Activating Cell Death Ligand Signaling Through Proteasome Inhibition

Pharmacologic inhibition of the 26S proteasome by the compound Velcade (PS-341/bortezomib) is a viable chemotherapeutic strategy that is currently approved to treat patients with refractory multiple myeloma. Additional clinical trials are currently underway to determine the efficacy of Velcade alone or in combination with other agents in hormone-refractory prostate cancer patients. As determined in a Phase 3 clinical trial, proteasome inhibition does have a positive anti-tumor effect, as 6% of refractory multiple myeloma patients exhibited a complete clinical response and an additional 39% a partial clinical response to Velcade. The prospect that this agent will lead to a clinical response in metastatic prostate cancer is encouraging, as 22-36% of patients with hormone refractory cancer have shown a decrease in markers of disease progression. Although response rates do show the promise of proteasome inhibition strategies, they also highlight the main problem with the use of Velcade, which is this agent only elicits a complete response in a small fraction of patients. Furthermore, it is unknown which patient population will have a clinical response. It is our long-term goal to improve the efficacy of proteasome inhibition strategies. One of the main obstacles in solving these problems is that the mechanisms by which Velcade leads to tumor cell death in vivo are poorly understood. The majority of studies have focused on angiogenesis or cellintrinsic changes that lead to apoptosis. In contrast, we have found that proteasome inhibition through the administration of MG132 or Velcade can sensitize multiple prostate cancer cell lines to apoptosis mediated by the cell death cytokines Fas ligand and TRAIL, while normal prostate epithelial cells require much higher cytokine levels to reach the same apoptotic index. The apoptosis induced by these cell-extrinsic ligands occurs at a much lower drug concentration than that required for cancer cell death with Velcade treatment alone. Furthermore, we have mechanistically dissected the death receptor signaling pathways and find that proteasome inhibition sensitizes cancer cells to the death ligands by conferring marked caspase-8 protein accumulation, which leads to robust enzymatic activity and apoptosis induction. These findings are clinically relevant for if tumor cells in vivo treated with Velcade can and do undergo apoptosis mediated by death ligands, then it may be possible to augment the efficacy of proteasome inhibition strategies by the administration of antibodies or proteins that activate cell death receptors. Furthermore, by defining the specific death receptor pathways and proteins that are targeted by proteasome inhibition that directly lead to apoptosis, it may be possible to identifY those tumors that will respond favorably to treatment and develop strategies that target specific components of the ubiquitin-proteasome pathway. It is our hypothesis that proteasome inhibition elicits anti-neoplastic effects in vivo by sensitizing cancer cells to apoptosis mediated through cell death ligand signaling. We will test this hypothesis through directing our efforts toward two Specific Aims. In Specific Aim 1, we will test the extent to which proteasome inhibition can act as an anticancer agent in vivo by sensitizing prostate cancer cells to death ligands in the tumor microenvironment. Using PC3 and LNCaP xenograft models, the ability of Velcade treatment alone to activate death receptor signaling pathways will be determined in addition to defining the efficacy of combining Velcade and TRAIL therapy in vivo. In Specific Aim 2, we will define the mechanism whereby proteasome inhibition enhances sensitivity to cell death ligand-induced apoptosis. The mechanism of caspase-8 activity induction by proteasome inhibition will be examined in prostate cancer cells. Specifically, regulation of cleaved caspase-8 protein levels by the 26S proteasome and the biological importance of this activity in mediating the efficacy ofVelcade will be tested.