TRAIL Inactivates the Mitotic Checkpoint and Potentiates Death Induced by Microtubule-Targeting Agents in Human Cancer Cells Mijin Kim, 1 Jessica Liao, 1 Melissa L. Dowling, 1 K. Ranh Voong, 1 Sharon E. Parker, 1 Shulin Wang, 2 Wafik S. El-Deiry, 2 and Gary D. Kao 1 1 Department of Radiation Oncology, University of Pennsylvania School of Medicine and the Philadelphia Veterans Affairs Medical Center, and 2 Departments of Medicine (Hematology/Oncology), Genetics, and Pharmacology, Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Abstract Tumor necrosis factor–related apoptosis–inducing ligand (TRAIL) has attracted interest as an anticancer treatment, when used in conjunction with standard chemotherapy. We investigated the mechanistic basis for combining low-dose TRAIL with microtubule-targeting agents that invoke the mitotic checkpoint. Treatment of T98G and HCT116 cells with nocodazole alone resulted in a robust mitotic block with initially little cell death; low levels of cell death were also seen with TRAIL alone at 10 ng/mL final concentration. In contrast, the addition of low-dose TRAIL to nocodazole was associated with maximally increased caspase-3, caspase-8, and caspase-9 activation, which efficiently abrogated the mitotic delay and markedly increased cell death. In contrast, the abrogation of mitotic checkpoint and increased cell death were blocked by inhibitors of caspase-8 and caspase-9 or pan-caspase inhibitor. The addition of TRAIL to either nocodazole or paclitaxel (Taxol) reduced levels of the mitotic checkpoint proteins BubR1 and Bub1. BubR1 mutated for the caspase cleavage sites, but not wild-type BubR1, was resistant to cleavage induced by TRAIL added to nocodazole, and partially blocked the checkpoint abrogation. These results suggest that adding a relatively low concentration of TRAIL to antimicrotubule agents markedly increases complete caspase activation. This in turn accentuates degradation of spindle checkpoint proteins such as BubR1 and Bub1, contributes to abrogation of the mitotic checkpoint, and induces cancer cell death. These results suggest that TRAIL may increase the anticancer efficacy of microtubule-targeting drugs. [Cancer Res 2008;68(9):3440–9] Introduction The tumor necrosis factor (TNF)–related apoptosis–inducing ligand (TRAIL), a member of the TNF family of death ligands, triggers apoptosis through interaction with the death receptors DR4 and DR5 (1–4). Many cancer cell lines express both DR4 and DR5, and each of these receptors can initiate apoptosis indepen- dently of the other (5, 6). Evidence suggests that endogenous TRAIL-mediated pathways contribute to anticancer surveillance (7). Although the mechanisms that are involved in this signaling pathway remain a topic of active investigation, one of the most attractive aspects of TRAIL is that it seems to specifically induce apoptosis in certain cancer cells while sparing most normal cells (8, 9). Evidence suggests that binding by TRAIL leads to trime- rization of its receptors, which in turn leads to recruitment of Fas-associated death domain, an adaptor molecule, which then recruits and activates caspase-8. Activated caspase-8, an ‘‘initiator’’ caspase, can directly cleave the proenzyme forms of ‘‘effector’’ caspases such as caspase-3, leading to activation of the latter (this pathway is often referred to as the type I ‘‘intrinsic’’ pathway of apoptosis signaling). Activation of caspase-8 may also induce apoptosis through mitochondrial pathways, referred to as type II ‘‘extrinsic’’ pathways. In an example of the latter, caspase-8 cleaves Bid, which in turn leads to release of cytochrome c from mitochondria, which in turn binds to and activates the adapter protein APAF-1. The resultant ‘‘apoptosome’’ in turn recruits, cleaves, and activates caspase-9, which in turn can also activate caspase-3. It should be noted that activation of caspase-9 may occur independent of caspase-8 activation. The extrinsic pathway of apoptosis signaling is thought to mediate many of the cytotoxic effects of DNA-damaging agents, independent of caspase-8 or receptor-mediated activation. Thus, depending on the cell line or stimulus, either the extrinsic or intrinsic pathway may be activated by TRAIL, but both pathways converge in activating effector caspases such as caspase-3 (reviewed in refs. 6, 10). The proapoptotic effects of TRAIL have led to interest in whether the anticancer efficacy of conventional chemotherapy, including microtubule-targeting agents such as the Vinca alkaloids or taxanes, might be enhanced when combined with TRAIL (11–14). The taxanes have emerged as part of the standard of care for certain solid tumors such as breast and prostate cancer (15–17), yet treatment resistance remains common and leads to morbidity and death. For other solid tumors such as colorectal cancer or glioblastoma multiforme, antimicrotubule agents have not been proven clinically useful. Microtubule-targeting drugs invoke the mitotic checkpoint by disrupting formation of the mitotic spindle. The resultant delay in progression of mitosis is mediated by a protein machinery that includes BubR1 and Bub1 (18). The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis, thereby ensuring that the daughter cells receive an equal complement of chromosomes. Because this helps ensure that aneuploidy or polyploidy does not occur, the mitotic checkpoint is thought to prevent genomic instability in untransformed cells. In cancer cells (many of which have already become aneuploid), the mitotic checkpoint potentially allows additional time to repair Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Gary D. Kao, Department of Radiation Oncology, University of Pennsylvania School of Medicine, John Morgan 180 H, Philadelphia, PA 19104. Phone: 215-573-5503; E-mail: kao@xrt.upenn.edu. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-0014 Cancer Res 2008; 68: (9). May 1, 2008 3440 www.aacrjournals.org Research Article Research. on March 4, 2016. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from