Depletion of the Human Ion Channel TRPM2 in Neuroblastoma Demonstrates Its Key Role in Cell Survival through Modulation of Mitochondrial Reactive Oxygen Species and Bioenergetics.
Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in modulating cell survival following oxidant injury and is highly expressed in many cancers including neuroblastoma. Here, in xenografts generated from neuroblastoma cells in which TRPM2 was depleted with CRISPR/Cas9 technology and in in vitro experiments, tumor growth was significantly inhibited and doxorubicin sensitivity increased. The hypoxia-inducible transcription factor 1/2α (HIF-1/2α) signaling cascade including proteins involved in oxidant stress, glycolysis, and mitochondrial function was suppressed by TRPM2 depletion. TRPM2-depleted SH-SY5Y neuroblastoma cells demonstrated reduced oxygen consumption and ATP production after doxorubicin, confirming impaired cellular bioenergetics. In cells in which TRPM2 was depleted, mitochondrial superoxide production was significantly increased, particularly following doxorubicin. Ectopic expression of superoxide dismutase 2 (SOD2) reduced ROS and preserved viability of TRPM2-depleted cells, however, failed to restore ATP levels. Mitochondrial reactive oxygen species (ROS) were also significantly increased in cells in which TRPM2 function was inhibited by TRPM2-S, and pretreatment of these cells with the antioxidant MitoTEMPO significantly reduced ROS levels in response to doxorubicin and protected cell viability. Expression of the TRPM2 pore mutant E960D, in which calcium entry through TRPM2 is abolished, also resulted in significantly increased mitochondrial ROS following doxorubicin treatment, showing the critical role of TRPM2-mediated calcium entry. These findings demonstrate the important function of TRPM2 in modulation of cell survival through mitochondrial ROS, and the potential of targeted inhibition of TRPM2 as a therapeutic approach to reduce cellular bioenergetics, tumor growth, and enhance susceptibility to chemotherapeutic agents.
Departments of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
Lei
L
Bao
Departments of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
Shu-Jen
SJ
Chen
Departments of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
Kathleen
K
Conrad
Departments of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
Kerry
K
Keefer
Neural and Behavioral Sciences and Microscopy Imaging Facility.
Thomas
T
Abraham
Departments of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
John P
JP
Lee
the Departments of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140; The Center of Translational Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
JuFang
J
Wang
the Departments of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140; The Center of Translational Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Xue-Qian
XQ
Zhang
Departments of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
Iwona
I
Hirschler-Laszkiewicz
Departments of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; Departments of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
Hong-Gang
HG
Wang
Departments of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.
Sinisa
S
Dovat
Departments of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
Brian
B
Gans
The Center of Translational Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140; Molecular Genetics and Medical Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Muniswamy
M
Madesh
the Departments of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140; The Center of Translational Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Joseph Y
JY
Cheung
Departments of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and. Electronic address: bmiller3@psu.edu.
Barbara A
BA
Miller
D019943:Amino Acid Substitution; D002118:Calcium; D020013:Calcium Signaling; D045744:Cell Line, Tumor; D002470:Cell Survival; D017353:Gene Deletion; D006019:Glycolysis; D006801:Humans; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism*; D000099156:Superoxide Dismutase 2
D002118:Calcium; D009363:Neoplasm Proteins; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase; D050053:TRPM Cation Channels; D000099156:Superoxide Dismutase 2; C121934:TRPM2 protein, human
TRPM2; mitochondria; neuroblastoma; oxidative stress; reactive oxygen species (ROS); transient receptor potential channels (TRP channels)
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