Investigating Glioblastoma Response to Hypoxia

Abstract

Glioblastoma (GB) is the most common and deadly type of primary malignant brain tumor with an average patient survival of only 15-17 months. GBs typically have hypoxic regions associated with aggressiveness and chemoresistance. Using patient derived GB cells, we characterized how GB responds to hypoxia. We noted a hypoxia-dependent glycolytic switch characterized by the up-regulation of HK2, PFKFB3, PFKFB4, LDHA, PDK1, SLC2A1/GLUT-1, CA9/CAIX, and SLC16A3/MCT-4. Moreover, many proangiogenic genes and proteins, including VEGFA, VEGFC, VEGFD, PGF/PlGF, ADM, ANGPTL4, and SERPINE1/PAI-1 were up-regulated during hypoxia. We detected the hypoxic induction of invasion proteins, including the plasminogen receptor, S100A10, and the urokinase plasminogen activator receptor, uPAR. Furthermore, we observed a hypoxia-dependent up-regulation of the autophagy genes, BNIP-3 and DDIT4 and of the multi-functional protein, NDRG1 associated with GB chemoresistance; and down-regulation of EGR1 and TFRC (Graphical abstract). Analysis of GB patient cohorts' revealed differential expression of these genes in patient samples (except SLC16A3) compared to non-neoplastic brain tissue. High expression of SLC2A1, LDHA, PDK1, PFKFB4, HK2, VEGFA, SERPINE1, TFRC, and ADM was associated with significantly lower overall survival. Together these data provide important information regarding GB response to hypoxia which could support the development of more effective treatments for GB patients.

Keywords: autophagy; glioblastoma; glycolysis; hypoxia; invasion; tumor angiogenesis.

Figure 1

Analysis of hypoxia related genes in SEBTA-023 and UP-029 cell lines. SEBTA-023 and UP-029 cells were either incubated under normoxic (NT) conditions (21% O₂) or under hypoxia (1% O₂) for the times indicated. (A) SEBTA-023 or (B) UP-029 cell lysates were prepared and 20 µg of each protein extract was subjected to SDS-PAGE and analyzed by Western blotting with the antibodies indicated. (C,D) RNA extraction was performed using the NZY Total RNA Isolation kit (Nzytech, Lisbon, Portugal). A panel of 86 ROS dependent genes was analyzed using the Hypoxia Signaling Pathway RT² Profiler PCR Array (QIAGEN, Manchester, UK). (C) Scatter plots for gene expression in hypoxia vs. normoxia in SEBTA-023 cells; (D) Scatter plots for gene expression in hypoxia vs. normoxia in UP-029 cells; (E) Venn diagram representing differential gene expression in hypoxia vs. normoxia in SEBTA-023 and UP-029 cells. (F) Table containing the genes that were differentially expressed in both SEBTA-023 and UP-029 cells throughout the hypoxia time-course. Yellow highlights unchanged genes, red highlights over-expressed genes and green highlights down-regulated genes in hypoxia compared to normoxic conditions.

Figure 2

Analysis of transcription factor genes and proteins in hypoxic Glioblastoma (GB) cells. (A) UP-007; (B) SEBTA-003; or (C) U87 cells were incubated under normoxia (21% O₂) or hypoxia (1% O₂) for the times indicated. Cells were lysed and 20 µg of each protein extract was subjected to SDS-PAGE and analyzed by Western blotting with the antibodies indicated. (D) UP-007; (E) UP-029; (F) SEBTA-003; (G) SEBTA-023; or (H) U87 cells were incubated under normoxia (21% O₂) or hypoxia (1% O₂) for the times indicated. RNA extraction was performed and gene expression was determined by RT-qPCR. Gene expression levels were normalized to RPLP0 mRNA using the 2^−ΔΔCT method [13]. Error bars represent the Standard Deviations obtained from the median value of at least three independent experiments, each performed in triplicate. Statistical analysis was evaluated using two-tailed Student’s t-test, comparing each hypoxia time-point to the respective normoxic control. In every case a p value of less than 0.05 (*), less than 0.01 (**), and 0.001 (***) was considered statistically significant.

Figure 3

Analysis of glycolysis related genes in hypoxic GB cells. (A) UP-007; (B) UP-029; (C) SEBTA-003; (D) SEBTA-023; or (E) U87 cells were incubated under normoxia (21% O₂) or hypoxia (1% O₂) for the times indicated. RNA extraction was performed and gene expression was determined by RT-qPCR. Gene expression levels were normalized to RPLP0 mRNA using the 2^−ΔΔCT method [13]. Error bars represent the Standard Deviations obtained from the median value of at least three independent experiments, performed in triplicate. Statistical analysis was done using two-tailed Student’s t-test, comparing each hypoxia time-point to the respective normoxic control. In every case a p value of less than 0.05 (*), less than 0.01 (**), and 0.001 (***) was considered statistically significant.

Figure 4

Analysis of angiogenic genes in hypoxic GB cells. (A) UP-007; (B) UP-029; (C) SEBTA-003; (D) SEBTA-023, or (E) U87 cells were incubated under normoxia (21% O₂) or hypoxia (1% O₂) for the times indicated. RNA extraction was performed and gene expression was determined by RT-qPCR. Gene expression levels were normalized to RPLP0 mRNA using the 2^-ΔΔCT method [13]. Error bars represent the standard deviations obtained from the median value of at least three independent experiments, each performed in triplicate. Statistical analysis was evaluated using two-tailed Student’s t-test, comparing each hypoxia time-point to the respective normoxic control. In every case a p value of less than 0.01 (**) and 0.001 (***) was considered statistically significant.

Figure 5

Analysis of invasion related genes in hypoxic GB cells. (A) UP-007; (B) UP-029; (C) SEBTA-003; (D) SEBTA-023; or (E) U87 cells were incubated under normoxia (21% O₂) or hypoxia (1% O₂) for the times indicated. RNA extraction was performed and gene expression was determined by RT-qPCR. Gene expression levels were normalized to RPLP0 mRNA using the 2^−ΔΔCT method [13]. Error bars represent the Standard Deviations obtained from the median value of at least three independent experiments, each performed in triplicate. Statistical analysis was evaluated using two-tailed Student’s t-test, comparing each hypoxia time-point to the respective normoxic control. In every case a p value of less than 0.05 (*), less than 0.01 (**) and 0.001 (***) was considered statistically significant. Relative gene expression compared to the housekeeping gene, RPLP0, under normoxic conditions for (F) UP-007; (G) UP-029; (H) SEBTA-003; (I) SEBTA-023; or (J) U87 cells. Relative expression was calculated by applying the following formula 2^−ΔΔCt, where ΔCt is obtained by subtracting RPLP0 Ct value to the Ct value of our genes of interest, as indicated. Error bars represent the Standard Deviations obtained from at least three independent experiments.

Figure 6

Analysis of varied functions genes in hypoxic GB cells. (A) UP-007; (B) UP-029; (C) SEBTA-003; (D) SEBTA-023; or (E) U87 cells were incubated under normoxia (21% O₂) or hypoxia (1% O₂) for the times indicated. RNA extraction was performed and gene expression was determined by RT-qPCR. Gene expression levels were normalized to RPLP0 mRNA using the 2^−ΔΔCT method [13]. Error bars represent the Standard Deviations from the median value of at least three independent experiments, performed in triplicate. Statistical analysis was evaluated using two-tailed Student’s t-test, comparing each hypoxia time-point to the respective normoxic control. In every case a p value of less than 0.05 (*), less than 0.01 (**) and 0.001 (***) was considered statistically significant.

Figure 7

Analysis of hypoxia related proteins in GB cells. UP-007; UP-029; SEBTA-003; SEBTA-023 or U87 cells were incubated under normoxia (21% O₂) or hypoxia (1% O₂) for the times indicated. Cells were lysed and 20 µg of each protein extract was subjected to SDS-PAGE, and analyzed by Western blotting with the antibodies indicated. NT- non treated/ normoxia.

Figure 8

Expression of hypoxia-related genes in GB patients. (A) Expression of selected hypoxia-related genes in normal brain and GB patient samples. Data sourced from the published study with gene expression microarrays generated from 7 normal brain and 217 GB tumors. Normalized expression values on log₂ scale are indicated on the y-axis. In the boxplots, the top, middle and bottom box delimiters represent the 75th, 50th, and 25th percentiles of the data, respectively. Top and bottom whiskers show the 75th percentile + 1.5*interquartile range and 25th percentile – 1.5*interquartile range, respectively. The ‘*’ above the boxplot indicates that the change in gene expression between normal and GB samples are significant at false discover rate, FDR < 0.05. (B) Expression of subset of hypoxia-related genes in non-neoplastic brain tissue samples (n = 17), contrast-enhancing GB core samples (n = 39) and non-enhancing GB invasive margin samples (n = 36). Normalized expression values on log2 scale are indicated on the y-axis and were obtained from the RNA-Seq data of the published study. The ‘*’ above the boxplot indicates that the change in gene expression between normal and GB core samples are significant at false discover rate, FDR < 0.05, whereas the “**” denotes the significance between normal and GB core as well as normal and GB invasive margin samples. (C–K) Kaplan–Meier survival curves of GB patients (n = 217) divided in high and low expression levels for each gene (split by median expression) as indicated. Survival curves were compared using the log-rank test. Genes that show significant association (p < 0.05) with the overall survival are shown.