The differing results are likely the result of several sig nificant differences in our screens. First, we performed the screen in a TNBC breast cancer cell line, whereas the prior study was performed in the cervical carcinoma HeLa cell line. It is likely sellekchem that the predominant regula tors of TRAIL induced apoptosis are different in different cell types. Second, our primary selection of genes whose LOF altered TRAIL activity was based on caspase 3 7 ac tivation 1 hour after the addition of TRAIL, whereas the previous study measured viability 20 hours after the addition Inhibitors,Modulators,Libraries of TRAIL. Thus, our screens were designed principally to identify regulators that affect early steps in TRAIL induced apoptosis, contributing to the dif ference noted.
Review of the putative negative regulators identified in our primary RNAi screens in MB231 revealed genes in volved in diverse cellular Inhibitors,Modulators,Libraries processes, including growth factor receptor signaling, cytoskeleton function, bio energetics, cell cycle regulation, transcrip tional regulation, and DNA repair. Also of note, several genes known to regulate apoptosis negatively were identified. The largest gene set in our RNAi library included the known kinases and kinase associated genes. Of the group of kinases that were identified Inhibitors,Modulators,Libraries as hits, the majority of them are serine threonine kinases, whereas fewer belonged to the tyrosine kinase, lipid kinase, or sugar metabolism kinase fam ilies. Interestingly, four kinases were identified, hexokinase 2, pyruvate kinase liver and red blood cells, and phosphofructose kinase liver which regulate irreversible steps of the glycolysis pathway.
Several studies have previously found that inhibition of glycolysis enhances TRAIL induced cell death. Based on the gene network analysis, four genes were identified that appear at central nodes of an interaction map generated Inhibitors,Modulators,Libraries by using the caspase 3 7 screening dataset, PDPK1, IKBKB, SRC, and BCL2L1. The caspase 8 and cell viability screening data confirmed these findings for BCL2L1 and PDPK1. PDPK1 phosphorylates and activates AKT. Constitutively active or overexpression of AKT has Inhibitors,Modulators,Libraries been shown to confer TRAIL resistance in several tumor types, including breast, lung, gastric, and prostate. Also, TRAIL can activate SRC, leading to AKT activation and TRAIL re sistance. Inhibition of the PI3 kinase AKT path way has been found to enhance TRAIL induced apoptosis. Therefore, identification of PDPK1 as one of the key nodes provides a rationale for pursuing studies on the combination especially of TRAIL with AKT inhib itors in treatment of TRAIL resistant tumors. NF ��B proteins are ubiquitously expressed proteins that can protect cells from apoptosis.
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