EGFR-targeted diphtheria toxin stimulates TRAIL killing of glioblastoma cells by depleting anti-apoptotic proteins

Abstract

Current treatments for Glioblastoma multiforme (GBM) involve surgery, radiotherapy, and cytotoxic chemotherapy; however, these treatments are not effective and there is an urgent need for better treatments. We investigated GBM cell killing by a novel drug combination involving DT-EGF, an Epidermal Growth Factor Receptor-targeted bacterial toxin, and Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) or antibodies that activate the TRAIL receptors DR4 and DR5. DT-EGF kills GBM cells by a non apoptotic mechanism whereas TRAIL kills by inducing apoptosis. GBM cells treated with DT-EGF and TRAIL were killed in a synergistic fashion in vitro and the combination was more effective than either treatment alone in vivo. Tumor cell death with the combination occurred by caspase activation and apoptosis due to DT-EGF positively regulating TRAIL killing by depleting FLIP, a selective inhibitor of TRAIL receptor-induced apoptosis. These data provide a mechanism-based rationale for combining targeted toxins and TRAIL receptor agonists to treat GBM.

Figure 1. DT-EGF kills U87MG cells by a caspase independent cell death mechanism

(A) Representative micrographs of U87MG cells treated with either DT-EGF (150ng ml⁻¹) or TRAIL (250ng ml⁻¹) depicting morphological differences in dying cells. (B) Cell viability of U87MG cells as determined by MTS assays after 24hours of treatment with DT-EGF (150ng ml⁻¹)(mean +/− SEM from 3 replicates). (C) Effector caspase activity was measured using a caspase 3/7 specific luminogenic substrate on U87MG cells treated with DT-EGF (150ng ml⁻¹) or TRAIL (250ng ml⁻¹) (mean +/− SEM from 3 replicates).

Figure 2. DT-EGF potentiates TRAIL receptor mediated killing in vitro and in vivo

(A) MTS assay quantifying cell viability of U87MG cells in response to DT-EGF and TRAIL either alone or in combination (mean +/− SEM from 3 replicates). (B) MTS assay quantifying cell viability of U87MG cells in response to DT-EGF and Lexatumumab either alone or in combination (mean +/− SEM from 3 replicates). (C) Table of combination doses between DT-EGF and the TRAIL receptor-targeted drugs. CI value for DT-EGF and TRAIL or Lexatumumab combinations calculated using Calcusyn. (D) U87MG tumor cells were implanted in the flanks of nude mice and tumors were allowed to grow for one week. Tumors were then treated with interstitial as previously described [12] with DT-EGF (0.5 μg), Lexatumumab (1.25 μg), or both in combination. Two-way repeated ANOVA showed a difference *p<0.001 for the combination compared to the single treatment groups (mean +/− SD from 3 replicates).

Figure 3. DT-EGF and TRAIL kill cells in a caspase dependent apoptotic fashion

(A) Representative micrographs of U87MG cells treated with either 1. untreated, 2. DT-EGF (150ng ml⁻¹), 3. TRAIL (250ng ml⁻¹), 4. DT-EGF (150ng ml⁻¹) + TRAIL (2ng ml⁻¹), 5. TRAIL (2ng ml⁻¹), 6. DT-EGF (150ng ml⁻¹) + TRAIL (2ng ml⁻¹) + zVADfmk (25 μM) depicting morphological differences in dying cells. (B) Western blot analysis of key apoptotic protein levels in U87MG cells in response to conditions 1-6 from Figure 3A. (C) Effecter caspase activity was measured using a caspase 3/7 specific luminogenic substrate on U87MG cells treated with DT-EGF (150ng ml⁻¹), TRAIL (2ng ml⁻¹), DT-EGF (150ng ml⁻¹) + TRAIL (2ng ml⁻¹), and TRAIL (250ng ml⁻¹) (mean +/− SEM from 3 replicates). (D) Effector caspase activity was measured using a caspase 3/7 specific luminogenic substrate on U87MG cells treated for 6 hours with DT-EGF (150ng ml⁻¹), TRAIL (20ng ml⁻¹), 0 (DT-EGF (150ng ml⁻¹) + TRAIL (20ng ml⁻¹)), 6 (pretreat with DT-EGF (150ng ml⁻¹) for 6 hours then treat with TRAIL (20ng ml⁻¹)), 24 (pretreat with DT-EGF (150ng ml⁻¹) for 24 hours then treat with TRAIL (20ng ml⁻¹)), (mean +/− SEM from 3 replicates).

Figure 4. DT-EGF potentiates TRAIL by depleting FLIP

(A) Western blot analysis of FLIP and XIAP protein levels in U87MG cells treated with DT-EGF (150ng ml⁻¹), TRAIL (2ng ml⁻¹), and Both (DT-EGF(150ng ml⁻¹) and TRAIL (2ng ml⁻¹)). (B) U87MG cells treated with siRNA targeting XIAP or FLIP for 48 and 72 hrs were harvested and western blot analysis was performed to determine XIAP or FLIP levels respectively. (C) Cells treated with control siRNAs or siRNA targeting XIAP or FLIP were treated with DT-EGF (150ng ml⁻¹), TRAIL (250ng ml⁻¹), Both 1 (DT-EGF (150ng ml⁻¹) + TRAIL (2ng ml⁻¹)), Both 2 (DT-EGF (10ng ml⁻¹) + TRAIL (2ng ml⁻¹)), and Both 3 (DT-EGF (2ng ml⁻¹) + TRAIL (2ng ml⁻¹)) and viability determined by MTS assay, data shows mean +/− SEM from 3 replicates, * indicates statistically significant difference by t-test p<0.05 between the sicontrol and siFLIP cells with low dose TRAIL. (D) Cells treated with siRNA's as in panel C then treated with with Chx (5 μg ml⁻¹), TRAIL (250ng ml⁻¹), Both 1 (Chx (5μg ml⁻¹) + TRAIL (2ng ml⁻¹)), Both 2 (Chx (0.15μg ml⁻¹) + TRAIL (2ng ml⁻¹)), and Both 3 (Chx (0.015μg ml⁻¹) + TRAIL (2ng ml⁻¹). Data shows mean +/− SEM from 3 replicates, * indicates statistically significant difference by t-test p<0.05 between the sicontrol and siFLIP cells with low dose TRAIL.

Figure 5. DT-EGF potentiates TRAIL by depleting FLIP in 42MGBA cells

(A) Cells treated with siRNA targeting FLIP or control cell were treated with DT-EGF (150ng ml⁻¹), TRAIL (250ng ml⁻¹), Both 1 (DT-EGF (150ng ml⁻¹) + TRAIL (2ng ml⁻¹)), Both 2 (DT-EGF (10ng ml⁻¹) + TRAIL (2ng ml⁻¹)), and Both 3 (DT-EGF (2ng ml⁻¹) + TRAIL (2ng ml⁻¹)). The data represent mean +/− SEM from 3 replicates. (B) Cells treated with siRNA targeting FLIP or control cell were treated with Chx (5μg ml⁻¹), TRAIL (250ng ml⁻¹), Both 1 (Chx (5μg ml⁻¹) + TRAIL (2ng ml⁻¹)), Both 2 (Chx (0.15μg ml⁻¹) + TRAIL (2ng ml⁻¹)), and Both 3 (Chx (0.015μg ml⁻¹) + TRAIL (2ng ml⁻¹)). The data represent mean +/− SEM from 3 replicates.