Functional genomic analysis of glioblastoma multiforme through short interfering RNA screening: a paradigm for therapeutic development

Abstract

<jats:p>Glioblastoma multiforme (GBM) is a high-grade brain malignancy arising from astrocytes. Despite aggressive surgical approaches, optimized radiation therapy regimens, and the application of cytotoxic chemotherapies, the median survival of patients with GBM from time of diagnosis remains less than 15 months, having changed little in decades. Approaches that target genes and biological pathways responsible for tumorigenesis or potentiate the activity of current therapeutic modalities could improve treatment efficacy. In this regard, several genomic and proteomic strategies promise to impact significantly on the drug discovery process. High-throughput genome-wide screening with short interfering RNA (siRNA) is one strategy for systematically exploring possible therapeutically relevant targets in GBM. Statistical methods and protein-protein interaction network databases can also be applied to the screening data to explore the genes and pathways that underlie the pathological basis and development of GBM. In this study, we highlight several genome-wide siRNA screens and implement these experimental concepts in the T98G GBM cell line to uncover the genes and pathways that regulate GBM cell death and survival. These studies will ultimately influence the development of a new avenue of neurosurgical therapy by placing the drug discovery process in the context of the entire biological system.</jats:p>

Identification of Druggable Targets for Radiation Mitigation Using a Small Interfering RNA Screening Assay

“…Data from the siRNA screening was obtained from experiments performed on 4 independent days. The resulting data were analyzed using our previously described methods (10, 11). Briefly, relative fluorescence units from each targeted siRNA well were normalized to in-plate scrambled negative control values, which allowed for plate-to-plate comparisons.…”

“…Protein lysates from NCCIT cells (~30 µg/well) were isolated as previously described (10) and, after separation on a 4–20% acrylamide gradient gel, the proteins were transferred to a nitrocellulose membrane with iBlot (Life Technologies). The membranes were blotted with caspase 3 antibody (Stressgen, New York, NY) at a 1:1000 dilution in 5% milk/Tris buffer saline (50 m M Tris • HCl, pH 7.4, 150 m M NaCl) with 0.1% Tween 20 at 4°C overnight.…”

Survivin inhibitor YM155 induces mitochondrial dysfunction, autophagy, DNA damage and apoptosis in Bcl‐xL silenced glioma cell lines

“…Targeted therapies affecting the Bcl-2 family of proteins as a strategy for overcoming chemotherapy drug resistance have been well characterized [2][3][4][5][6][7]. Using a large-scale siRNA screening approach to identify critical "nodes" for cell death signaling, we identified several targets, including nuclear factor kB (NF-kB) and the proteasome, as well as Akt and Bcl-xL that, when inhibited, promoted apoptotic signaling in glioma cells [8][9][10][11][12]. Therefore, we hypothesized that the functional blockade of Bcl-xL (genetic inhibition) should sensitize these cancer cells to chemotherapies or signaling inhibitors by restoring the apoptotic process.…”

Cucurbitacin-I inhibits Aurora kinase A, Aurora kinase B and survivin, induces defects in cell cycle progression and promotes ABT-737-induced cell death in a caspase-independent manner in malignant human glioma cells

“…[20][21][22][23][24] Bcl -2 antagonists can induce apoptosis as single agents only in cancer cells dependent on Bcl -2 and/or Bcl -xL for survival. As a single agent ABT-737, a novel Bcl -2/ Bcl -xL inhibitor triggers apoptosis in various types of human cancers including multiple myeloma, leukemia, lymphoma and small cell lung cancer.…”