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

Lei Bao, Shu-jen Chen, Kathleen Conrad, Kerry Keefer, Thomas Abraham, John P. Lee, Ju Fang Wang, Xue Qian Zhang, Iwona Hirschler-Laszkiewicz, Hong-Gang Wang, Sinisa Dovat, Brian Gans, Muniswamy Madesh, Joseph Y. Cheung, Barbara Miller

Research output: Contribution to journalArticle

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Abstract

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 Mito TEMPO 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.

Original languageEnglish (US)
Pages (from-to)24449-24464
Number of pages16
JournalJournal of Biological Chemistry
Volume291
Issue number47
DOIs
StatePublished - Nov 18 2016

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Neuroblastoma
Ion Channels
Energy Metabolism
Reactive Oxygen Species
Cell Survival
Doxorubicin
Cells
Modulation
Oxidants
Clustered Regularly Interspaced Short Palindromic Repeats
Adenosine Triphosphate
Calcium
Hypoxia-Inducible Factor 1
Neoplasms
Glycolysis
Growth
Heterografts
Oxygen Consumption
Superoxides
Transcription Factors

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "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",
abstract = "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 Mito TEMPO 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.",
author = "Lei Bao and Shu-jen Chen and Kathleen Conrad and Kerry Keefer and Thomas Abraham and Lee, {John P.} and Wang, {Ju Fang} and Zhang, {Xue Qian} and Iwona Hirschler-Laszkiewicz and Hong-Gang Wang and Sinisa Dovat and Brian Gans and Muniswamy Madesh and Cheung, {Joseph Y.} and Barbara Miller",
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language = "English (US)",
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pages = "24449--24464",
journal = "Journal of Biological Chemistry",
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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. / Bao, Lei; Chen, Shu-jen; Conrad, Kathleen; Keefer, Kerry; Abraham, Thomas; Lee, John P.; Wang, Ju Fang; Zhang, Xue Qian; Hirschler-Laszkiewicz, Iwona; Wang, Hong-Gang; Dovat, Sinisa; Gans, Brian; Madesh, Muniswamy; Cheung, Joseph Y.; Miller, Barbara.

In: Journal of Biological Chemistry, Vol. 291, No. 47, 18.11.2016, p. 24449-24464.

Research output: Contribution to journalArticle

TY - JOUR

T1 - 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

AU - Bao, Lei

AU - Chen, Shu-jen

AU - Conrad, Kathleen

AU - Keefer, Kerry

AU - Abraham, Thomas

AU - Lee, John P.

AU - Wang, Ju Fang

AU - Zhang, Xue Qian

AU - Hirschler-Laszkiewicz, Iwona

AU - Wang, Hong-Gang

AU - Dovat, Sinisa

AU - Gans, Brian

AU - Madesh, Muniswamy

AU - Cheung, Joseph Y.

AU - Miller, Barbara

PY - 2016/11/18

Y1 - 2016/11/18

N2 - 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 Mito TEMPO 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.

AB - 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 Mito TEMPO 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.

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