Epigenetic Silencing of Death Receptor 4 Mediates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Resistance in Gliomas

Abstract

Purpose: To identify and characterize epigenetically regulated genes able to predict sensitivity or resistance to currently tested chemotherapeutic agents in glioma therapy. Experimental Design: We used methylation-sensitive BeadArray technology to identify novel epigenetically regulated genes associated with apoptosis and with potential therapeutic targets in glioma therapy. To elucidate the functional consequences of promoter methylation in the identified target death receptor 4 (DR4), we investigated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated and anti-DR4-mediated apoptosis in glioma cell lines (U373 and A172) with loss of DR4 and one glioma cell line (LN18) with robust DR4 expression. Results: In human astrocytic tumors, we detected DR4 promoter hypermethylation in 60% (n = 5) of diffuse astrocytomas WHO grade 2, in 75% (n = 8) of anaplastic astrocytomas WHO grade 3, and in 70% of glioblastomas WHO grade 4 (n = 33). DR4 is a cell surface protein restricted to glioma cells and is targeted by TRAIL. Glioma cell lines U373 and A172 harbored heavily methylated DR4 promoters, and 5-aza-2-deoxycytidinemediated demethylation reconstituted DR4 expression in these cell lines. Functional knockdown of DR4 by DR4-specific small interfering RNA in TRAIL-sensitive glioma cell line LN18 significantly mitigated apoptosis induced by an agonistic anti-DR4 antibody. 5-Aza-2-deoxycytidine-mediated demethylation resulted in a functional reconstitution of DR4 on the cell surface of TRAIL-resistant glioma cell line U373 and sensitized U373 to TRAIL-mediated apoptosis. Suppression of DR4 by small interfering RNA in demethylated U373 successfully reestablished the TRAIL-resistant phenotype of U373. Conclusions: DR4 promoter methylation is frequent in human astrocytic gliomas, and epigenetic silencing of DR4 mediates resistance to TRAIL/DR4-based glioma therapies. Epigenetic modifications contribute to tumorigenesis. Examples are epigenetically silenced tumor suppressor genes (1). However, the epigenome becomes crucial clinical relevance when epigenetically regulated proteins are identified that themselves constitute therapeutic targets or are responsible for the development of therapy resistance of the tumor. An example for the latter is O 6 -methylguanine DNA methyltransferase (MGMT) in glioma therapy. Epigenetic silencing of MGMT abolishes its capability of chemotherapy resistance and predicts therapeutic response to alkylating drugs in glioma therapy (2-4). Caspase-8 may exemplify a therapeutically targeted epigenetically silenced molecule in gliomas. A downstream target for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis, loss of caspase-8 expression, has been associated with epigenetic silencing and TRAIL resistance in these tumors (5). We were interested in identifying additional epigenetically silenced proteins or receptor molecules that are already targeted in glioma therapy. To address this, we implemented single-nucleotide polymorphism-based BeadArra...

The effect of epigenetic regulation of fucosylation on TRAIL-induced apoptosis

“…c After HL60, PC3, T24, HT29, SW620, SW834, and KOC7C cells were pretreated with zebularine and then treated with TRAIL, expression of cleaved PARP was examined by Western blotting cancer cells resistant to TRAIL-induced apoptosis. Treatment with 5-aza-2′-deoxycitidine reversed hypermethylation of the DR4 [30][31][32] and caspase-8 [33][34][35][36][37] genes, resulting in restoration of these molecules and activation of TRAILinduced apoptosis. However, no alteration of the expression of the DR4, DR5, and caspase-8 genes was observed in MDA-MB-231 cells treated with or without zebularine.…”

Apoptosis-reactivating agents for targeted anticancer therapy

“…Адекватному функционированию TRAIL-R1 и TRAIL-R2 могут также препятствовать эпигенетические изменения. Например, для клеток астроцитарной глиомы и рака молочной железы характерно гиперметилирование промоторов генов TRAIL-R1 или TRAIL-R2 [8,9]. Другой механизм, с помощью которого опухолевые клетки способны избегать апоптоза, вызываемого TRAIL, связан с аномальной экспрессией "рецепторов-ловушек".…”

The novel Raf inhibitor Raf265 decreases Bcl-2 levels and confers TRAIL-sensitivity to neuroendocrine tumour cells

“…However, promoter hypermethylation may result in complete loss of TRAIL receptors and TRAIL resistance (Horak et al 2005, Elias et al 2009). Using RT-PCR, we could show that all NET cells (pancreatic BON1 and CM cells, bronchus NCI-H727 cells, and midgut GOT1 cells) express mRNA of DR4 and DR5 (Fig.…”

“…This opens perspectives for novel tailor-made cancer therapies, as defining and targeting the genetic aberration underlying apoptosis resistance may restore tumour sensitivity to TRAIL and other apoptotic stimuli. While complete loss of TRAILR1 or TRAILR2 may result in TRAIL resistance (Horak et al 2005, Elias et al 2009, there is no clear correlation between TRAIL sensitivity and the expression level of TRAIL receptors. Instead, TRAIL resistance seems to be the result of alteration in the pathway linking TRAIL receptors to the apoptotic machinery.…”