Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma.

Brian Cunniff,Arti Shukla,Alexina C Haynes,Kelly Butnor,Kheng Newick,Bruce Leavitt,Andrew M James,Kimberly J Nelson,Edward T Chouchani,Michael P Murphy,Stacie Beuschel,W Todd Lowther,Andreas Koenig,Anant Bhave,Alexandra N Wozniak,Nicholas H Heintz,Luis Eduardo Soares Netto

doi:10.1371/journal.pone.0127310

Abstract

Dysregulation of signaling pathways and energy metabolism in cancer cells enhances production of mitochondrial hydrogen peroxide that supports tumorigenesis through multiple mechanisms. To counteract the adverse effects of mitochondrial peroxide many solid tumor types up-regulate the mitochondrial thioredoxin reductase 2 - thioredoxin 2 (TRX2) - peroxiredoxin 3 (PRX3) antioxidant network. Using malignant mesothelioma cells as a model, we show that thiostrepton (TS) irreversibly disables PRX3 via covalent crosslinking of peroxidatic and resolving cysteine residues in homodimers, and that targeting the oxidoreductase TRX2 with the triphenylmethane gentian violet (GV) potentiates adduction by increasing levels of disulfide-bonded PRX3 dimers. Due to the fact that activity of the PRX3 catalytic cycle dictates the rate of adduction by TS, immortalized and primary human mesothelial cells are significantly less sensitive to both compounds. Moreover, stable knockdown of PRX3 reduces mesothelioma cell proliferation and sensitivity to TS. Expression of catalase in shPRX3 mesothelioma cells restores defects in cell proliferation but not sensitivity to TS. In a SCID mouse xenograft model of human mesothelioma, administration of TS and GV together reduced tumor burden more effectively than either agent alone. Because increased production of mitochondrial hydrogen peroxide is a common phenotype of malignant cells, and TS and GV are well tolerated in mammals, we propose that targeting PRX3 is a feasible redox-dependent strategy for managing mesothelioma and other intractable human malignancies.

Highlights

Altered redox balance in tumor cells, characterized by an increase in the production of reactive oxygen species (ROS) and changes in antioxidant gene expression, supports a pro-proliferative state and evasion from apoptosis [1]
Treatment of recombinant peroxiredoxin 3 (PRX3) with TS resulted in the dose-dependent formation of rPRX3 species with retarded electrophoretic mobility on reducing SDS-PAGE (Fig 1A, lanes 2–3)
We investigated TS-induced modifications to rPRX3 mutants where specific cysteine residues corresponding to cysteine at position 108 (Cys108), cysteine at position 229 (Cys229) and cysteine at position 127 (Cys127) were replaced with serine

Summary

Introduction

Altered redox balance in tumor cells, characterized by an increase in the production of reactive oxygen species (ROS) and changes in antioxidant gene expression, supports a pro-proliferative state and evasion from apoptosis [1]. Tumor cells escape from redox-dependent cytotoxic responses via loss of tumor suppressor genes and/or up-regulation of antioxidant enzymes and stress response factors, allowing tumor cells to prosper in a pro-oxidative state [9] Because this phenotypic adaptation is not limited to a specific subset of oncogenes and tumor suppressor genes, exploiting perturbations in the metabolism of mitochondrial and cytosolic-derived oxidants has been proposed to be a viable therapeutic target in a variety of human cancers [10,11]. The pro-oxidant state induced in mitochondria by activated K-RAS is counterbalanced through increased expression of Forkhead Box M1 (FOXM1), a redox-responsive transcription factor that promotes expression of the mitochondrial antioxidant enzymes manganese superoxide dismutase (MnSOD or SOD2) and PRX3, permitting cells to escape from ROS-induced senescence [27]. Evaluation of the effects of TS and GV, alone or together, in a SCID mouse xenograft model of human MM indicates combinatorial targeting of the PRX3 antioxidant network is a feasible strategy for managing a wide variety of tumors characterized by dysregulation of mitochondrial metabolism that results in high oxidant production

Results

Discussion

Materials and Methods