TNF-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in transformed cells by activating the extrinsic apoptotic pathway via its cognate receptors on the cell surface, TRAIL receptor 1 and TRAIL receptor 2. Triple negative breast cancer (TNBC) cells (so-called because TNBC lacks estrogen and progesterone receptor expression and Her-2 amplification) have been found to be sensitive to TRAIL while breast cancer cells of other subtypes of disease remain relatively resistant. Unfortunately, the mechanisms that govern sensitivity to TRAIL are not yet understood. The identification and characterization of novel regulators of the TRAIL pathway would provide new insights into the mechanisms that regulate TRAIL and potentially provide therapeutically exploitable molecular targets for the enhancement of TRAIL-based cancer treatments. In order to identify candidate regulators of the TRAIL pathway, our lab carried out a high-throughput RNAi-mediated screen of ∼1300 genes using the TNBC cell line MB231. One hundred fifty candidate regulators were identified. The RING finger ubiquitin ligase gp78 (also known as AMFR) was identified as a candidate negative regulator of TRAIL sensitivity. gp78 is an endoplasmic reticulum (ER)-residing protein that helps facilitate the retrotranslocation of substrates across the ER membrane into the cytosol during ER-associated protein degradation. This process is critical to maintaining cellular homeostasis by promoting the proteasomal elimination of misfolded proteins and is integral to cell survival. Interestingly, gp78 has previously been found to promote metastasis in a mouse sarcoma model, and in this study, we have further characterized gp78 as an inhibitor of apoptosis. The initial findings from the RNAi screen were confirmed by carrying out siRNA-mediated knockdown of gp78 in MB231 cells. The cells with inhibited gp78 expression were found to be significantly sensitive to TRAIL-induced caspase-3/7 activity and loss in viability. Knockdown of gp78 using in total 11 independent siRNAs demonstrated a reduction in gp78 expression is associated with TRAIL sensitization. Furthermore, pan-caspase inhibition with the pharmacologic inhibitor ZVAD-FMK completely abrogated sensitization to TRAIL with gp78 knockdown, demonstrating that loss in viability is caspase dependent. These results were further characterized by knocking down gp78 alone or along with one of the initiator caspases, 2, 8, 9, or 10. Loss of the initiator caspases 2 and 8 reduced sensitivity to TRAIL with gp78 knockdown, suggesting that pathways involved in the activation of these caspases in particular may be conferring sensitivity to TRAIL. In summary, our lab has identified and characterized gp78 as a regulator of TRAIL sensitivity through a caspase-mediated mechanism in breast cancer cells. Continued study is warranted to fully elucidate the molecular mechanisms by which gp78 inhibits TRAIL-induced apoptosis.
