Identification and impact of Topoisomerase II β induced DNA double-strand breaks in Glioblastoma multiforme: NMDA-receptor signaling pathway as target structure
Glioblastoma multiforme (GBM) is a highly lethal and incurable cancer of the central nervous system and current therapies are challenged by GBMs invasive growth and chemo-radioresistance. Ca2+-permeable N-Methyl-D-aspartate receptors (NMDARs) are important for synaptic transmission of excitatory neu...
| Summary: | Glioblastoma multiforme (GBM) is a highly lethal and incurable cancer of the central nervous system and current therapies are challenged by GBMs invasive growth and chemo-radioresistance. Ca2+-permeable N-Methyl-D-aspartate receptors (NMDARs) are important for synaptic transmission of excitatory neurons and essentially regulate the plasticity of our brain via activation of several NMDAR-dependent signaling pathways. However, NMDARs have been shown to contribute to GBMs malignancy by promoting growth, survival and migration. Although the impact of NMDARs on GBM has been clearly demonstrated, the particular signaling pathways used by GBM are poorly known. The identification of the NMDAR signaling pathways used by GBM cells might therefore help to develop NMDAR-targeted cancer therapies which highly impact GBM cells but do not disrupt synaptic transmission in neurons.
The NMDAR-dependent expression of early-response genes (ERGs) upon neuronal activity is essential for synaptic plasticity and the formation of long term memory. The expression of ERGs depends on NMDAR-induced DNA-double strand breaks (DSBs) in the transcriptional start site of these genes. Some neuronal ERGs encode for proto-oncogenes like cFos, suggesting that GBM cells might hijack NMDAR signaling pathways to promote proto-oncogenes expression.
In order to investigate the impact of NMDAR-dependent ERG expression in GBM cells we intended to identify the hallmark of this NMDAR signaling pathway: The induction of NMDAR-dependent and Topoisomerase II β (Top2β) mediated DSBs in GBM cells.
For this task we validated the expression of NMDARs and functional Ca2+ signaling in the LN229 GBM cell line, which revealed functional NMDAR signaling in LN229 cells. Immunofluorescence staining of the DSB marker 53BP1 showed that NMDARs activation induces DSBs in a subpopulation of GBM cells and that DSB induction depends on Top2β activity, which demonstrates an analogues NMDAR signaling pathway in GBM cells and neurons.
Analysis of ERG expression revealed that NMDARs, the cAMP-responsive element binding transcription factor (CREB) and Top2β all contribute to the expression of cFos and the brain-derived neurotrophic factor (BDNF) in GBM cells. Inhibition of Top2β or NMDARs also impaired the expression of cFos in a primary GBM cell line. In a clonogenic survival assay knock-down of Top2β with siRNAs and inhibition of NMDARs decreased LN229 cells resistance to X-rays. Additionally, a newly discovered interplay of NMDAR signaling and IR damage response on the expression of BDNF and cFos might explain the high impact of NMDAR inhibition on radiosensitivity. Interestingly, inhibition of DNA-dependent protein kinase indicates that NMDAR-mediated transcription involves factors required in DSB repair, suggesting an important role for DNA repair in NMDAR-mediated transcriptional regulation.
The results presented in this work demonstrate a functional Top2β-dependent NMDAR signaling pathway in GBM cells. The radiosensitizing effect of Top2β and NMDAR inhibition reveals that targeting NMDAR-dependent and Top2β-mediated ERG expression might be a promising strategy for GBM therapy. |
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