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. 2015 Apr;17(4):555-65.
doi: 10.1093/neuonc/nou282. Epub 2014 Oct 10.

Activation of the NRF2 pathway and its impact on the prognosis of anaplastic glioma patients

Affiliations

Activation of the NRF2 pathway and its impact on the prognosis of anaplastic glioma patients

Masayuki Kanamori et al. Neuro Oncol. 2015 Apr.

Abstract

Background: Nuclear factor erythroid 2-related factor 2 (NRF2) plays pivotal roles in cytoprotection. We aimed at clarifying the contribution of the NRF2 pathway to malignant glioma pathology.

Methods: NRF2 target gene expression and its association with prognosis were examined in 95 anaplastic gliomas with or without isocitrate dehydrogenase (IDH) 1/2 gene mutations and 52 glioblastomas. To explore mechanisms for the altered activity of the NRF2 pathway, we examined somatic mutations and expressions of the NRF2 gene and those encoding NRF2 regulators, Kelch-like ECH-associated protein 1 (KEAP1) and p62/SQSTSM. To clarify the functional interaction between IDH1 mutations and the NRF2 pathway, we introduced a mutant IDH1 to T98 glioblastoma-derived cells and examined the NRF2 activity in these cells.

Results: NRF2 target genes were elevated in 13.7% and 32.7% of anaplastic gliomas and glioblastomas, respectively. Upregulation of NRF2 target genes correlated with poor prognosis in anaplastic gliomas but not in glioblastomas. Neither somatic mutations of NRF2/KEAP1 nor dysregulated expression of KEAP1/p62 explained the increased expression of NRF2 target genes. In most cases of anaplastic glioma with mutated IDH1/2, NRF2 and its target genes were downregulated. This was reproducible in IDH1 R132H-expressing T98 cells. In minor cases of IDH1/2-mutant anaplastic gliomas with increased expression of NRF2 target genes, the clinical outcomes were significantly poor.

Conclusions: The NRF2 activity is increased in a significant proportion of malignant gliomas in general but decreased in the majority of IDH1/2-mutant anaplastic gliomas. It is plausible that the NRF2 pathway plays an important role in tumor progression of anaplastic gliomas with IDH1/2 mutations.

Keywords: IDH1/2 gene; Keap1-NRF2 system; malignant glioma; prognosis.

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Figures

Fig. 1.
Fig. 1.
Expression of NRF2 target genes NQO1 and GCLM in anaplastic gliomas and glioblastomas. (A and B) Scattergrams demonstrating the correlations between NQO1 and GCLM expression in (A) anaplastic gliomas and (B) glioblastomas. (C) NQO1 and GCLM expression in anaplastic gliomas and glioblastomas relative to the normal brain. Data represent means ± SD. Each sample was analyzed in triplicate. *P < .05. (D and E) Scattergrams demonstrating the correlations between NQO1 and KEAP1 expression in (D) anaplastic gliomas and (E) glioblastomas. (F–I) Kaplan–Meier analysis demonstrating (F and G) PFS and (H and I) OS rates (according to the NQO1 and GCLM expression levels in [F and H] anaplastic gliomas and [G and I] glioblastomas). P-values were calculated with the log-rank test.
Fig. 2.
Fig. 2.
Expression of NRF2 target genes in anaplastic gliomas with and without mutated IDH1/2. (A) Heat map showing the expression of the 5 NRF2 target genes in anaplastic gliomas with (cases 1–6) and without (cases 7–12) mutated IDH1/2. A sample with a median value was defined as the control. The gray scale indicates the level of fold change in gene expression relative to the control value. The darkest and the brightest colors indicate a 4-fold increase and decrease, respectively. (B) Expression of NQO1 and GCLM in anaplastic gliomas with and without IDH1/2 mutations and glioblastomas relative to the normal brain. Data represent means ± SD. Each sample was analyzed in triplicate. *P < .05. (C) Protein abundance of NQO1 and GCLM in anaplastic gliomas with and without IDH1/2 mutations. Band intensities of NQO1 and GCLM were quantified and normalized with those of alpha-tubulin. Values of case 1 were set as one. (D–F) Profiles of CpG methylation ratio in the promoter regions of (D) MGMT, (E) NQO1, and (F) GCLM in anaplastic gliomas with mutated (n = 64) and wild-type IDH1/2 (n = 24). Data are expressed as the averages and SDs of the methylation ratios of each CpG unit. *P < .05. (G) Scattergrams demonstrating the correlations between NQO1 and NRF2 expression and between GCLM and NRF2 expression in anaplastic gliomas. (H) Expression of NRF2 in anaplastic gliomas with mutated and wild-type IDH1/2 genes relative to the normal brain. Data represent means ± SD. Each sample was analyzed in triplicate. *P < .05.
Fig. 3.
Fig. 3.
Effect of IDH1 R132H expression on NRF2 activity in T98 glioblastoma cells. (A) Expression levels of exogenously introduced IDH1 and IDH1 R132H. Exogenous IDH1 and IDH1 R132H were detected with anti-FLAG antibody. Lamin B was used as a loading control. (B and C) Relative mRNA levels of (B) NQO1, GCLC, and GCLM and (C) NRF2. Data represent the means ± SD of 5 independent experiments, and each sample was analyzed in triplicate. The expression levels in IDH1-expressing cells were set as one. **P < .005, ***P < .001. (D) NRF2 protein abundance in nuclear extracts. Lamin B was detected as a loading control. Band intensities of NRF2 were quantified and normalized with those of Lamin B. A value of IDH1-expressing cells was set as one. (E) Location and sequence of AREs in NQO1, GCLC, and GCLM gene loci. (F) ChIP assay with anti-NRF2 antibody in IDH1- and IDH1 R132H–expressing cells. In the control samples, rabbit IgG was added instead of anti-NRF2 antibody. NQO1 promoter, GCLC enhancer, and GCLM promoter, which contain AREs, and NQO1 exon 2, which does not contain AREs, were examined. Data represent the means ± SD of triplicate samples. A representative result of 3 independent experiments is shown. (G) Metabolomic profiling. Data represent the means ± SD of 3 independent samples. The expression levels in IDH1-expressing cells were set as one. P-values were calculated with Student's t-test. *P < .05, **P < .005, ***P < .001. Promoted and inhibited processes in IDH1 R132H–expressing cells are indicated with red and blue arrows, respectively. Abbreviations of the metabolites are described in the Supplementary information.
Fig. 4.
Fig. 4.
Impact of NRF2 activity on drug sensitivity and prognosis under the influence of IDH1/2 mutation status. (A and B) BCNU sensitivity of T98 cells expressing (A) IDH1 or IDH1 R132H and (B) T98 cells with NRF2 knockdown. Cells were treated with increasing concentration of BCNU, and their viability was examined at 48 h after the addition of BCNU. All samples were prepared in triplicate. Three independent experiments were conducted, and the means and SDs were calculated. *P < .05, ***P < .001 ([A] and control vs NRF2 siRNA-1 in [B]), ##P < .005, ###P < .001 (control vs NRF2 siRNA-2 in [B]). (C–F) Kaplan–Meier analyses demonstrating (C and D) the PFS and (E and F) OS rates according to NQO1/GCLM expression levels in anaplastic gliomas with (C and E) mutated and (D and F) wild-type IDH1/2 genes. P-values were calculated with the log-rank test.

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