Identification of aurintricarboxylic acid as a selective inhibitor of the TWEAK-Fn14 signaling pathway in glioblastoma cells

Abstract

The survival of patients diagnosed with glioblastoma (GBM), the most deadly form of brain cancer, is compromised by the proclivity for local invasion into the surrounding normal brain, which prevents complete surgical resection and contributes to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) superfamily, can stimulate glioma cell invasion and survival via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the transcription factor NF-κB. To discover small molecule inhibitors that disrupt the TWEAK-Fn14 signaling axis, we utilized a cell-based drug-screening assay using HEK293 cells engineered to express both Fn14 and a NF-κB-driven firefly luciferase reporter protein. Focusing on the LOPAC1280 library of 1280 pharmacologically active compounds, we identified aurintricarboxylic acid (ATA) as an agent that suppressed TWEAK-Fn14-NF-κB dependent signaling, but not TNFα-TNFR-NF-κB driven signaling. We demonstrated that ATA repressed TWEAK-induced glioma cell chemotactic migration and invasion via inhibition of Rac1 activation but had no effect on cell viability or Fn14 expression. In addition, ATA treatment enhanced glioma cell sensitivity to both the chemotherapeutic agent temozolomide (TMZ) and radiation-induced cell death. In summary, this work reports a repurposed use of a small molecule inhibitor that targets the TWEAK-Fn14 signaling axis, which could potentially be developed as a new therapeutic agent for treatment of GBM patients.

Keywords: Fn14; aurintricarboxylic acid; glioblastoma; invasion; survival.

Figure 1. ATA inhibited TWEAK-Fn14-mediated NF-κB activation

A. Schematic drawing of TWEAK-Fn14 signaling pathway leading to NF-κB-driven luciferase expression in reporter cell lines. B. Structure of ATA. C. Dose response curve of inhibitory activity of ATA in NF-κB-Luc and NF-κB-Luc/Fn14 cells following TWEAK or TNFα stimulation. D. ATA effects on NF-κB-Luc and NF-κB-Luc/Fn14 cell growth as measured by CellTiterGlo® assay.

Figure 2. ATA suppressed TWEAK-Fn14 downstream pathway activation in glioma cells

A. T98G, A172, and GBM44 glioma cells were treated with ATA (10 μM) for the indicated times, lysed, and Western blot analysis was performed using Fn14 and α-tubulin antibodies. B. T98G, A172, and GBM44 glioma cells were stimulated with TWEAK (100 ng/mL) in presence or absence of ATA (10 μM) for 10 min, lysed, and Western blot analysis was performed using Phospho NF-κB (p65), Total NF-κB (p65), Pan Phospho Src, Total Src, Phospho AKT, Total AKT, and α-tubulin antibodies.

Figure 3. ATA blocked TWEAK-stimulated Rac1 activation and TRAF2 recruitment to Fn14 cytoplasmic domain in glioma cells

A. T98G and A172 glioma cells were stimulated with TWEAK (100 ng/mL) in presence or absence of ATA (10 μM) for 10 min and lysed. Rac1 activity was determined using Rac1 activity assay kit. B. A172 and GBM44 glioma cells were stimulated with TWEAK (100 ng/mL) in presence or absence of ATA (10 μM) for 2 min and lysed. Cell lysates were immunoprecipitated using TRAF2 or IgG control antibody and then Western blot analysis was conducted using Fn14 and TRAF2 antibodies.

Figure 4. ATA repressed TWEAK-stimulated glioma cell migration and invasion without causing cell cytotoxicity

A & B. A172, GBM44, and T98G glioma cells were added to the top well of a modified transwell chamber pre-coated with collagen in serum-free DMEM + 0.1% BSA to assess migration (A) or 10 mg/mL growth factor-free Matrigel to assess invasion (B). Either TWEAK alone (100 ng/mL) or TWEAK and ATA (10 μM) was added to the lower wells and the number of cells migrated to the bottom chamber quantitated after 5 hr. Values are mean ± standard deviation of triplicate measurements (*, p < 0.01). C. Glioma cells were seeded in 96-well plates and after 24 hr of incubation either vehicle (DMSO) or ATA at indicated concentration was added to each well. After 72 hr of incubation viability of the cells was measured using CellTiterGlo assay kit. Values are mean ± standard deviation of six separate measurements.

Figure 5. ATA suppressed TWEAK-stimulated glioma cell survival after TMZ and radiation therapy

A. T98G, A172, and GBM44 glioma cells were treated with ATA (10 μM), TMZ (250 μM), ATA + TMZ, Radiation (2 Gy), and ATA + Radiation in presence or absence of TWEAK (100 ng/mL) for 48 hr and lysed. Western blot analysis was performed using cleaved PARP and α-tubulin antibodies. B & C. A172, GBM44, and T98G glioma cells were treated with ATA (10 μM), TMZ (500 μM), ATA + TMZ, Radiation (2Gy), and Radiation + ATA in presence or absence of TWEAK (100 ng/mL) for 24 hr. Cells were trypsinized and 250 cells were seeded in triplicate in 35 mm dishes and allowed to form colonies. At the end of the assay cells were fixed in PFA and stained with crystal violet, and number of colonies were counted. Values are mean ± standard deviation of three separate measurements (**p<0.01)

Figure 6. Knockdown of Fn14 expression concurrent with TMZ treatment significantly increases animal survival

A. Kaplan-Meier survival curves of athymic nude mice with intracranial xenografts of GBM44 cells transduced with a control shRNA or shRNA targeting Fn14 and treated with/without TMZ(control shRNA + TMZ vs. Fn14 shRNA + TMZ p<0.0008). B. a, c, e, g = control shRNA; b, d, f, h = Fn14 shRNA. a, b) H&E staining of GBM44 PDX tumor (T) and normal (N) brain tissue. Bar=300 uM. c, d) pH2AX staining of vehicle-treated brain tissue.e, f) pH2AX staining of TMZ-treated brain tissue. g, h) Cleaved Caspase 3 staining of TMZ-treated tumor tissue. Arrows highlight apoptotic cells. Bars for c-h=100 uM. C. Quantification of γH2AX positive nuclei in tumors expressing a control shRNA or Fn14 shRNA and treated with the vehicle alone or TMZ. D. Quantification of cleaved caspase 3 positive nuclei in control shRNA or Fn14 shRNA tumors treated with TMZ.