![]() ![]() siRNA was dispensed using a Caliper Sciclone ALH3000 (PerkinElmer). After 24 hours, the media was replaced and at 72 hours 10 μL CellTiter-Glo was added to each well (final concentration 1/5), shaken on an orbital shaker for 2 minutes and incubated for a further 30 minutes at room temperature before measuring luminescence using a Synergy H4 microplate reader (Biotek). Cellprofiler identify postive cells free#The following controls were included in each plate: the death controls si EGFR and si PLK1 (4 wells each), a synthetic lethal control si CTNNB1 (6 wells) and two negative controls, siRISC free and mock (lipid only, 9 wells each). Cells were reverse transfected and seeded onto the siRNA cocktail at a density of 700 MCF10A cells per well and 900 CDH1 −/− cells/well to enable the two cell lines to reach confluence at the same time point (72 hours after seeding). Each reaction, in a white walled, clear-bottom 384-well plate format, contained 0.125% DharmaFECT 3 (0.05 μL), 27.4% OptiMEM (Invitrogen), and 40 nmol/L of the siRNA SMARTpool (total volume 37.5 μL). Each SMARTpool contained four siRNAs that targeted different regions of each gene in one well. ©2015 AACR.Ĭells were transfected with siRNAs from the Dharmacon SMARTpool whole genome protein-coding siRNA library (RefSeq 27) housed in the Victorian Center for Functional Genomics. Together, these results demonstrate that E-cadherin loss creates druggable vulnerabilities that have the potential to improve the management of both sporadic and familial LBC and DGC. These included multiple histone deacetylase inhibitors, including vorinostat and entinostat, PI3K inhibitors, and the tyrosine kinase inhibitors crizotinib and saracatinib. Next, we conducted a 4,057 known drug screen and time course studies on the CDH1 isogenic MCF10A cell lines and identified additional drug classes with linkages to GPCR signaling and cytoskeletal function that showed evidence of E-cadherin synthetic lethality. These broad classes of E-cadherin synthetic lethal hits were validated using both lentiviral-mediated shRNA knockdown and specific antagonists, including the JAK inhibitor LY2784544, Pertussis toxin, and the aurora kinase inhibitors alisertib and danusertib. Diverse families of cytoskeletal proteins were also frequently represented. Gene ontology analysis demonstrated that G-protein–coupled receptor (GPCR) signaling proteins were highly enriched among the synthetic lethal candidates. We first conducted a genome-wide siRNA screen of isogenic MCF10A cells with and without CDH1 expression. To overcome this, we have taken a synthetic lethal approach to determine whether the loss of E-cadherin creates druggable vulnerabilities. However, because E-cadherin is a tumor suppressor protein and lost from the cancer cell, it is not a conventional drug target. The CDH1 gene, which encodes the cell-to-cell adhesion protein E-cadherin, is frequently mutated in lobular breast cancer (LBC) and diffuse gastric cancer (DGC). ![]()
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