NMDA Receptor Signaling Mediates cFos Expression via Top2β-Induced DSBs in Glioblastoma Cells

Abstract

The activation of Ca2+-permeable N-methyl-D-aspartic acid (NMDA) receptor channels (NMDARs) is crucial for the development and survival of neurons, but many cancers use NMDAR-mediated signaling as well, enhancing the growth and invasiveness of tumors. Thus, NMDAR-dependent pathways emerge as a promising target in cancer therapy. Here, we use the LN229 and U-87MG glioblastoma multiforme (GBM) cells and immunofluorescence staining of 53BP1 to analyze NMDAR-induced DNA double-strand breaks (DSBs), which represent an important step in the NMDAR signaling pathway in neurons by facilitating the expression of early response genes. Our results show that NMDAR activation leads to the induction of DSBs in a subpopulation of glioma cells. In a further analogy to neurons, our results demonstrate that the induction of DSBs in LN229 cells is dependent on the activity of topoisomerase IIβ (Top2β). Western blot analysis revealed that the inhibition of NMDARs, cAMP-responsive element binding transcription factor (CREB) and Top2β decreased the expression of the proto-oncogene cFos. Knockdown of Top2β with siRNAs resulted in a downregulation of cFos and increased the radiosensitivity of LN229 cells in clonogenic survival. We also observed impaired cFos expression upon NMDAR and Top2β inhibition in a primary GBM cell line, suggesting that NMDAR signaling may be widely used by GBMs, demonstrating the potential of targeting NMDAR signaling proteins for GBM therapy.

NMDA receptor modulation of glutamate release in activated neutrophils

“…Since mature neutrophils release more ROS, we examined whether GluN2B expression changed when HL60 cells are differentiated into neutrophil-like cells. Undifferentiated HL60 cells showed low levels of GluN2B subunit expression, similar to the low quantities known to be expressed in the human neuroblastoma LN229 cell line ([21]; Fig. 3A).…”

“…Gene expression was quantified by quantitative PCR performed with SYBR Green Supermix (Roche) on an Eppendorf Realplex system. Gene expression was analyzed using the ΔΔCt method, related to expression in the LN229 neuroblastoma cell line [21] (see Supplementary data for primer details).…”

FMRP modulates the Wnt signalling pathway in glioblastoma

“…5B). Among the transcription factors regulated by ERK1/2 and involved in cell proliferation, CREB and ETS1 are known to modulate malignant features of glioma cells [63] controlling transcription of GBM genes [64,65]. Consistently, we observed a decrease of CREB and ETS1 phosphorylation in GSCs silenced for FMR1 mRNA (Fig.…”

NMDA Receptor-Mediated Signaling Pathways Enhance Radiation Resistance, Survival and Migration in Glioblastoma Cells—A Potential Target for Adjuvant Radiotherapy

“…All chemicals were added prior to irradiation. X-ray irradiation was performed at 90 kV and 19 mA with an aluminum filter with single doses of 2, 4 and 6 Gy using an x-ray tube equipped with a tungsten-anode (Philips, Amsterdam, Netherlands) as described previously [55]. X-ray treatment was performed using a power of 19 mA, 90 kV voltage and 30 cm distance to the IR source, which makes an applied dose of 1.96 Gy/min, established by Ficke-dosimetry.…”