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...

Supplementary Data from Epigenetic Silencing of <i>Death Receptor 4</i> Mediates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Resistance in Gliomas

Supplementary Data from Epigenetic Silencing of <i>Death Receptor 4</i> Mediates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Resistance in Gliomas

<div>Abstract<p><b>Purpose:</b> To identify and characterize epigenetically regulated genes able to predict sensitivity or resistance to currently tested chemotherapeutic agents in glioma therapy.</p><p><b>Experimental Design:</b> 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 <i>death receptor 4 (DR4)</i>, 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.</p><p><b>Results:</b> In human astrocytic tumors, we detected <i>DR4</i> promoter hypermethylation in 60% (<i>n</i> = 5) of diffuse astrocytomas WHO grade 2, in 75% (<i>n</i> = 8) of anaplastic astrocytomas WHO grade 3, and in 70% of glioblastomas WHO grade 4 (<i>n</i> = 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 <i>DR4</i> promoters, and 5-aza-2-deoxycytidine–mediated 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.</p><p><b>Conclusions:</b> <i>DR4</i> promoter methylation is frequent in human astrocytic gliomas, and epigenetic silencing of <i>DR4</i> mediates resistance to TRAIL/DR4-based glioma therapies. (Clin Cancer Res 2009;15(17):5457–65)</p></div>