Reprogramming of arachidonate metabolism confers temozolomide resistance to glioblastoma through enhancing mitochondrial activity in fatty acid oxidation

Abstract

Background: Sp1 is involved in the recurrence of glioblastoma (GBM) due to the acquirement of resistance to temozolomide (TMZ). Particularly, the role of Sp1 in metabolic reprogramming for drug resistance remains unknown.

Methods: RNA-Seq and mass spectrometry were used to analyze gene expression and metabolites amounts in paired GBM specimens (primary vs. recurrent) and in paired GBM cells (sensitive vs. resistant). ω-3/6 fatty acid and arachidonic acid (AA) metabolism in GBM patients were analyzed by targeted metabolome. Mitochondrial functions were determined by Seahorse XF Mito Stress Test, RNA-Seq, metabolome and substrate utilization for producing ATP. Therapeutic options targeting prostaglandin (PG) E2 in TMZ-resistant GBM were validated in vitro and in vivo.

Results: Among the metabolic pathways, Sp1 increased the prostaglandin-endoperoxide synthase 2 expression and PGE2 production in TMZ-resistant GBM. Mitochondrial genes and metabolites were obviously increased by PGE2, and these characteristics were required for developing resistance in GBM cells. For inducing TMZ resistance, PGE2 activated mitochondrial functions, including fatty acid β-oxidation (FAO) and tricarboxylic acid (TCA) cycle progression, through PGE2 receptors, E-type prostanoid (EP)1 and EP3. Additionally, EP1 antagonist ONO-8713 inhibited the survival of TMZ-resistant GBM synergistically with TMZ.

Conclusion: Sp1-regulated PGE2 production activates FAO and TCA cycle in mitochondria, through EP1 and EP3 receptors, resulting in TMZ resistance in GBM. These results will provide us a new strategy to attenuate drug resistance or to re-sensitize recurred GBM.

Keywords: Fatty acid β-oxidation; Mitochondria; PGE2; Sp1; TMZ-resistant GBM.

Fig. 1

Regulation of phospholipid and AA metabolism by Sp1. a Sp1-regulated metabolic genes. After Sp1 knockdown by siRNA, U87MG cell RNA was collected and subjected to RNA-Seq. b AA-related metabolite alteration determined via UHPLC-QTOF-MS. U87MG and U87MG-Sp1 cKO cells were subjected to mass spectrometry-based metabolomics analysis. The alteration of metabolite levels was illustrated by performing two-tailed unpaired Student’s t test. The chemical structures were obtained from PubChem, National Library of Medicine. The complete definitions of the different abbreviations are provided in Additional file 1: Table S5

Fig. 2

The Sp1-regulated COX2/PTGS pathway in recurrent glioblastoma. Effects of multiple inhibitors, including a Pyrrophenone (left) and celecoxib (right), b Zileuton (left) and ML-355 (right) on the viability of U87MG-R cells. The cells were treated with TMZ in the presence of the indicated inhibitors for 72 h, and cell viability was determined via MTT assay. c Paired primary and recurrent glioblastoma specimens. Samples were collected from 14 patients and subjected to RNA-Seq. The red arrow-marked genes play important roles in the metabolic pathway involving the synthesis of prostaglandins from AA (the COX pathway) as shown on the right panel. d Defined promoter regions of PLA2G5, ABHD8, and PTGS2. The binding regions of Sp1 were determined via ChIP-Seq. e Sp1-induced activities of pGL2-conjugated promoter constructs, including PLA2G5, ABHD8, and PTGS2. Luciferase reporter assay was employed to analyse the promoter activities in wild type and TMZ-resistant glioblastoma cells. Data were analysed by performing two-tailed unpaired Student’s t test

Fig. 3

Effect of Sp1-regulated PGE2 production and secretion on TMZ-resistant glioblastoma. a Targeted ω-6 fatty acid metabolome analysis for paired primary and recurrent glioblastoma specimens from five patients based on UHPLC-QTOF-MS. Data in bar plots were analysed by performing two-tailed unpaired Student’s t test. The alteration in prostanoids from each patient was expressed as a heatmap. The full definitions of the different abbreviations are provided in Additional file 1: Table S6. b Effect of PGE2 on glioblastoma cell viability. After treatment with TMZ in the presence of PGE2 for four days, cell viability was estimated via MTT assay. c, d Level of PGE2 in culture media estimated via ELISA. e, f Cell lysates after treatment with TMZ for the indicated time interval analysed via Western blotting. The level of PGE2 in the culture media was determined via ELISA. Data and the quantifications were analysed by performing two-tailed unpaired Student’s t test

Fig. 4

Effect of PGE2 on mitochondrial function. a RNA expression in cell line from patient with glioblastoma and paired glioblastoma specimens. b Mitochondrial morphology was fluorescently labelled by the pDsRed 2-Mito expression clone. Statistical data were analysed by performing two-tailed unpaired Student’s t test. Scale bar is 50 μm. c Effect of TMZ on mitochondrial fusion-related protein expression. d Mitochondrial activities of U87MG, U87MG-R, and U87MG-Sp1KO cells determined by performing Seahorse XF Mito Stress tests. e Mitochondrial activity determined via Seahorse XF Mito Stress tests after treatment with PGE2 for four days. f Mitochondrial activity after treatment with TMZ in the presence or absence of PGE2 for four days. g Mitochondrial activity after treatment with PGE2 in the presence of each antagonist for four days. Statistical data were analysed by performing two-tailed unpaired Student’s t test

Fig. 5

Effect of Sp1-regulated PGE2 on FAO and TCA cycles. a RNA expression in paired glioblastoma specimens. b Expression of mRNA in Pt#3 and Pt#3-R cells determined via real-time PCR. c Cell lysates after treatment with PGE2 for four days based on Western blotting. d Percentage of FAO measured by performing Seahorse XF Mito Stress Tests. e After transfection with Sp1, PTGS2, or CPT1A siRNA for three days, mitochondrial activity of the TMZ-resistant U87MG-R and Pt#3-R was determined. f left panel: FAO metabolic pathway; right panel: wild type Pt#3 and TMZ-resistant Pt#3-R cells subjected to metabolomics analysis. g Pt#3 cells after treatment with PGE2 for four days based on targeted metabolomics analysis. The alteration of the levels of the different metabolites is illustrated using bar plots. Data were analysed by performing two-tailed unpaired Student’s t test. h A172 and A172R cells after treatment with PGE2 for four days based on MitoPlate assay for 2 h. Data were analysed by performing two-tailed unpaired Student’s t test

Fig. 6

Therapeutic effect of EP antagonists on TMZ-resistant glioblastoma. a, b Effect of EP1 antagonist, ONO-8713, or EP3 antagonist, ONO-AE3-240, on glioblastoma cells. The cells were treated with ONO-8713 or ONO-AE3-240 in the presence of TMZ and PGE2 for 96 h. Cell viability was determined via MTT assay. c TMZ-resistant glioblastoma cells treated with different compounds in the presence of TMZ for 96 h. Cell viability was determined by performing MTT assay. (# implies a significant difference compared with the TMZ-treated group). d After treatment with ONO-8713 for four days, cell lysates were prepared for Western blotting. e, f After treatment for 48 h, migratory and invasive activities of TMZ-resistant GBM cells were determined by wound-healing and transwell invasion assays, respectively. g, h Effect of ONO-8713 or celecoxib on the growth of TMZ-resistant cell-derived tumour in xenograft mouse model. The difference between the control and experimental groups was analysed by performing two-way ANOVA. The scale bar is 1 cm

Fig. 7

Schematic representation of the working model. According to established gene expression profiles and ω-3/6 fatty acid metabolome, we found that Sp1 increased the PTGS2 expression and PGE2 production in recurrent GBM patients and in TMZ-resistant GBM cells. For inducing TMZ resistance, PGE2 activated mitochondrial FAO and TCA cycle progression through enhancing mitochondrial fusion. These results showing the role of PGE2 metabolism in GBM provide us a new strategy to attenuate drug resistance or to re-sensitize recurred glioblastoma