Calabrese, Lucrezia (2021) Identification and characterization of the role played by the neuronal NCX2 in glioblastoma progression and malignity. [Tesi di dottorato]

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Glioblastoma multiforme (GBM) is one of the most malignant primary brain tumors with an aggressive phenotype and poor lines of evidence in genetic or environmental risk factors. In addition, glioblastoma patients show an ineffective treatment and a very short survival rate. For these reasons, there is a great demand of novel pharmacological targets for new effective strategies of treatment. A common feature of all GBM sub-types is the alteration in the activity of pathways controlling the expression of several transcription factors involved in the up- or down-regulation of pro-oncogenes or anti-oncogenes, respectively. Among the downregulated genes there is Slc8a2 encoding for the brain Sodium Calcium Exchanger 2 (NCX2). Recent data suggest that this antiporter can represent a possible anti-oncogene for GBM since it is silenced in all glioma stages. To explore the genetic and epigenetic mechanisms leading to NCX2 downregulation, we identified, cloned and analyzed both rat and human slc8a2 promoters in two cell lines PC12 and U87 expressing high or low level of this antiporter, respectively. In addition, we selected several transcription factors (TFs) able to modify both rat and human promoter activity of NCX2 in PC12 and U87 cell lines. However, TFs were able to increase mRNA expression of endogenous NCX2 only in PC12 cells. Interestingly, pharmacological inhibition of EGF pathway at different points restored NCX2 mRNA expression levels and increased its promoter activity in U87 cells. Moreover, transfection of NFkB, a downstream transcription factor of EGF pathway, downregulated NCX2 expression even in the presence of an inhibitor of EGF pathway. In addition, the blockage of this receptor-dependent cascade, or the stably transfection of NCX2, or the other isoform NCX1, hampered cell growth of U87 cells. In a prospective therapeutic approach, we also analyzed the effect of two compounds, namely neurounina-1 and CN-PYB2, on the vitality and cell growth of U87 cell line. Neurounina-1 is a stimulator of both NCX1 and NCX2 activity, whereas CN-PYB2 is a selective stimulator of NCX1 activity. Results showed that these compounds hampered in a concentration- and time-dependent manner cell growth of U87 cell line, whereas they were ineffective in other cell lines, including BHK, SH-SH5Y and PC12. Altogether, these data suggest that glioblastoma silences NCX2 by an EGF pathway via NFkB, and the increase of NCX expression via EGF-pathway inhibition, or the increase of NCX activity, slows-down glioblastoma cell growth and thus might exert a tumor suppressor effect.

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