I��B proteins block nuclear localization signals of func tional NF ��B dimers by binding to dimerization domains and sequestering the dimers in the cytoplasm. I��B ki nases phosphorylate I��B on a serine residue, tar geting them for proteasomal degradation, thereby activating NF ��B, which protects cells by increasing the expression of antiapoptotic proteins. Previously, most we showed Inhibitors,Modulators,Libraries that inhibition of NF ��B increases TRAIL sensitivity in breast cancer cell lines. Similar results were reported in other cancer cell lines. Again, our findings in this article that I��BKB LOF leads to enhanced TRAIL induced caspase activation provide support for further studies of NF ��B in hibitors in combination with TRAIL.

Further to confirm our primary screen results, Inhibitors,Modulators,Libraries we per formed a secondary screen of 16 genes identified as negative regulators of TRAIL induced caspase activation in four cell lines representing different subtypes of breast cancer. We selected 16 genes that were included in the network analysis in Figure 4 and that both increased TRAIL induced caspase 3 7 activity and enhanced TRAIL induced toxicity in a viability assay. In MB231, 13 of 16 genes scored positive in this assay, and 15 of 16 genes scored positive at a lower stringency cutoff. This high level of reproducibility between the primary and secondary screen in MB231 sup ports the validity of the primary screen. All of the 16 genes scored positive by using the high stringency criterion in MB468. The Inhibitors,Modulators,Libraries TNBC basal A MB468 cell line is most closely related to the TNBC basal B MB231 cell line by cDNA microarray expression analysis, and thus the high degree of overlap between the two cell lines in this screen is not surprising.

By contrast, fewer of the 16 genes were scored positive in T47D and SKBR3. The T47D cell line is an ER positive luminal breast cancer cell line, and the SKBR3 cell line is an HER2 amplified luminal breast cancer cell line. Thus they are more distantly related to the MB231 cell line. The only gene that scored positive in our screen at high stringency in Inhibitors,Modulators,Libraries all four cell lines is alpha actinin 4. ACTN4 is a cytoskeletal protein that has been found to interact with signaling molecules, chromatin remodeling factors, and transcription factors. Of note, ACTN4 can serve as a scaffold to pro mote AKT activation, and it has been shown to interact with NF ��B in breast cancer cells.

Thus, it is plausible that by modulating activity through these two antiapoptotic pathways, ACTN4 might serve as a negative regulator of TRAIL induced apoptosis. The mech anisms by which ACTN4 regulate TRAIL induced apoptosis in breast cancer Inhibitors,Modulators,Libraries cells will require further investigation. LOF of BCL2L1 enhanced TRAIL GW572016 induced caspase 3 7 activation in three of the four cell lines at high stringency and in all four cell lines when a lower stringency was used. Expanded screening of five BCL2L1 siRNAs confirmed that BCL2L1 LOF results in enhanced TRAIL activity in four breast cancer cell lines.