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,Jufang Wang,Xue-Qian Zhang,Iwona Hirschler-Laszkiewicz,Hong-Gang Wang,Sinisa Dovat,Brian Gans,Muniswamy Madesh,Joseph Y Cheung,Barbara A Miller

doi:10.1074/jbc.m116.747147

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 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.

Highlights

D (Tx’d) with empty vector, SOD1, SOD2 or both
Recent studies have shown that TRPM2 is highly expressed in a number of malignancies including melanoma, lung, neuroblastoma, and breast cancer [39, 41, 42], suggesting that it plays a role in promoting tumor growth, and that inhibition may be a novel therapeutic approach
TRPM2 was depleted in neuroblastoma cells with CRISPR/Cas9 technology as a model to explore its role in tumor growth and cell survival after doxorubicin

Summary

Introduction

D (Tx’d) with empty vector, SOD1, SOD2 or both. Blots were probed with anti-SOD1 or SOD2 antibodies. TRPM2-depleted SH-SY5Y cells loaded with MitoSOX Red showed a small but significantly greater level of mitochondrial ROS compared with scrambled control cells (Fig. 7). Without doxorubicin treatment, TRPM2depleted SH-SY5Y cells transfected with empty vector showed significantly greater levels of mitochondrial ROS compared

Results

Conclusion