Reciprocal regulation of the cholinic phenotype and epithelial-mesenchymal transition in glioblastoma cells

Glioblastoma (GBM) is the most malignant brain tumor with very limited therapeutic options. Standard multimodal treatments, including surgical resection and combined radio-chemotherapy do not target the most aggressive subtype of glioma cells, brain tumor stem cells (BTSCs). BTSCs are thought to be responsible for tumor initiation, progression, and relapse. Furthermore, they have been associated with the expression of mesenchymal features as a result of epithelial-mesenchymal transition (EMT) thereby inducing tumor dissemination and chemo resistance. Using high resolution proton nuclear magnetic resonance spectroscopy (1H NMR) on GBM cell cultures we provide evidence that the expression of well-known EMT activators of the ZEB, TWIST and SNAI families and EMT target genes N-cadherin and VIMENTIN is associated with aberrant choline metabolism. The cholinic phenotype is characterized by high intracellular levels of phosphocholine and total choline derivatives and was associated with malignancy in various cancers. Both genetic and pharmacological inhibition of the cardinal choline metabolism regulator choline kinase alpha (CHKα) significantly reduces the cell viability, invasiveness, clonogenicity, and expression of EMT associated genes in GBM cells. Moreover, in some cell lines synergetic cytotoxic effects were observed when combining the standard of care chemotherapeutic temozolomide with the CHKα inhibitor V-11-0711. Taken together, specific inhibition of the enzymatic activity of CHKα is a powerful strategy to suppress EMT which opens the possibility to target chemo-resistant BTSCs through impairing their mesenchymal transdifferentiation. Moreover, the newly identified EMT-oncometabolic network may be helpful to monitor the invasive properties of glioblastomas and the success of anti-EMT therapy

Enzymatic Activity of CD73 Modulates Invasion of Gliomas via Epithelial–Mesenchymal Transition-Like Reprogramming

Glioblastoma (GBM) is the most aggressive malignant primary brain tumour in adulthood. Despite strong research efforts current treatment options have a limited impact on glioma stem-like cells (GSCs) which contribute to GBM formation, progression and chemoresistance. Invasive growth of GSCs is in part associated with epithelial–mesenchymal-like transition (EMT), a mechanism associated with CD73 in several cancers. Here, we show that CD73 regulates the EMT activator SNAIL1 and further investigate the role of enzymatic and non-enzymatic CD73 activity in GBM progression. Reduction of CD73 protein resulted in significant suppression of GSC viability, proliferation and clonogenicity, whereas CD73 enzymatic activity exhibited negative effects only on GSC invasion involving impaired downstream adenosine (ADO) signalling. Furthermore, application of phosphodiesterase inhibitor pentoxifylline, a potent immunomodulator, effectively inhibited ZEB1 and CD73 expression and significantly decreased viability, clonogenicity, and invasion of GSC in vitro cultures. Given the involvement of adenosine and A3 adenosine receptor in GSC invasion, we investigated the effect of the pharmacological inhibition of A3AR on GSC maintenance. Direct A3AR inhibition promoted apoptotic cell death and impaired the clonogenicity of GSC cultures. Taken together, our data indicate that CD73 is an exciting novel target in GBM therapy. Moreover, pharmacological interference, resulting in disturbed ADO signalling, provides new opportunities to innovate GBM therapy

The effect of neurosphere culture conditions on the cellular metabolism of glioma cells

Glioblastoma (GBM) is the most common and lethal adult glial brain tumour, with a mean overall survival of 16-19 months after primary diagnosis under the current standard-of-care treatment scheme [26]. Despite enormous research efforts towards early diagnosis and more efficient treatment, the prognosis of GBMs remains dismal.The influence of culture conditions has been wi­dely investigated in the field of glioma research, suggesting that neurosphere cultures, compared to adherent growth, more closely resemble the original patient’s tumour [29] showing high stem cell compartment [1] and therefore are more suitable for testing of novel therapeutic spectras approaches [30]. In this report we describe altered relative concentrations of the cholines, creatine, myo-inositol, and glycine in the human GBM cell line U87 propagated under stem cell conditions as compared to classical monolayer culture. Furthermore, U87 neurospheres showed significant higher levels of the putative GBM stem cell marker CD133 as their serum-propagated counterparts. Detection and targeting of miss-regulated choline-, myo-inosi­tol-, creatine-, and glycine-metabolism has been de­s­cribed to have potential utility in the diagnosis and treatment of malignant gliomas [2-4,13,16,19].This is, to our knowledge, the hitherto first link of changes in those oncometabolites [4,25] to variations in cell culture conditions of glioma cells. Inter spectral co-analysis of metabolite concentrations under the two propagation conditions identified reductions in ratios of phosphocholine to glycerophosphocholine (PC/GPC) and glycine to total choline (Gly/tCho) but increases in the quotient of total choline to total creatine (tCho/tCre) and PC/tCre, as well as Gly/myo-inositol (Gly/myo). This work should draw the attention of the scientific community on possible in vitro artefacts and on the need for appropriate models most closely resembling the in vivo biology of investigated tumours