cells Article Dihydroartemisinin Inhibits mTORC1 Signaling by Activating the AMPK Pathway in Rhabdomyosarcoma Tumor Cells Jun Luo 1,2,† , Yoshinobu Odaka 1,†,‡ , Zhu Huang 1,3,† , Bing Cheng 1 , Wang Liu 1 , Lin Li 1 , Chaowei Shang 1 , Chao Zhang 1,4,5 , Yang Wu 1,6 , Yan Luo 1,6 , Shengyong Yang 6 , Peter J. Houghton 7 , Xiaofeng Guo 2, * and Shile Huang 1,8, *   Citation: Luo, J.; Odaka, Y.; Huang, Z.; Cheng, B.; Liu, W.; Li, L.; Shang, C.; Zhang, C.; Wu, Y.; Luo, Y.; et al. Dihydroartemisinin Inhibits mTORC1 Signaling by Activating the AMPK Pathway in Rhabdomyosarcoma Tumor Cells. Cells 2021, 10, 1363. https://doi.org/10.3390/cells10061363 Academic Editors: Stephen Yarwood and Alexander E. Kalyuzhny Received: 19 March 2021 Accepted: 29 May 2021 Published: 1 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA; junluo@scau.edu.cn (J.L.); odakayu@ucmail.uc.edu (Y.O.); huangzhu@xmu.edu.cn (Z.H.); bing.cheng@lsuhs.edu (B.C.); wliu6@kumc.edu (W.L.); lin.li@lsuhs.edu (L.L.); chaowei.shang@lsuhs.edu (C.S.); zhangchao@ahmu.edu.cn (C.Z.); wuyang@scu.edu.cn (Y.W.); yan.luo@mayo.edu (Y.L.) 2 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China 3 Research Center of Aquatic Organism Conservation and Water Ecosystem Restoration in Anhui Province, Anqing Normal University, Anqing 246011, China 4 Key Laboratory of National Health and Family Planning Commission on Parasitic Disease Control and Prevention, Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China 5 Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China 6 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China; yangsy@scu.edu.cn 7 Greehey Children’s Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX 78229-3000, USA; houghtonp@uthscsa.edu 8 Department of Hematology and Oncology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA * Correspondence: xfguo@scau.edu.cn (X.G.); shile.huang@lsuhs.edu (S.H.); Tel.: +86-20-38295980 (X.G.); +1-318-675-7759 (S.H.) † These authors contributed equally to this work. ‡ Current address: Biology Department, University of Cincinnati Blue Ash College, Blue Ash, OH 45236, USA. Abstract: Dihydroartemisinin (DHA), an anti-malarial drug, has been shown to possess potent anti- cancer activity, partly by inhibiting the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling. However, how DHA inhibits mTORC1 is still unknown. Here, using rhabdomyosarcoma (RMS) as a model, we found that DHA reduced cell proliferation and viability in RMS cells, but not those in normal cells, which was associated with inhibition of mTORC1. Mechanistically, DHA did not bind to mTOR or FK506 binding protein 12 (FKBP12). In addition, DHA neither inhibited insulin-like growth factor-1 receptor (IGF-1R), phosphoinositide 3-kinase (PI3K), and extracellular signal-regulated kinase 1 2 (Erk1/2), nor activated phosphatase and tensin homolog (PTEN) in the cells. Rather, DHA activated AMP-activated protein kinase (AMPK). Pharmacological inhibition of AMPK, ectopic expression dominant negative or kinase-dead AMPK, or knockdown of AMPKα at- tenuated the inhibitory effect of DHA on mTORC1 in the cells. Additionally, DHA was able to induce dissociation of regulatory-associated protein of mTOR (raptor) from mTOR and inhibit mTORC1 activity. Moreover, treatment with artesunate, a prodrug of DHA, dose-dependently inhibited tumor growth and concurrently activated AMPK and suppressed mTORC1 in RMS xenografts. The results indicated that DHA inhibits mTORC1 by activating AMPK in tumor cells. Our finding supports that DHA or artesunate has a great potential to be repositioned for treatment of RMS. Keywords: dihydroartemisinin; rhabdomyosarcoma; mTOR; AMPK; PTEN; raptor 1. Introduction Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma, which often occurs in the head, neck, bladder, vagina, uterus, arms, legs, and trunk [1]. Approximately Cells 2021, 10, 1363. https://doi.org/10.3390/cells10061363 https://www.mdpi.com/journal/cells