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

Henrik Lutz,Juliane Joswig,Thy Nguyen,Bodo Laube,Kerstin Rau

doi:10.3390/cancers11030306

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.

Highlights

Glutamate (Glu), the major excitatory neurotransmitter in the vertebrate central nervous system (CNS), operates via two types of receptors: Metabotropic glutamate receptors and ionotropic glutamate receptors
It has been shown in neurons that Glu can induce double-strand breaks (DSBs) upon the specific activation of N-methyl-D-aspartic acid receptors (NMDARs) [9]
These results show that NMDAR signaling regulates cFos expression in LN229 cells transcription of genes in glioblastoma multiforme (GBM) cells, we chose to analyze the impact of NMDAR signaling and Top2β and support our idea of Top2β-mediated DSBs regulating gene expression in GBM

Summary

Introduction

Glutamate (Glu), the major excitatory neurotransmitter in the vertebrate central nervous system (CNS), operates via two types of receptors: Metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs). Ca2+ -permeable N-methyl-D-aspartic acid receptors (NMDARs) especially, as part of the iGluR family, have gained particular attention because of their crucial roles in brain development, synaptic plasticity and memory formation, and in neurotoxicity [1,2]. NMDARs are composed as tetrameric assemblies with two essential GluN1 subunits and varying contributions of two GluN2A-D subunits or possibly GluN3 subunits [3,4]. The specific expression of the subunits differs between brain regions as well as during development and determines the functional specificity of the NMDAR subtypes [5]. NMDAR-mediated Ca2+ -influx regulates fundamental cellular processes, including the proliferation, migration and survival of neurons [6], playing a lifelong essential role in the formation of synaptic plasticity [2]. NMDAR-signaling acts by activating two major downstream signaling pathways: The

Results

Discussion

Conclusion