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. 2018 Jan 8;9(7):7541-7556.
doi: 10.18632/oncotarget.24045. eCollection 2018 Jan 26.

Dual targeting of mitochondrial function and mTOR pathway as a therapeutic strategy for diffuse intrinsic pontine glioma

Maria Tsoli  1 , Jie Liu  1 , Laura Franshaw  1 , Han Shen  1 , Cecilia Cheng  1 , MoonSun Jung  2 , Swapna Joshi  1 , Anahid Ehteda  1 , Aaminah Khan  1 , Angel Montero-Carcabosso  3 , Pierre J Dilda  4 , Philip Hogg  5 , David S Ziegler  1   6
Affiliations

Dual targeting of mitochondrial function and mTOR pathway as a therapeutic strategy for diffuse intrinsic pontine glioma

Maria Tsoli et al. Oncotarget. .

Abstract

Diffuse Intrinsic Pontine Gliomas (DIPG) are the most devastating of all pediatric brain tumors. They mostly affect young children and, as there are no effective treatments, almost all patients with DIPG will die of their tumor within 12 months of diagnosis. A key feature of this devastating tumor is its intrinsic resistance to all clinically available therapies. It has been shown that glioma development is associated with metabolic reprogramming, redox state disruption and resistance to apoptotic pathways. The mitochondrion is an attractive target as a key organelle that facilitates these critical processes. PENAO is a novel anti-cancer compound that targets mitochondrial function by inhibiting adenine nucleotide translocase (ANT). Here we found that DIPG neurosphere cultures express high levels of ANT2 protein and are sensitive to the mitochondrial inhibitor PENAO through oxidative stress, while its apoptotic effects were found to be further enhanced upon co-treatment with mTOR inhibitor temsirolimus. This combination therapy was found to act through inhibition of PI3K/AKT/mTOR pathway, HSP90 and activation of AMPK. In vivo experiments employing an orthotopic model of DIPG showed a marginal anti-tumour effect likely due to poor penetration of the inhibitors into the brain. Further testing of this anti-DIPG strategy with compounds that penetrate the BBB is warranted.

Keywords: DIPG; PDGFR; mTOR; mitochondria; paediatric brain tumour.

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Conflict of interest statement

CONFLICTS OF INTEREST No conflicts of interest.

Figures

Figure 1
Figure 1. ANT2 is a potential therapeutic target for DIPG
(A) Representative western blot image of ANT2 protein in a panel of DIPG cells and normal astrocytes (NHA); (B) Cytotoxic efficacy of PENAO tested for 72 h against a panel of neurosphere-forming DIPG cells. Viability was assessed by resasurin assay and presented as a percentage viability compared to untreated cells; Experiment was replicated 3 times; (C) Flow cytometric analysis of HSJD-DIPG007 cells for production of mitochondrial ROS. DIPG cells were treated with increasing concentration of PENAO (P) for 18 h and subsequently stained with MitosoxRed and analysed with FACS Canto B. Data represent average and SD of 3 determinations; untreated vs PENAO p < 0.001 for all concentrations tested; For positive control DIPG cells were treated with 100 uM antimycin for 1 h and analysed. (D) Flow cytometric analysis of HSJD-DIPG007 cells for mitochondrial depolarisation. DIPG cells were treated with increasing concentration of PENAO for 18 h and subsequently stained with JC1 and analysed with FACS Canto B. Data represent average and SD of 3 determinations; untreated vs PENAO p < 0.01 for all concentrations tested. For positive control DIPG cells were treated with 50 uM CCCP for 2 h and analysed.
Figure 2
Figure 2. PENAO induces apoptotic cell death and affects major oncogenic pathways in DIPG cells
(A) Flow cytometric analysis of HSJD-DIPG007 cells for apoptotic cell death. HSJD-DIPG007 cells were treated with increasing concentration of PENAO (P) for 42 h and subsequently stained with AnnexinV-FITC and 7AAD and analysed with FACS Canto B. Data represent average and SD of 3 determinations; untreated vs PENAO p < 0.01 for all concentrations tested. (B) Representative western blot images of key players involved in RTK/PI3K/mTOR pathway in HSJD-DIPG007 cells treated with PENAO as a function of time. DIPG cells were treated with 3 µM of PENAO for 24 h, 48, 72 h and subsequently lysed pellets were examined by western blotting.
Figure 3
Figure 3. Inhibition of mTOR with temsirolimus enhances cytotoxic activity of PENAO and decreases clonogenic ability of DIPG cells
(A) Representative western blot image of phosphorylated mTOR at Ser2448 as well as total mTOR levels in a panel of primary DIPGs and NHA cells (A), and patient DIPG autopsy samples with matched cerebellum (cer); (B–D) Cytotoxic efficacy of PENAO combined with temsirolimus tested at IC50 fractions in 3 neurosphere-forming DIPG cell lines. Viability was assessed by resasurin assay and presented as percentage viability compared to untreated cells; Synergy scores (CI) are included for each cell line. Experiment was replicated twice; each time n = 10. (E–F) Soft-agar colony assays performed in two DIPG cell lines show enhancement of irradiation upon combination with PENAO/temsirolimus; SU-DIPG-VI cells were treated with 25 nM of PENAO and 25 nM of temsirolimus, HSJD-DIPG007 cells were treated with 200 nM of PENAO and 50 nM of temsirolimus; Colonies were stained with MTT after 2 weeks and counted. Data represent surviving fractions; Experiment was performed twice n = 4 each time. SU-DIPG-VI, P/4Gy vs P/T/4Gy p < 0.01, T/4Gy vs P/T/4Gy p < 0.01; 4Gy vs P/T/4Gy p < 0.01; HSJD-DIPG007 P/4Gy vs P/T/4Gy p < 0.001, T/4Gy vs P/T/4Gy p < 0.05, 4Gy/P/T/4Gy p < 0.001.
Figure 4
Figure 4. Combination of PENAO with temsirolimus increases mitochondrial dysfunction and enhances apoptosis
(A) Flowcytometric analysis of HSJD-DIPG007 cells for production of mitochondrial ROS. DIPG cells were treated with 0.25 µM of PENAO, 2.5 µM temsirolimus, combined agents for 18 h and subsequently stained with MitosoxRed and analysed with FACS Canto B. Data represent average and SD of 3 determinations, PENAO vs Combination p < 0.001; temsirolimus vs Combination p < 0.001; (B) Flow cytometric analysis of HSJD-DIPG007 cells for mitochondrial depolarisation. DIPG cells were treated with 0.25 µM of PENAO, 2.5 µM temsirolimus, combined agents for 18 h and subsequently stained with JC1 and analysed with FACS Canto B. Data represent average and SD of 3 determinations; PENAO vs combination p < 0.05; temsirolimus vs combination p < 0.01; (C) Flow cytometric analysis of HSJD-DIPG007 cells for apoptotic cell death. DIPG cells were treated with 5 µM of PENAO, 10 µM temsirolimus, combined agents for 48 h and subsequently stained with AnnexinV-FITC and 7AAD and analysed with FACS Canto B. Data represent averages and SD of 3 determinations. PENAO vs combination p < 0.001; temsirolimus vs combination p < 0.001.
Figure 5
Figure 5. Combination of PENAO with temsirolimus affects PDGFRa/PI3K/mTOR pathway, HSP90 and activates AMPK
(A) Representative image from western blot analysis of PDGFRa/PI3K/mTOR pathway in HSJD-DIPG007 cells treated with PENAO, temsirolimus and dual therapy for 48 h. Significant decrease is observed in protein levels in PDGFR, PI3K subunits, mTOR and its targets (B) Expression analysis of a panel of genes involved in AMPK pathway in HSJD-DIPG007 cells treated with PENAO, temsirolimus and dual therapy for 48h. Significant increase in mRNA levels of AMPK subunits and its targets Tsc1, and Deptor is observed, (p values for: PRKAB1: V vs C < 0.05, P vs C < 0.05, T vs C < 0.01, PRKAB2: V vs C < 0.01, P vs C < 0.05, T vs C< 0.05; PRKAG1, V vs C ns, P vs C ns, T vs C ns; PRKAG2, V vs C < 0.01, P vs C < 0.05, T vs C < 0.01; PRKAG3, V vs C < 0.05, P vs C < 0.05, T vs C < 0.05; TSC1, V vs C < 0.05, P vs C < 0.05, T vs C < 0.05; DEPTOR, V vs C < 0.01, P vs C < 0.05, T vs C < 0.001; TEL2, V vs C < 0.05, P vs C < 0.05, T vs C < 0.05); ULK1 V vs C < 0.05, P vs C < 0.05, T vs C < 0.05; (C) Representative image from western blot analysis of mTOR regulators AMPK and HSP90 in HSJD-DIPG007 cells treated with PENAO, temsirolimus and dual therapy for 48 h. Significant decrease is observed in phosphorylation of AMPK indicating activation and protein levels of HSP90. (D) ATP determination in HSJD-DIPG007 cells treated with PENAO, temsirolimus and dual therapy for 48h. Significant decrease in ATP levels is observed in PENAO and combination treatments; Experiment performed twice each time N = 5; P vs C p < 0.001, T vs C p < 0.001.
Figure 6
Figure 6. Therapeutic efficacy of PENAO, temsirolimus and combination in the HSJD-DIPG007 orthotopic animal model
(A) Survival curve of orthotopicaly-injected DIPG animals treated with vehicle, PENAO administered by alzet Pump (3 mg/kg/day 7 days/week, 4 weeks), temsirolimus (10 mg/kg/day 5 days/week 2 weeks and 5 mg/kg/day 5 days/week 2 weeks) and combination. Each treatment cohort consisted of 12 animals and tumour engraftment was assessed by neurological symptoms and/or weight loss. At endpoint animals were sacrificed and brains were examine histologically with H&E and Ki67 stains. (B) Histological analysis of harvested brains from each treatment cohort. Ki67 positive cells were quantified in 6 separate fields of view in the pons region from 3–4 animals per cohort. (C) Mass spectrometric analysis of arsenic levels in brains from vehicle and PENAO-treated DIPG xenografts. (D) Survival curve of orthotopicaly-injected DIPG animals treated with vehicle, PENAO administered intraperitonealy (10 mg/kg/day 5 days/week), temsirolimus (10 mg/kg/day 5 days/ week) and combination for 4 weeks. Each treatment cohort consisted of 12 animals and tumor engraftment was assessed by neurological symptoms and/or weight loss.
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