Background: TRPM2 channels play an essential role in cell death following oxidative stress. Results: Dominant negative TRPM2-S decreases growth of neuroblastoma xenografts and increases doxorubicin sensitivity through modulation of HIF-1/2␣ expression, mitophagy, and mitochondrial function. Conclusion: TRPM2 is important for neuroblastoma growth and viability through modulation of HIF-1/2␣. Significance: Modulation of TRPM2 may be a novel approach in cancer therapeutics.

Edited by Xiao-Fan WangTransient 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 1/2␣ (HIF-1/2␣) 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.

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca(2+) influx that was dependent on extracellular Ca(2+), was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/dt between WT and KO hearts. At 2-3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/dt compared with WT hearts. Compared with WT I/R myocytes, expression of the Na(+)/Ca(2+) exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na(+)-K(+)-ATPase and Na(+) pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca(2+) concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.