Dexamethasone-mediated oncogenicity in vitro and in an animal model of glioblastoma

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OBJECTIVE

Dexamethasone, a known regulator of mesenchymal programming in glioblastoma (GBM), is routinely used to manage edema in GBM patients. Dexamethasone also activates the expression of genes, such as CEBPB, in GBM stem cells (GSCs). However, the drug’s impact on invasion, proliferation, and angiogenesis in GBM remains unclear. To determine whether dexamethasone induces invasion, proliferation, and angiogenesis in GBM, the authors investigated the drug’s impact in vitro, in vivo, and in clinical information derived from The Cancer Genome Atlas (TCGA) cohort.

METHODS

Expression profiles of patients from the TCGA cohort with mesenchymal GBM (n = 155) were compared with patients with proneural GBM by comparative marker selection. To obtain robust data, GSCs with IDH1 wild-type (GSC3) and with IDH1 mutant (GSC6) status were exposed to dexamethasone in vitro and in vivo and analyzed for invasion (Boyden chamber, human-specific nucleolin), proliferation (Ki-67), and angiogenesis (CD31). Ex vivo tumor cells from dexamethasone-treated and control mice were isolated by fluorescence activated cell sorting and profiled using Affymetrix chips for mRNA (HTA 2.0) and microRNAs (miRNA 4.0). A pathway analysis was performed to identify a dexamethasone-regulated gene signature, and its relationship with overall survival (OS) was assessed using Kaplan-Meier analysis in the entire GBM TCGA cohort (n = 520).

RESULTS

The mesenchymal subgroup, when compared with the proneural subgroup, had significant upregulation of a dexamethasone-regulated gene network, as well as canonical pathways of proliferation, invasion, and angiogenesis. Dexamethasone-treated GSC3 demonstrated a significant increase in invasion, both in vitro and in vivo, whereas GSC6 demonstrated a modest increase. Furthermore, dexamethasone treatment of both GSC3 and GSC6 lines resulted in significantly elevated cell proliferation and angiogenesis in vivo. Patients with mesenchymal GBM had significant upregulation of dexamethasone-regulated pathways when compared with patients with proneural GBM. A prognostic (p = 0.0007) 33-gene signature was derived from the ex vivo expression profile analyses and used to dichotomize the entire TCGA cohort by high (median OS 12.65 months) or low (median OS 14.91 months) dexamethasone signature.

CONCLUSIONS

The authors present evidence that furthers the understanding of the complex effects of dexamethasone on biological characteristics of GBM. The results suggest that the drug increases invasion, proliferation, and angiogenesis in human GSC-derived orthotopic tumors, potentially worsening GBM patients’ prognoses. The authors believe that careful investigation is needed to determine how to minimize these deleterious dexamethasone-associated side effects in GBM.

ABBREVIATIONS APC = allophycocyanin; FACS = fluorescence-activated cell sorting; FDR = false discovery rate; FSC-A = forward scatter absorption; GBM = glioblastoma; GSC = glioblastoma stem cell; GSEA = gene set enrichment analysis; IPA = Ingenuity Pathway Analysis; miRNA = microRNA; TCGA = The Cancer Genome Atlas.

Article Information

Correspondence Pascal O. Zinn: Baylor College of Medicine, Houston, TX. zinn@bcm.edu.

INCLUDE WHEN CITING Published online January 12, 2018; DOI: 10.3171/2017.7.JNS17668.

Disclosures Dr. Sulman: non–study-related clinical support from Novocure and AbbVie. Funding was provided by the following. Dr. Luedi: The Mach-Gaensslen Foundation Neurology Grant 2017 from the Mach-Gaensslen Foundation, Unteraegeri, Switzerland; Alfred & Anneliese Sutter-Stoettner Foundation, Muenchwilen, Switzerland; and Foundation for Research in Anesthesiology and Intensive Care Medicine, Bern, Switzerland. Dr. Colen: John S. Dunn Sr. Distinguished Chair in Diagnostic Imaging and The University of Texas MD Anderson Cancer Center Start-up Fund. Dr. Zinn: Neurosurgery Research & Education Foundation.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Dexamethasone promotes a protumorigenic gene signature in GBM. A: Heat map depicting z-scores of activation (red) and inhibition (blue) for various drugs, illustrating the status of these various drugs and their downstream regulated gene networks in mesenchymal (MES) GBM patients compared with proneural (PN) GBM patients. Fold-change cutoffs from left to right of the heat map are 5, 6, and 8 in MES versus PN comparisons. B: Activated dexamethasone-regulated gene network contributing to the prediction of the activated z-score for the dexamethasone-regulated gene network and its differential expression in MES compared with PN GBM patients. The z-score and the p value of activation are shown below the network. C: Venn diagram of genes from the MES versus PN comparison showing association of differentially regulated genes in MES vs PN analysis to various activated cellular functions, i.e., proliferation, invasion, and angiogenesis. The z-score and the p value of activation are presented under each cellular function. The numbers represent the numbers of genes involved. D: Schematic representation of the experimental plan to investigate the role of dexamethasone in GSC-derived orthotopic tumors, as well as downstream ex vivo analyses. E and F: Ex vivo enrichment of GSC3-derived cells of human origin from control (E) and dexamethasone-treated (F) mouse brains. G: Gene set enrichment plot for epithelial to mesenchymal transition in ex vivo GSC3 dexamethasone-treated samples. Values for the enrichment score, normalized enrichment score, nominal p value, FDR, and class identification are shown under the plot. H: Venn diagram of altered genes in dexamethasone-treated versus control ex vivo cells showing their cellular function association. The z-score and p value of activation are presented under each cellular function.

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    Dexamethasone promotes cell invasion in vitro and in vivo. A and B: Dexamethasone promotes cell invasion in GSC3 in vitro. Representative micrographs of control and dexamethasone-treated H & E–stained Boyden chamber membranes show cells (arrowheads) that have invaded through the Matrigel layer and are present on the other side of membrane (A). Quantification of invading cells in control and dexamethasone-treated membranes is shown in panel B. Error bars represent the standard deviation around the mean value, and the p value is shown above the graph. C and D: Dexamethasone treatment promotes the in vivo invasion of GSC3-derived orthotopic tumors. Micrographs of human-specific nucleolin-stained mouse brains with orthotopic tumors, including a representative low magnification (left) and high magnification (right; boxed in red within the left panel) to show the tumor margin (C). The bulk of the tumor is marked with “T” in the left panels. Scale bars are shown in the micrograph. Quantification of invading human nucleolin-positive cells in normal mouse brain away from the bulk of the tumor (D). Error bars represent the standard deviation around the mean value, and the p value is shown above the graph. The dots and squares represent normalized numbers of invading cells.

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    Dexamethasone promotes cell proliferation and angiogenesis in vivo. A and C: Representative micrographs of proliferation marker Ki-67–stained orthotopic control and dexamethasone-treated tumors derived from GSC3 (A) and GSC6 (C). Bar = 100 µm in A and C. B and D: Quantification of ratio of Ki-67–positive/total cells in control and dexamethasone-treated GSC3 (B) and GSC6 (D) tumors. Error bars represent the standard deviation around the mean value, and the p value is shown above the graph. E and G: Representative micrographs of endothelial marker CD31-stained orthotopic control and dexamethasone-treated tumors derived from GSC3 (E) and GSC6 (G). Bar = 200 µm in E and G. F and H: Quantification of CD31-positive cells in control and dexamethasone-treated GSC3 (F) and GSC6 (H) tumors. Error bars represent the standard deviation around the mean value. The triangles represent normalized numbers of invading cells. *p = 1.16E-07 in F and 9.49E-09 in H.

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    Dexamethasone (dexa)–promoted gene signature has prognostic value for GBM patients. A: Schematic representation of the derivation of the dexamethasone-regulated gene signature from an ex vivo expression profile analysis, subgrouping of TCGA patients, and a survival analysis. B: Heat map showing the expression levels of 33 genes among TCGA patients (n = 520) with a high or low dexamethasone signature. The color index bar is shown above the heat map. C. Kaplan-Meier curve analysis of patients with high and low dexamethasone signatures. The median survival durations in months are shown within the plot.

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