Wnt/beta-catenin synergises with FOXG1 to drive exit from quiescence in neural stem cells, including glioblastoma stem cells


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Faye Robertson1,Harry Bulstrode1,Maria-Angeles Marques Torrejon1,Eoghan O'Duibhir1,Steve Pollard1
1University of Edinburgh

Abstract

Background

Glioblastoma is a malignant brain tumour which is universally fatal. Stem cells within the tumour exist in a quiescent state, evade destruction and reactivate, causing relapse. These stem cells are known to overexpress the transcription factor FOXG1.

Method

We used an in vitro model of quiescence in mouse neural stem cells, incorporating a conditional human FOXG1 overexpression cassette, to identify, through high content pharmacological screening, a synergistic relationship between high FOXG1 expression and inhibition of glycogen synthase kinase 3 (GSK3) in driving cells into an active, proliferative state. We quantified this effect using EdU incorporation and colony forming assays. Wnt inhibitors were used to abrogate the effect and a genetic approach, using a constitutively active beta-catenin cassette, was used to elucidate the nature of the synergy. Patient-derived human glioblastoma stem cells (GSCs), with or without CRISPR-Cas9 excision of FOXG1, were used to confirm the relevance of the effect.

Results

EdU incorporation, following a 2 hour pulse, was increased from 4% in growth factors alone, 5.9% with FOXG1 overexpression, 5.5% with GSK3 inhibition, to 31.3% with FOXG1 overexpression and GSK3 inhibition combined. Colony forming assays confirm high efficiency cell cycle re-entry in cells with FOXG1 overexpression treated with a GSK3 inhibitor.

We subsequently show that this effect is present in patient-derived human GSCs and is abolished by excision of FOXG1.

The effect of GSK3 inhibition can be phenocopied both by a ligand of the canonical Wnt signalling pathway and by inducible constitutively active beta-catenin, suggesting that the synergy is effected through beta-catenin, the key downstream effector of canonical Wnt signalling.

Furthermore, the combined effect of FOXG1 overexpression and GSK3 inhibition on exit from quiescence can be abrogated by Wnt inhibitors.

Conclusion

Targeting the synergistic relationship between FOXG1 and beta-catenin may provide an exciting therapeutic opportunity in preventing relapse and improving the prognosis of glioblastoma.