The human ion channel TRPM2 modulates neuroblastoma cell survival and mitochondrial function through Pyk2, CREB, and MCU activation

Abstract

Transient receptor potential melastatin channel subfamily member 2 (TRPM2) has an essential function in cell survival and is highly expressed in many cancers. Inhibition of TRPM2 in neuroblastoma by depletion with CRISPR technology or expression of dominant negative TRPM2-S has been shown to significantly reduce cell viability. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in TRPM2 modulation of neuroblastoma viability was explored. In TRPM2-depleted cells, phosphorylation and expression of Pyk2 and cAMP-responsive element-binding protein (CREB), a downstream target, were significantly reduced after application of the chemotherapeutic agent doxorubicin. Overexpression of wild-type Pyk2 rescued cell viability. Reduction of Pyk2 expression with shRNA decreased cell viability and CREB phosphorylation and expression, demonstrating Pyk2 modulates CREB activation. TRPM2 depletion impaired phosphorylation of Src, an activator of Pyk2, and this may be a mechanism to reduce Pyk2 phosphorylation. TRPM2 inhibition was previously demonstrated to decrease mitochondrial function. Here, CREB, Pyk2, and phosphorylated Src were reduced in mitochondria of TRPM2-depleted cells, consistent with their role in modulating expression and activation of mitochondrial proteins. Phosphorylated Src and phosphorylated and total CREB were reduced in TRPM2-depleted nuclei. Expression and function of mitochondrial calcium uniporter (MCU), a target of phosphorylated Pyk2 and CREB, were significantly reduced. Wild-type TRPM2 but not Ca2+-impermeable mutant E960D reconstituted phosphorylation and expression of Pyk2 and CREB in TRPM2-depleted cells exposed to doxorubicin. Results demonstrate that TRPM2 expression protects the viability of neuroblastoma through Src, Pyk2, CREB, and MCU activation, which play key roles in maintaining mitochondrial function and cellular bioenergetics.

TRPM2 channel-mediated cell death: An important mechanism linking oxidative stress-inducing pathological factors to associated pathological conditions

“…The TRPM2-S protein is of particular interest in that while short of channel-forming ability, it is capable of co-assembling with the full-length TRPM2 protein or channel and imposing dominant negative inhibition of the TRPM2 channel. Due to limited pharmacological tools for investigating the TRPM2 channel as discussed below, this dominant negative attribute of TRPM2-S has been employed in in vitro studies to interrogate the role of the TRPM2 channel in mediating ROS-induced Ca 2+ signalling and regulation of cellular functions by overexpression of TRPM2-S to suppress the formation or activity of functional channels by the endogenous full-length TRPM2 protein [ 33 , [74] , [75] , [76] , [77] ].…”

Increasing the TRPM2 Channel Expression in Human Neuroblastoma SH-SY5Y Cells Augments the Susceptibility to ROS-Induced Cell Death

“…Human neuroblastoma SH-SY5Y cells have been widely used as a human neuronal cell model in the study of the molecular and signaling mechanisms for neurodegeneration, particularly those related to PD because of the catecholaminergic, albeit strictly speaking not dopaminergic, neuronal properties [31]. Studies from other groups and us have consistently documented functional expression of the TRPM2 channel in SH-SY5Y cells [21,23,24,32,33,34], but the findings regarding the role of the TRPM2 channel in SH-SY5Y cells are intriguing. A recent study using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay has shown that concentration-dependent reduction in cell viability after exposure to 10 to 400 µM H 2 O 2 or 125 to 500 µM MPP via inducing H 2 O 2 generation [23].…”

Roles of NAD+ and Its Metabolites Regulated Calcium Channels in Cancer

“…However, the data in cancer models mostly supports the conclusion that TRPM2 expression and function have an important role in preserving cancer cell viability and survival. Consistent with this view, the TRPM2 channel has been found to be highly expressed in numerous cancers including bladder, breast, head and neck, lung, pancreatic, prostate, melanoma, and neuroblastoma [ 51 ], among which most studies were focused on the neuroblastoma [ 58 , 59 , 60 , 61 , 62 , 63 , 64 ] ( Figure 2 ). The higher levels of ROS in cancer cells impel the enhancement of their anti-oxidant capacity to detoxify ROS and preserve cells viability.…”

“…Ca 2+ influx via TRPM2 results in the activation of phosphorylation of Pyk2, which regulates the cell survival and tumor growth of various cancers through the CREB pathway, leading to increased expression of phosphorylated and total CREB. When TRPM2 was inhibited, pPyk2, Pyk2, pCREB, and CREB were reduced and mitochondrial function as well as mitochondrial Ca 2+ uptake were impaired, together with more mitochondrial and cellular ROS, reducing cells survival and tumor growth [ 62 ]. Taken together, all studies in neuroblastoma show the critical role of TRPM2 that modulates both ROS production and the antioxidant response through the Ca 2+ entry via the channel activation.…”