Cancer Therapy: Preclinical Bortezomib Primes Glioblastoma, Including Glioblastoma Stem Cells, for TRAIL by Increasing tBid Stability and Mitochondrial Apoptosis Thomas Unterkircher 1 , Silvia Cristofanon 2 , Sri Hari Krishna Vellanki 1 , Lisa Nonnenmacher 1 , Georg Karpel-Massler 3 , Christian Rainer Wirtz 3 , Klaus-Michael Debatin 1 , and Simone Fulda 2 Abstract Purpose: Searching for novel approaches to sensitize glioblastoma for cell death, we investigated the proteasome inhibitor bortezomib. Experimental Design: The effect of bortezomib on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)–induced apoptosis signaling pathways was analyzed in glioblastoma cell lines, primary glioblastoma cultures, and in an in vivo model. Results: Bortezomib and TRAIL synergistically trigger cell death and reduce colony formation of glioblastoma cells (combination index < 0.1). Investigations into the underlying molecular mechanisms reveal that bortezomib and TRAIL act in concert to cause accumulation of tBid, the active cleavage product of Bid. Also, the stability of TRAIL-derived tBid markedly increases on proteasome inhibition. Notably, knockdown of Bid significantly decreases bortezomib- and TRAIL-mediated cell death. By comparison, silencing of Noxa, which is also upregulated by bortezomib, does not confer protection. Coinciding with tBid accumulation, the activation of Bax/Bak and loss of mitochondrial membrane potential are strongly increased in cotreated cells. Overexpression of Bcl-2 significantly reduces mitochondrial perturbations and cell death, underscoring the functional relevance of the mitochondrial pathway. In addition, bortezomib cooperates with TRAIL to reduce colony formation of glioblastoma cells, showing an effect on long-term survival. Of note, bortezomib profoundly enhances TRAIL-triggered cell death in primary cultured glioblastoma cells and in patient-derived glioblastoma stem cells, underlining the clinical relevance. Importantly, bortezomib cooperates with TRAIL to suppress tumor growth in an in vivo glioblastoma model. Conclusion: These findings provide compelling evidence that the combination of bortezomib and TRAIL presents a promising novel strategy to trigger cell death in glioblastoma, including glioblastoma stem cells, which warrants further investigation. Clin Cancer Res; 17(12); 4019–30. Ó2011 AACR. Introduction Glioblastoma is the most common primary brain tumor (1). Even though aggressive treatment protocols are used, therapy response is still very poor and median survival is only 14.6 months (2). Thus, it is crucial to develop alter- native treatment strategies. Human cancers often show inherent resistance to apop- tosis (3). As most current treatment approaches, like chemo- and radiotherapy, primarily act by inducing cell death in cancer cells (4), defects in cell death induction can lead to treatment resistance. Apoptotic cell death is mediated by two distinct pathways. The extrinsic pathway is initiated by stimulation of receptors of the tumor necro- sis factor (TNF) superfamily, such as TNF-related apopto- sis-inducing ligand (TRAIL) receptors resulting in the formation of the death-inducing signaling complex (DISC), activation of caspase-8 and subsequently effector caspases such as caspase-3 (5). The mitochondrial pathway is initiated by the permeabilization of the outer mitochon- drial membrane, followed by the release of cytochrome c and other apoptosis-promoting factors from the mitochon- drial intermembrane space into the cytosol, resulting in the formation of the cytochrome c/Apaf-1/caspase-9 contain- ing apoptosome complex, which promotes the activation of caspase-9 and -3 (6). The release of apoptotic factors from mitochondria is tightly controlled by the action of pro- and antiapoptotic proteins of the Bcl-2 family (7). Both pathways are linked by the Bcl-2 family protein Bid, which initiates the mitochondrial pathway on its cleavage to tBid by caspases (7). Authors' Affiliations: 1 University Children's Hospital, Ulm, Germany; 2 Institute for Experimental Cancer Research in Pediatrics, Goethe-Uni- versity, Frankfurt, Germany; and 3 Department of Neurosurgery, Ulm Uni- versity, Ulm, Germany Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Simone Fulda, Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, Komturstr. 3a, 60528 Frankfurt, Germany. Phone: 49-69-67866557; Fax: 49-69- 6786659157; E-mail: simone.fulda@kgu.de doi: 10.1158/1078-0432.CCR-11-0075 Ó2011 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 4019