We also thank Dr William Gillette and Ralph Hopkins of the Protein Expression Laboratory (NCICFrederick) for purification of the MD5-1 antibody

We also thank Dr William Gillette and Ralph Hopkins of the Protein Expression Laboratory (NCICFrederick) for purification of the MD5-1 antibody. The funders had no role in the design of the study; the collection, analysis, and interpretation of the data; the decision to submit the manuscript for publication; or the writing of the manuscript. Footnotes PD318088 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.. (20 nM) sensitized Renca-FLAG and 4T1 cells to TRAIL-mediated apoptosis (mean percent decrease in numbers of viable cells, bortezomib + TRAIL vs TRAIL: Renca-FLAG, 95% vs 34%, difference = 61%, 95% confidence interval [CI] = 52% to 69%, .001; 4T1, 85% vs 20%, difference = 65%, 95% CI = 62% to 69%, .001). Sensitization involved activation of caspase-8 and caspase-3 but not mitochondrial membrane depolarization, suggesting an amplified signaling of the extrinsic cell death pathway. Treatment with bortezomib and MD5-1 reduced lung metastases in mice carrying Renca and 4T1 tumors (mean number of metastases, bortezomib + MD5-1 vs MD5-1: Renca-FLAG, 1 vs 8, difference = 7, 95% CI = 5 to 9, .001; 4T1, 1 vs 12, difference = 11, 95% CI = 9 to 12, .001) and increased median survival of mice bearing Renca-FLAG tumors (bortezomib + MD5-1 vs bortezomib + control isotype antibody: 22 of PD318088 30 [73%] were still alive at day 180 vs median survival of 42 days [95% PD318088 CI = 41 to 44 days, .001]) in the absence of obvious toxicity. Conclusion Bortezomib combined with DR5 agonist monoclonal antibody may be a useful treatment for metastatic solid tumors. Resistance of tumor cells to signals that trigger cell death is a major impediment in the treatment of cancer. Tumor necrosis factor (TNF)Crelated apoptosis-inducing ligand (TRAIL/Apo2L) (1), which is implicated in host immunosurveillance against tumor development and metastasis (2C6), has tumoricidal activity in various models of human xenogeneic tumors in immunodeficient mice (7C11) without toxicity to most nontransformed cells (7,12,13). Agonist antibodies to the TRAIL death receptors have also been tested as potential therapeutic agents. These antibodies may offer distinct therapeutic advantages over TRAIL itself due to their long half-life and their lack of binding to decoy receptors on target cells (14). However, treatment with an agonist monoclonal antibody to the mouse TRAIL death PD318088 receptor (DR5, TRAIL-R2, or CD262) only delays tumor development and does not improve the survival of mice bearing R331 renal carcinomas or 4T1 breast carcinomas (15,16), although the R331 clone is highly sensitive to TRAIL-mediated apoptosis in contrast to the parental Renca tumor (5,17). Bortezomib, a specific and reversible inhibitor of the proteasome function that is crucial for protein degradation (18), has been approved by the Food and Drug Administration for the therapy of multiple myeloma (19) and has been shown by us and others (20C22) to sensitize tumors to TRAIL. However, to date no experimental evidence exists as to whether bortezomib can be combined with TRAIL or TRAIL receptor agonists in vivo for therapy of preexisting tumors within acceptable limits of toxicity. Here we examined the therapeutic potential and the toxicity of the combination of bortezomib and the TRAIL receptor DR5 agonist monoclonal antibody MD5-1 in mice bearing Renca and 4T1 carcinomas. The molecular basis of bortezomib sensitization of tumor cells to TRAIL-mediated apoptosis was also investigated. CONTEXT AND CAVEATS Prior knowledgeTumor necrosis factorCrelated apoptosis-inducing ligand (TRAIL) is involved in host immunosurveillance against tumor development and metastasis and been shown to have RNF49 antitumor activity in various mouse models of human cancers. Bortezomib is an inhibitor of protein degradation and has been approved by the Food and Drug Administration for the treatment of multiple myeloma. Study designCell viability and signaling of apoptotic death of mouse renal adenocarcinoma and mammary carcinoma cells were assayed after treatment with TRAIL and/or bortezomib. Development of lung metastases and survival of mice carrying tumors derived from these cells were measured after treatment with bortezomib and/or MD5-1, a TRAIL receptor agonist antibody. ContributionsMore cells underwent apoptotic death after treatment with bortezomib and TRAIL than with TRAIL alone. Mice treated with bortezomib and MD5-1 lived longer and developed fewer lung metastases than mice treated with MD5-1 alone. ImplicationsThe combination of PD318088 bortezomib and a TRAIL agonist antibody may be useful in the treatment of solid metastatic tumors. LimitationsIt is unknown whether results will be similar in humans or what nonspecific toxic effects a similar drug combination might have in humans. Methods Tumor Cell Lines The renal cell adenocarcinoma (Renca) cell line of BALB/c origin was provided by Dr Robert H. Wiltrout (National Cancer Institute [NCI]CFrederick). To obtain Renca cells that were sensitive or relatively resistant to a TRAIL-mediated death signal, steady cell lines of TRAIL-sensitive Renca-FLAG and TRAIL-resistant Renca-FLIP had been produced by transfecting complementary DNA.