P53 suppresses ribonucleotide reductase via inhibiting mTORC1

// Zhengfu He 1 , Xing Hu 2 , Weijin Liu 2 , Adrienne Dorrance 3 , Ramiro Garzon 3 , Peter J. Houghton 4 and Changxian Shen 3 1 Department of Thoracic Surgery, Sir Run Run Shaw Hospital, College of Medicine Zhejiang University, Hangzhou, Zhejiang Province, China 2 College of Biology and Food Engineering, Huaihua University, Huaihua, Hunan Province, China 3 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA 4 The Greehey Children’s Cancer Research Institute, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA Correspondence to: Changxian Shen, email: shen.637@osu.edu Keywords: mammalian target of rapamycin (mTOR), deoxyribonucleotides (dNTPs), ribonucleotide reductase (RNR), p53, nutlin-3 Received: September 27, 2016      Accepted: April 11, 2017      Published: April 26, 2017 ABSTRACT Balanced deoxyribonucleotides pools are essential for cell survival and genome stability. Ribonucleotide reductase is the rate-limiting enzyme for the production of deoxyribonucleotides. We report here that p53 suppresses ribonucleotide reductase subunit 1 (RRM1) and 2 (RRM2) via inhibiting mammalian target of rapamycin complex 1 (mTORC1). In vitro , cancer cell lines and mouse embryonic fibroblast cells were treated with different concentrations of pharmacological inhibitors for different times. In vivo , rhabdomyosarcoma Rh30 cell tumor-bearing mice were treated with rapamycin or AZD8055. Protein levels and phosphorylation status were assessed by immunoblotting and mRNA levels were determined by real time RT-PCR. Pharmacological inhibition of mTORC1 with rapamycin, mTOR kinase with AZD8055 or protein kinase B with MK2206 resulted in decrease of RRM1 and RRM2 in Rh30 cells both in vitro and in mouse tumor xenografts. Moreover, eukaryotic translational initiation factor 4E-binding proteins 1 and 2 double knockout mouse embryonic fibroblast cells demonstrated an elevation of RRM1 and RRM2. Furthermore, down-regulation of mTOR-protein kinase B signaling or cyclin dependent kinase 4 led to decrease of RRM1 and RRM2 mRNAs. In addition, TP53 mutant cancer cells had elevation of RRM1 and RRM2, which was reduced by rapamycin. Importantly, human double minute 2 inhibitor nutlin-3 decreased RRM1 and RRM2 in TP53 wild type rhabdomyosarcoma Rh18 but not in TP53 mutated Rh30 cells. Our data demonstrated that mTOR enhances the cap-dependent protein translation and gene transcription of RRM1 and RRM2. Our findings might provide an additional mechanism by which p53 maintains genome stability.