TNF-related apoptosis inducing ligand (TRAIL) is a member of the tumor necrosis factor super family and can induce apoptotic cell death upon binding to its cognate receptors, TRAIL Receptor 1 (TRAIL-R1) and TRAIL Receptor 2 (TRAIL-R2). TRAIL has been found to induce selectively cell death in triple negative breast cancer (TNBC) cell lines (so called because the breast cancer cells lack estrogen and progesterone receptor expression and Her-2 amplification). Other subtypes of breast cancer are relatively resistant to TRAIL-induced apoptosis. However, the mechanisms that underlie the sensitivity of TNBCs to TRAIL-induced apoptosis are not yet understood. To identify regulators of TRAIL-induced apoptosis in the TNBC/mesenchymal cell line MB231, an siRNA screen of the kinome, phosphatome, and other potential regulators (~1,300 genes) of the TRAIL pathway was carried out to measure the effects of loss of function of these genes on TRAIL-induced caspase-3/7 activation, capase-8 activation, and cell death. One hundred fifty negative regulators of the TRAIL pathway were identified, including 83 kinases, 4 phosphatases, and 63 non-kinases. The identified regulators were involved in diverse cell processes, including apoptosis, transcriptional regulation, growth factor receptor signaling, DNA repair, cell metabolism, and cell cycle regulation. Interestingly, no positive regulators were identified. A subset of the 150 candidates were rescreened in MB231 and three additional cell lines representative of other subytpes of breast cancer, including MB468 (TNBC/basal), SKBR3 (Her-2 amplified), and T47D (estrogen receptor positive). Interestingly, the TNBC/basal cell line MB468 was sensitized to TRAIL-induced caspase-3/7 activation by all of the candidates, whereas T47D and SKBR3 were sensitized to TRAIL by only two and three of the candidates, respectively. These findings demonstrate that breast cancer cell lines are varyingly sensitized to the effects of TRAIL. The anti-apoptotic protein Bcl-xL and the tyrosine kinase Src were also identified as negative regulators of TRAIL-induced apoptosis. We have further shown that pharmacologic inhibition of Bcl-xL or Src sensitize a wide range of breast cancer cell lines to TRAIL-induced apoptosis. These findings lend further support to the screen. Overall, the siRNA screen has identified many regulators of TRAIL-induced apoptosis in the TNBC cell line MB231 and a smaller subset of regulators in breast cancer cell lines representative of other subtypes. These findings suggest targets for the rational development of combination therapy in breast cancer treatment.
