MEOX2 Regulates the Growth and Survival of Glioblastoma Stem Cells by Modulating Genes of the Glycolytic Pathway and Response to Hypoxia

Abstract

The most widely accepted hypothesis for the development of glioblastoma suggests that glioblastoma stem-like cells (GSCs) are crucially involved in tumor initiation and recurrence as well as in the occurrence of chemo- and radio-resistance. Mesenchyme homeobox 2 (MEOX2) is a transcription factor overexpressed in glioblastoma, whose expression is negatively correlated with patient survival. Starting from our observation that MEOX2 expression is strongly enhanced in six GSC lines, we performed shRNA-mediated knock-down experiments in two different GSC lines and found that MEOX2 depletion resulted in the inhibition of cell growth and sphere-forming ability and an increase in apoptotic cell death. By a deep transcriptome analysis, we identified a core group of genes modulated in response to MEOX2 knock-down. Among these genes, the repressed ones are largely enriched in genes involved in the hypoxic response and glycolytic pathway, two strictly related pathways that contribute to the resistance of high-grade gliomas to therapies. An in silico study of the regulatory regions of genes differentially expressed by MEOX2 knock-down revealed that they mainly consisted of GC-rich regions enriched for Sp1 and Klf4 binding motifs, two main regulators of metabolism in glioblastoma. Our results show, for the first time, the involvement of MEOX2 in the regulation of genes of GSC metabolism, which is essential for the survival and growth of these cells.

Keywords: MEOX2; glioblastoma stem cells; glycolytic enzymes; sphere formation.

Figure 1

MEOX2 depletion inhibits the sphere-forming and the growth ability of glioblastoma stem cells BT273 and BT379. (a,b) MEOX2 qRT-PCR analysis of BT273 (a) or BT379 (b) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. The values were reported in relation to cells transduced with ctrl vector set as = 1 and normalized to PPP2CA mRNA expression (n = 3; mean ± SD). (c,d) MEOX2 Western blot analysis of BT273 (c) or BT379 (d) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. For BT273 and BT379, α-tubulin and β-actin were used as the internal loading controls, respectively. Representative images are shown. The bottom histograms show the quantification of MEOX2 in relation to α-tubulin and β-actin. (n = 3; mean ± SD). (e,f) Sphere-forming assay of BT273 (e) and BT379 (f) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. Histograms show the percentage of cells capable of re-forming a neurosphere seven days after dissociation (n = 3; mean ± SD). Representative micrographs are shown. (g,h) Growth curves of BT273 (g) and BT379 (h) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. (n = 3; mean ± SD). Differences between two groups were assessed using unpaired Student’s t-test (two-tailed). Significance was defined as * p < 0.05; ** p < 0.01; *** p < 0.001, statistical difference compared to the control.

Figure 2

MEOX2 depletion induces the apoptosis of glioblastoma stem cells BT273 and BT379. (a). Cleaved Caspase-3 Western blot analysis of BT273 and BT379 cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. Total Caspase-3 is shown. β-actin was used as the internal loading control. (n = 3; mean ± SD). One representative image is shown. The histograms on the right show the quantification of Cleaved Caspase-3 in relation to β-actin. (Differences between two groups were assessed using unpaired Student’s t-test (two-tailed). Significance was defined as * p < 0.05; ** p < 0.01, statistical difference compared to the control). (b). One representative dot plot of the percentage of Annexin V/7AAD positive and negative cells analysed by Flow cytometry analysis; the mean values ± s.d. of three independent experiments performed were reported in Table 1. (c). FACS analysis based on Propidium Iodide staining of hypodiploid nuclei fraction (%) in either BT273 or BT379 GSCs transduced with shRNA18 or shRNA53. Data are presented as mean values ± s.d. Differences between two groups were assessed using unpaired Student’s t-test (two-tailed). Significance was defined as ** p ≤ 0.01.

Figure 3

MEOX2 knock down in glioblastoma stem cells differentially modulates gene expression in different cells, but consistently affects some subsets. (a,b) Venn diagrams showing the numbers and percentages of DEGs (absolute log2 fold change ≥ 0.7, adjusted p-value ≤ 0.05) in BT273 or BT379 cells transduced with either shRNA18 or shRNA53, compared to the same cell types transduced with a negative control. Panel a shows the downregulated genes, and panel b the upregulated ones. Each diagram is flanked, on the left, by the list of genes consistently modulated in all three conditions, i.e., BT273 shRNA 18, BT273 shRNA53, BT379 shRNA18.

Figure 4

Enrichment analysis of transcription factor binding motifs in the regulatory regions of genes modulated by MEOX2 knock-down. (a) All DEGs (both down- and upregulated, in both BT273 and BT379 cells, by MEOX2 KD) were subjected to enrichment analysis of TF binding motifs using oPOSSUM-3 software. (b) Only downregulated DEGs analyzed as in (a). (c) Only upregulated DEGs analyzed as in (a). The names of the significantly enriched transcription factor binding motifs (Z score > mean + 2 SD) are shown.