protein, which phosphorylates diversified downstream substrates including the mammalian target of rapamycin (mTOR) [2], a master regulator of protein translation. Moreover, PI3K and its downstream components cross-talk with a number of other pathways, such as the Raf/mitogen-activated protein kinase kinase/extracellular signal–regulated kinase (MEK/ERK)–activated pathways [3]. PI3K-mediated signaling network is important in tumorigenesis and progression, which is frequently hyper-activated in a variety of human cancers, as a result of gain-of-function in PI3K, inactivation of the lipid phosphatase PTEN or deregulated growth factor signaling [1,3,4]. As such, the PI3K-Akt-mTOR pathway has been validated as a tumor therapeutic target of increasing interests. Multiple efforts are under way in academia and industry to develop clinically relevant inhibitors of this signaling pathway. Temsirolimus (ToriselH, Wyeth pharmaceuticals), an analog of rapamycin targeting mTOR [5], represents the most successful PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4881