Abstract 4106: A functional genomic approach to identify novel molecular regulators of the TRAIL pathway in breast cancer

Abstract

Abstract Tumor Necrosis Factor (TNF)-Related Apoptosis Inducing Ligand (TRAIL) is a member of the TNF family. The ligand binds to its receptors TRAIL-R1 or TRAIL-R2 leading to formation of the death-inducing signaling complex (DISC) which cleaves the initiator pro-caspase 8 to active caspase 8. This cleaved caspase 8 triggers the activation of the downstream executioner caspases (3/7) resulting in apoptosis. Earlier, we showed that triple-negative breast cancer cell lines with mesenchymal phenotype were sensitive to TRAIL while other breast cancer cell lines were resistant. To date the underlying mechanisms that control TRAIL sensitivity or resistance in breast cancer cells are not well understood. To identify potential molecular regulators of the TRAIL pathway, we carried out RNAi screens of the human kinome (691 genes), phosphatome (320 genes), and ≈300 additional genes in MB231, a triple-negative mesenchymal breast cancer cell line. Parallel RNAi screens targeting each gene with four different synthetic siRNAs in an arrayed 384 well plate format (one siRNA per well) were performed. Forty-eight hours post siRNA transfection, one screen received vehicle only, one a low concentration of TRAIL (62.5ng/ml), and one a high concentration of TRAIL (1000ng/ml). One hour after addition of TRAIL, caspase 3/7 activation was measured. The fold change in caspase activity was calculated relative to negative control-siRNA (siNeg) transfected cells. siRNAs corresponding to CASP8 and FLIP were used as assay specific controls. The silencing of CASP8 suppresses caspase 3/7 activation and FLIP enhances it. A gene was considered with high confidence as a potential regulator of TRAIL if three or more siRNAs induced a change in caspase 3/7 levels more than one standard deviation of the siNeg treated wells. These screens identified 45 genes, including 11 kinases and 34 non-kinases, where all four siRNAs induced a phenotype consistent with the targeted gene acting as a negative regulator of TRAIL. These effects were most evident at the high TRAIL dose. Of the 34 non-kinase genes, several known regulators of the TRAIL pathway including BCL2L1, BCL2L2, and BIRC2 were identified. Also, three of the four siRNAs corresponding to a further 126 genes were identified as potential negative regulators of TRAIL. These included 69 kinase genes, 33 phosphatase genes, and 24 genes encoding proteins with a variety of functions. High confidence hits (three or four siRNAs per gene) representing potential positive regulators of TRAIL were not identified, but two of four siRNAs corresponding to four kinases, eight phosphatases and one additional gene did suppress TRAIL activation more than one standard deviation. These screens suggest that a large number of genes act as negative regulators of TRAIL activity while there are fewer positive regulators. Validation of candidate regulators of the TRAIL pathway identified by these screens is in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4106. doi:10.1158/1538-7445.AM2011-4106