Abstract
Glioblastoma is the most common malignant brain tumour in adults. The failure of current therapies can be ascribed to glioma stem cells (GSCs), which can rapidly repopulate the tumour following the initial treatment. The study of histone deacetylase inhibitors, such as valproic acid (VPA), is becoming an attractive field in cancer research. However, the exact mechanisms underlying its anti-cancer effect remain to be elucidated due to its pleiotropic effects on several cell-signalling pathways. Ingenuity Pathway Analysis (IPA) bioinformatics analysis was performed on genome-wide data regarding GSCs methylome to identify the signalling pathways mainly affected by methylation changes induced by VPA. Real time PCR and luciferase reporter assay were used to better investigate VPA effects on Wnt/β-catenin signalling pathway. VPA effect on GSC proliferation was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Trypan blue assays. Finally, VPA impact on GSC motility was demonstrated by Boyden chamber assay and further confirmed evaluating the expression levels or localisation, through western blot or immunofluorescence, of Twist1, Snail1, E-Cadherin and N-Cadherin. The bioinformatics analyses performed on GSCs methylome highlighted that Wnt/β-catenin signalling was affected by the methylation changes induced by VPA, which could influence its activation status. In particular, we pointed out a general activation of this pathway after VPA exposure, which was accompanied by an inhibitory potential on GSCs proliferation. Finally, we also proved VPA’s ability to inhibit GSCs invasion through Snail1 and Twist1 downregulation and E-Cadherin relocalisation. VPA treatment may represent a new, interesting therapeutic approach to affect GSC proliferation and motility, but further investigations are certainly needed.
Highlights
Glioblastoma (GBM) is the most common and aggressive type of primary brain tumour
With regards to the GBM2 cell line, Wnt signalling pathway modulation by valproic acid (VPA) was shown explicitly by Ingenuity Pathway Analysis (IPA) analysis (Figure S1), while in the G144, this was proven through the presence of a more generic “Glioblastoma multiforme signalling” (Figure S2A), which includes the Wnt signalling pathway (Figure S2B)
Z-score values, calculated by IPA through an algorithm that compared the dataset of genes that changed their methylation status after treatment with the expected canonical pathway patterns, gave us a prediction of the activation state of the pathways affected by methylation changes after VPA exposure
Summary
Glioblastoma (GBM) is the most common and aggressive type of primary brain tumour. Despite several biological and clinical studies, the median survival of GBM patients is still about 15 months post-diagnosis, with high risk of recurrence [1]. Increasing evidence has suggested that GBM contains a small subpopulation of tumour cells with stem-like properties, called “glioma stem cells”. (GSCs) [2,3,4,5], characterised by self-renewal capacity, multilineages differentiation potential, enhanced invasive behaviour and tumorigenicity in vivo. New therapeutic strategies are needed to completely eradicate this tumour. Epigenetic mechanisms have being exploited as new potential therapeutic targets and increasing evidence suggests that epigenetic aberrations, apart from genetic alterations, may play a key role in the development and/or progression of gliomas [7]. The reversibility of epigenetic alterations encourages the use of epigenetic drugs, such as histone deacetylase inhibitors (HDACi), as an attractive approach to “reset” the abnormal cancer epigenome [8]
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