CREB5 promotes the proliferation and self-renewal ability of glioma stem cells

Abstract

Glioblastoma multiforme (GBM) is the most fatal form of brain cancer in humans, with a dismal prognosis and a median overall survival rate of less than 15 months upon diagnosis. Glioma stem cells (GSCs), have recently been identified as key contributors in both tumor initiation and therapeutic resistance in GBM. Both public dataset analysis and direct differentiation experiments on GSCs have demonstrated that CREB5 is more highly expressed in undifferentiated GSCs than in differentiated GSCs. Additionally, gene silencing by short hairpin RNA (shRNA) of CREB5 has prevented the proliferation and self-renewal ability of GSCs in vitro and decreased their tumor forming ability in vivo. Meanwhile, RNA-sequencing, luciferase reporter assay, and ChIP assay have all demonstrated the closely association between CREB5 and OLIG2. These findings suggest that targeting CREB5 could be an effective approach to overcoming GSCs.

Fig. 1. CREB5 mRNA is expressed higher in the undifferentiated GSC.

A, B The comparison of CREB Family expression in GSCs cultured under NBE and Serum conditions according to multiple probes set of the GSE4536 dataset. A 0308 cells; B 1228 cells. Data are means ± SEM (NBE, n = 10 or 11; FBS, n = 11 or 10). ***p < 0.001.

Fig. 2. CREB5 is expressed highly expressed in GSC.

A RT-qPCR analysis of CREB5 mRNA expression in NHA, non-stem cell glioma cells, and GSCs. B Western blot analysis of CREB5 protein in GSCs and differentiated GSCs. C RT-qPCR analysis of mRNA expressions in GSCs and differentiated GSCs. Data are means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. Suppressing of CREB5 inhibits proliferation and self-renewal ability in GSCs.

A RT-qPCR analysis showing CREB5 knockdown after transduction with shRNA in GSCs. B Cell proliferation shCREB5 treated GSCs. C Annexin-V/propidium iodide staining of GSCs after transduction with shCREB5. D Representative images showing cell spheres. Scale bars = 100 μm. E An in vitro limiting dilution assay using gradually decreasing cell seeding density shows the cell sphere forming ability of GSCs transduced with shCREB5. Data are means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4. The inhibition of CREB5 reduces the tumorigenic potential of GSCs in vivo.

A In vivo bioluminescent imaging was performed on nude mice bearing intracranial xenografts derived from shNT (Non-target) and shCREB5 transduced GSC11. Data are means ± SEM (n = 6). ***p < 0.001. B Representative images of H&E stained coronal sections of tumor bearing brains harvested after implantation of shNT and shCREB5 treated GSC11. Scale bars represent 2 mm. C Kaplan–Meier survival curves of nude mice bearing intracranial tumors derived from shNT and shCREB5 treated GSC11.

Fig. 5. RNA-Sequencing reveals pathways and genes downregulated by shCREB5.

A Heat map and volcano plots of transcriptional regulation patterns of shNT and shCREB5 transduced GSCs. B Dot plots present enrichment pathways of genes downregulated with CREB5 in shCREB5 transduced GSCs. Data are means ± SEM.

Fig. 6. CREB5 binds to the AP-1 sites within the OLIG2 promoter.

A RT-qPCR validation of downregulated genes after CREB5 inhibition. B Luciferase assay using OLIG2 promoter after indicated treatment. C ChIP analysis of CREB5 binding to the OLIG2 promoter in HEK293T. Data are means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.