P53 suppresses ribonucleotide reductase via inhibiting mTORC1.

Zhengfu He,Changxian Shen,Adrienne Dorrance,Ramiro Garzon,Weijin Liu,Xing Hu,Peter J. Houghton

doi:10.18632/oncotarget.17440

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.

Highlights

Increased or imbalanced deoxyribonucleotides lead to genome instability, a hallmark of cancer cells [1], while decreased dNTP level impairs cell survival [2,3,4]
As mammalian target of rapamycin complex 1 (mTORC1) is a downstream effector of the mTORC2-AKT signaling pathway [19], our data clearly indicate that pharmacological inhibition of mTORC1 is sufficient to reduce RRM1 and RRM2 regardless of the its negative feedback activation of the upstream AKT signaling, and suggest that mTORC1/eukaryotic translational initiation factor 4E (eIF-4E) cap-dependent protein translation may be required to maintain the protein levels of RRM1 and RRM2 (Figure 2A)
In comparison to wild type MEFs, 4E-BP DKO MEFs demonstrated elevated RRM1 and RRM2, but not p53R2 (Figure 2B). These data suggest that mTORC1/eIF-4E cap-dependent protein translation plays an important role in the control of both RRM1 and RRM2 (Figure 2A)

Summary

Introduction

Increased or imbalanced deoxyribonucleotides (dNTPs) lead to genome instability, a hallmark of cancer cells [1], while decreased dNTP level impairs cell survival [2,3,4]. The mammalian RNR www.impactjournals.com/oncotarget is composed of two identical ribonucleotide reductase large subunit 1 (RRM1) and two small subunits of either RRM2 or p53R2. Balanced dNTPs pools are essential for the accurate and efficient DNA synthesis for replication and repair, defects of which lead to cell death, genome instability, or anti-cancer drug resistance [3,4,5]. Proliferating tumor cells encounter frequent metabolic stress due to the transient and long-term lack of nutrients, oxygen, and growth factors [1, 7, 8]; the mechanisms by which cancer cells maintain the activity of RNR, and thereby the level of dNTPs under ever-changing microenvironment is not fully understood

Methods

Results

Conclusion