Abstract B14: Wnt signaling circuits in glioblastoma multiforme

Abstract

Abstract Glioblastoma multiforme (GBM) is the most common malignant tumour in the central nervous system with a prevalence of 2-3 cases per 100 000 people. Although the standard treatment of surgery, chemotherapy and radiotherapy improve survival, the median survival continues to remain at only 15 months with a 5-year survival rate of under 10%. Glioma neural stem-like (GNS) cells have been identified in GBM and have the capability of regenerating the tumour. Treatment strategies that target the majority of the tumour may be incapable of also targeting GNS cells and thus characterization of GNS cells may provide insight into additional treatment options. The Wnt signalling pathway has been linked to several cancers including GBM. Wnt signalling involves the secretion of Wnt ligand proteins that bind to specific Frizzled (FZD) receptor complexes on the cell surface of Wnt-responding cells to activate intracellular signalling cascades. The transcriptional and epigenetic regulation in GNS cells is the focus of several recent studies. The transcription factor ASCL1 was identified to be overexpressed and to lead to Wnt signalling activation by repressing Dickkopf (DKK1, Wnt inhibitor). Using microarray data we analyzed the expression of FZD receptors and Wnt target genes in over 50 primary GNS cell lines cultured in serum free conditions in order to maintain the GIC population and identified a subgroup of glioma lines with activated Wnt signalling. To determine the requirement of autocrine Wnt signalling for GNS cell renewal we inhibited Wnt secretion, using the porcupine inhibitor LGK974, and measured self-renewal using a limited dilution assay. There was a significant reduction in GNS cell frequency with LGK974 (1uM) treatment in four out of eight lines tested (G432NS, G472NS, G511NS and G523NS). Furthermore, a secondary sphere assay with G523NS cells also showed a significant reduction in GNS cell frequency. When G511NS and G523NS cells were treated with LGK974 (1uM) over a two week period, there was a significant increase in the percentage of GFAP (astrocytic marker) expressing cells whereas a two week treatment with Bio (1uM, Wnt activator), significantly increased the percentage of Tuj1 (neuronal marker) expressing cells. This finding suggest that these cells require a specific amount of Wnt signalling for self-renewal and Wnt inhibition or activation may lead to differentiation. RNAseq analysis comparing the four LGK974 responsive lines with the four LGK974 unresponsive lines identified ASCL1 to be highly expressed in the responsive lines. Gene set enrichment analysis identified four gene sets significantly enriched in the responsive group including the Glioblastoma Proneural gene set whereas genes from the Glioblastoma Mesenchymal gene set were significantly enriched in the unresponsive group. We have identified a subset of GNS cells that are dependent on Wnt secretion for self-renewal and RNAseq analysis suggests that GBMs that fall under the Proneural subtype may be sensitive to Wnt inhibition. Citation Format: Nishani Rajakulendran, Hayden Selvadurai, Katherine Rowland, Nicole Park, Nizar Batada, Peter Dirks, Stephane Angers. Wnt signaling circuits in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B14.