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. 2014 Jan;32(1):301-12.
doi: 10.1002/stem.1528.

A high Notch pathway activation predicts response to γ secretase inhibitors in proneural subtype of glioma tumor-initiating cells

Norihiko Saito  1 , Jun Fu, Siyuan Zheng, Jun Yao, Shuzhen Wang, Diane D Liu, Ying Yuan, Erik P Sulman, Frederick F Lang, Howard Colman, Roel G Verhaak, W K Alfred Yung, Dimpy Koul
Affiliations

A high Notch pathway activation predicts response to γ secretase inhibitors in proneural subtype of glioma tumor-initiating cells

Norihiko Saito et al. Stem Cells. 2014 Jan.

Abstract

Genomic, transcriptional, and proteomic analyses of brain tumors reveal subtypes that differ in pathway activity, progression, and response to therapy. However, a number of small molecule inhibitors under development vary in strength of subset and pathway-specificity, with molecularly targeted experimental agents tending toward stronger specificity. The Notch signaling pathway is an evolutionarily conserved pathway that plays an important role in multiple cellular and developmental processes. We investigated the effects of Notch pathway inhibition in glioma tumor-initiating cell (GIC, hereafter GIC) populations using γ secretase inhibitors. Drug cytotoxicity testing of 16 GICs showed differential growth responses to the inhibitors, stratifying GICs into responders and nonresponders. Responder GICs had an enriched proneural gene signature in comparison to nonresponders. Also gene set enrichment analysis revealed 17 genes set representing active Notch signaling components NOTCH1, NOTCH3, HES1, MAML1, DLL-3, JAG2, and so on, enriched in responder group. Analysis of The Cancer Genome Atlas expression dataset identified a group (43.9%) of tumors with proneural signature showing high Notch pathway activation suggesting γ secretase inhibitors might be of potential value to treat that particular group of proneural glioblastoma (GBM). Inhibition of Notch pathway by γ secretase inhibitor treatment attenuated proliferation and self-renewal of responder GICs and induces both neuronal and astrocytic differentiation. In vivo evaluation demonstrated prolongation of median survival in an intracranial mouse model. Our results suggest that proneural GBM characterized by high Notch pathway activation may exhibit greater sensitivity to γ secretase inhibitor treatment, holding a promise to improve the efficiency of current glioma therapy.

Keywords: Glioma; Notch activation; Proneural genes; γ Secretase inhibitors.

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Figures

Figure 1
Figure 1. γ Secretase inhibitors effectively showed dose-dependent growth inhibition of GICs
A) A panel of GICS lines was treated with various concentrations of γ secretase inhibitors DAPT, BMS-708163 and RO4929097. Cells were treated with increasing concentrations of γ secretase inhibitors in triplicate wells for 72 hours, and cell viability was assessed by CellTiter-Blue assay as described in Materials and Methods. The results shown are of a single experiment with 3 independent replicates cell viability was measured by CellTiter-Blue assay. The graph depicts cell viability at 72 hours. Cell viability in the vehicle control was considered as to be 100%. B). Waterfall diagram of IC50 of 16 GICs. *p<0.001 responder GICs vs non-responder GICs.
Figure 2
Figure 2. Enrichment of Notch pathway components and proneural signature in responder GICs
A) Hierarchical cluster analysis on gene expression data from 16 GICs identified several genes highly expressed in the responder group and classified 16 GICs into two major groups with unique gene signatures. The proneural gene signature defined by Verhaak et al was projected to responder and non-responder GICs. (B) RT-PCR validating expression of proneural marker genes Olig2 and Sox2 in responder GICs. (C) Up regulation of Notch pathway in responder cell lines by Gene Set Enrichment Analysis. X-axis represents gene ordered by expression changes between responders and non-responders and Y-axis represents cumulative enrichment score. (D) Expression pattern of NOTCH pathway genes in the GIC microarray data set showing enrichment of Notch pathway components mainly NOTCH1, NOTCH3, HES1, MAML1, JAG2, and DLL3 in responder GICs. (E) RT-PCR data validated expression of the Notch pathway genes Notch1, Notch3, Hes1, Hes3 and Hes5 in the responder GICs.
Figure 3
Figure 3. γ Secretase inhibitors suppresses Notch signaling pathway in a dose- or time-dependent manner
A) Western blotting was performed to analyze the cellular protein levels of Notch signaling pathway in 16 GICs. Activated Notch-1 (NICD) and Notch-1 pathway components Hes1, Hes3 and Hes5, were all expressed in the responder GICs. β-Actin was used as loading control. B) A panel of GICs was treated with the indicated doses of DAPT, BMS-708163 and RO4929097 for indicated time intervals. All the γ secretase inhibitors inhibited expression of NICD, Hes1, Hes3 and Hes5 in a time-dependent manner. The decrease in NICD was followed by a decrease in Hes1, Hes3 and Hes5 expression, whereas the expression of Notch-1 did not change. C) A panel of GICs was treated with the indicated doses of DAPT, BMS-708163 and RO4929097 for 48 hours. γ Secretase inhibitors inhibited expression of NICD, Hes1, Hes3 and Hes5 in a dose-dependent manner. D) Western blot analysis confirmed the knockdown effect of Notch shRNA lentivirus on GSC35. shRNA knockdown of Notch-1 in GICs decreased Hes1 and Hes5 protein expression.
Figure 4
Figure 4. γ Secretase inhibitors decrease self-renewal capacity of responder GICs
A) GICs were treated with of γ secretase inhibitors DAPT, BMS-708163 and RO4929097 at doses described in Materials and Methods for 7 days in a neurosphere formation assay and numbers of neurospheres were counted in 10 separate fields using a microscope. All the tested γ secretase inhibitors reduce the number and size of neurosphere after 7 days of treatment in responder GICs. Micrographs showed the representative neurosphere results of for GSC35 (Responder) and GSC20 (Non-Responder). Bars, 250 μm. B) We performed primary neurosphere assay to show the effect on proliferation. All the tested γ secretase inhibitors significantly reduce the number of neurosphere after 7 days of treatment in responder GICs (p<0.001). Neurosphere formation in the vehicle control was considered to be 100%.C) For secondary neurosphere assay to show the effect on self-renewal, neurospheres after 7 days of treatment as indicated above were dissociated in to single cells and grown as single cells into a 96-well plate. Cells were grown for another 7 days after which cells as spheres were counted for self-renewal capacity. The bar graph depicts secondary neurosphere formation after 7 days of replating. Neurosphere formation in the vehicle control was considered as to be 100%.
Figure 5
Figure 5. Notch inhibition induces neuronal and astrocytic differentiation
A)GICs were treated with indicated doses of DAPT, BMS-708163 and RO4929097 for 5 days. Cells were stained with CNPase (an oligodendrocytic marker), GFAP (an astrocytic marker), TuJ1 (a neuronal marker), and Nestin (a neural stem cell marker). Nuclei were stained with DAPI. Micrographs showed the representative staining results of for GSC35 (Responder) and GSC20 (Non-Responder) cells. Bars, 100 μm. DAPT, BMS-708163 and RO4929097 induce neuronal and astrocytic differentiation in responder GICs (GSC35). n=4, p<0.05, as compared to marker staining positivity in vehicle control. B) Knockdown of Notch-1 by shRNA decreased the expression of only Nestin and increased that of TuJ1 showing that Notch knockdown induces only neuronal differentiation. Bars, 100 μm.
Figure 6
Figure 6. RO4929097 extends survival in an intracranial animal model
GSC35 cells were implanted intracranially in nude mice (n=6 in each group), and treatment was commenced 4 days later. RO4929097 (10 and 20 mg/kg/day) was administered orally 5 times a week for 4 weeks. Control group mice were treated with 1.0% carboxymethyl cellulose with 0.2% Tween 80. Mice were sacrificed at morbidity, and survival curves were compared using Prism 5 software. A)Representative H&E-stained whole brain sections at 4 weeks post-treatment showing the tumor mass. Bars, 1 mm B). Kaplan-Meier survival probability plots of tumor-bearing mice in vehicle or RO4929097 treatment groups (n = 6), using the log-rank method to test for a difference between groups. Treatment of RO4929097 (10 and 20 mg/kg/day) showed a statistically significant improvement over control (p= 0.05, log-rank test for both experiments). C) Immunostaining of the brain sections of animals treated with RO4929097 for 4 weeks. The tissue section was incubated with antibodies against NICD, Hes1, Nestin, TuJ1 and Ki67. Bars, 100 μm.
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