The concept of killing cancer cells without adverse. Editorials, continued

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1 RAC1 are associated with ulcerative colitis. Gastroenterology 2011;141: Ullman TA, Itzkowitz SH. Intestinal inflammation and cancer. Gastroenterology 2011;140: Eblen ST, Slack JK, Weber MJ, et al. Rac-PAK signaling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes. Mol Cell Biol 2002; 22: Fernandez-Zapico ME, Gonzalez-Paz NC, Weiss E, et al. Ectopic expression of VAV1 reveals an unexpected role in pancreatic cancer tumorigenesis. Cancer Cell 2005;7: Yang C, Liu Y, Leskow FC, et al. Rac-GAP-dependent inhibition of breast cancer cell proliferation by {beta}2-chimerin. J Biol Chem 2005;280: Jordan P, Brazao R, Boavida MG, et al. Cloning of a novel human Rac1b splice variant with increased expression in colorectal tumors. Oncogene 1999;18: Klimstra DS, Longnecker DS. K-ras mutations in pancreatic ductal proliferative lesions. Am J Pathol 1994;145: Morris JPt, Wang SC, Hebrok M. KRAS, Hedgehog, Wnt and the twisted developmental biology of pancreatic ductal adenocarcinoma. Nat Rev Cancer;10: Farrow B, Sugiyama Y, Chen A, et al. Inflammatory mechanisms contributing to pancreatic cancer development. Ann Surg 2004; 239: Reid-Lombardo KM, Fridley BL, Cunningham JM, et al. Inflammation-related gene variants as risk factors for pancreatic cancer. Cancer Epidemiol Biomarkers Prev 2011;20: Scotti ML, Bamlet WR, Smyrk TC, et al. Protein kinase Ciota is required for pancreatic cancer cell transformed growth and tumorigenesis. Cancer Res 2010;70: Rodriguez-Viciana P, Warne PH, Khwaja A, et al. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 1997;89: Tsianos EV, Katsanos K. Do we really understand what the immunological disturbances in inflammatory bowel disease mean? World J Gastroenterol 2009;15: Zhang H, Sun C, Glogauer M, et al. Human neutrophils coordinate chemotaxis by differential activation of Rac1 and Rac2. J Immunol 2009;183: Glogauer M, Marchal CC, Zhu F, et al. Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions. J Immunol 2003;170: Reprint requests Address requests for reprints to: Marcelo G. Kazanietz, PhD, Department of Pharmacology, University of Pennsylvania School of Medicine, 1256 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania marcelog@upenn.edu; fax: (215) Conflicts of interest The authors disclose no conflicts by the AGA Institute /$36.00 doi: /j.gastro Oxaliplatin Uses JNK to Restore TRAIL Sensitivity in Cancer Cells Through Bcl-xL Inactivation See Oxaliplatin sensitizes human colon cancer cells to TRAIL through JNK-dependent phosphorylation of Bcl-xL, by El Fajoui Z, Toscano F, Jacquemin G, et al, on page 663. The concept of killing cancer cells without adverse effects on normal cells is a long-held ideal of cancer therapy. An epitome of such therapeutic index, TRAIL (TNF-related apoptosis-inducing ligand; Apo2L) is a protein involved in the immune surveillance of cancer that selectively induces apoptosis in cancer cells. 1 This property of TRAIL has led to several clinical trials in a range of malignancies using recombinant TRAIL and TRAIL-receptor agonist antibodies. 2 The sensitivity of cancer cells to TRAIL-induced apoptosis and the molecular determinants that confer this sensitivity are heterogeneous. This differential sensitivity of cancer cells to TRAIL-induced apoptosis cannot be superficially explained by expression levels of TRAIL receptors in cancer cells, which include 2 proapoptotic receptors (DR4 and DR5) and 2 decoy receptors (DcR1 and DcR2). From a therapeutic perspective, reversing cancer cell resistance to TRAIL is a priority and has resulted in a number of synergistic combinations with TRAIL and other cancer therapeutics have been identified including sorafenib, bortezomib, tamoxifen, and DNA-damaging agents such as oxaliplatin. In some cases, the molecular basis of these synergistic combinations have been in part elucidated, for example, the down-regulation of Mcl-1 and ciap2 by sorafenib, 3 whereas other combinations remain unexplained. In this issue of GASTROENTEROLOGY, El Fajoui et al 4 have determined a mechanism by which oxaliplatin induces TRAIL sensitization. The authors found that this synergistic combination depends exclusively on caspase-9 dependent, mitochondrial-mediated apoptosis and inactivates Bcl-xL by phosphorylation at serine 62 by c-jun N-terminal kinase (JNK). This phosphorylation disrupts its inhibitory binding to the potent proapoptotic Bcl-2 protein Bax, and is a critical aspect of restoring TRAIL sensitivity. TRAIL-induced trimerization of its receptors upon binding colocalizes their intracellular death domains and recruits the Fas-associated death domain (FADD) and pro caspase-8, forming the death-inducing signaling complex. The death-inducing signaling complex activates caspase-8 by autocatalytic cleavage, although the ensuing signaling events are cell-type dependent. In type I cells, apoptosis is initiated through the extrinsic 430

2 death pathway by caspase-8 directly triggering the cascade of effector caspases-3, -6, and -7. Alternatively, type II cells engage the intrinsic death pathway by caspase-8 mediated cleavage of Bid to tbid that ultimately disrupts the mitochondrial membrane integrity and causes formation of the apoptosome that executes apoptosis. 5 Poration of the mitochondrial membrane is regulated by the Bcl-2 family of proteins. This family includes proapoptotic members (Bak, Bok, and Bax), antiapoptotic members that effectively sequester the proapoptotic members (A1, Bcl-2, Bcl-w, Bcl-xL, and Mcl-1), and BH3-only proteins that bind and antagonize these antiapoptotic members (Bad, Bid, Bik, Bim, Bmf, Hrk, Noxa, and Puma). Although the exact details that control mitochondrial membrane disruption are still debated, it seems to be directly controlled by oligomerization of proapoptotic Bcl-2 proteins, particularly Bax, which can be promoted by tbid 5 and antagonized by antiapoptotic Bcl-2 proteins. The regulation of Bax seems to involve its localization as well as a conformation-dependent insertion into the mitochondrial membrane. 6,7 Several molecules that impact the extrinsic and intrinsic cell death pathways have been found to modulate TRAIL sensitivity at the intracellular level such as c-flip, XIAP, Mcl-1, ciap2, caspase-8 expression, and Bcl-2 family proteins. In light of these cell type dependent cascades of events that control TRAIL-induced apoptosis and associated regulators of proteins within these pathways, it is perhaps unsurprising that TRAIL resistance is a multifactorial and context-dependent phenomenon. In accordance with its role in mitochondria-mediated apoptosis, overexpression of Bcl-xL antagonizes TRAILinduced apoptosis specifically in type II cells Sensitization to TRAIL-induced apoptosis by oxaliplatin has been reported in chemoresistant Jurkat cells that overexpress either Bcl-2 or Bcl-xL that was caspase-8 independent. 12 Previously, the authors reported that TRAILresistant, type II colon cancer cells could be sensitized by oxaliplatin. 13 However, this sensitization in wildtype p53 cells was inhibited by a p53-dependent upregulation of a TRAIL decoy receptor that we previously described as mechanism of protection from p53-dependent apoptosis. 14 Given the role of the Bcl-2 family in the intrinsic death pathway, it is logical that these proteins play a critical role in TRAIL sensitivity and therefore the synergy of TRAIL with chemotherapies in type II cells. Whereas regulation of these Bcl-2 family members may be conferred at the expression level, phosphorylation of these proteins is an alternative and frequently utilized mechanism of controlling apoptosis by the intrinsic death pathway. Inhibition of Bcl-2 by direct phosphorylation 15 occurs in response to several stimuli including interleukin-3 and apoptosis-inducing chemotherapies such as taxol and etoposide. 16,17 Although many kinases have since been found to phosphorylate Bcl-2, JNK is thought to be a major regulator of Bcl-2 mediated apoptosis and autophagy through multiple phosphorylation sites JNK is a stress-induced MAPK family member that is activated in response to a variety of stimuli including cytokines, ultraviolet radiation, environmental stresses, and chemotherapies. 22 This kinase plays an essential role in the intrinsic death pathway 23 and has been shown to phosphorylate and antagonize Bcl-2, Bcl-xL, and Mcl-1. 20,24,25 Oxaliplatin causes DNA strand breaks and activation of JNK independently of DNA mismatch repair proteins, contrary to cisplatin, 26 and subsequent apoptosis that involves PUMA. 27 The exact molecular details of oxaliplatin-induced JNK activation are unclear. The identification of JNK-dependent phosphorylation of serine 62 of Bcl-xL by El Fajoui et al 4 adds another avenue by which JNK regulates the mitochondrial death pathway through the Bcl-2 family and provides a molecular explanation for oxaliplatin-induced sensitization of cancer cells to TRAIL. Integrating this finding with our current understanding of TRAIL- and oxaliplatin-induced cellular events, it is clear that oxaliplatin-induced apoptosis and sensitization to TRAIL is conferred by JNK-dependent phosphorylation of Bcl-2 family members (Figure 1). Oxaliplatin inhibits DNA replication by forming platinum- DNA adducts 28 and subsequently activates JNK, which phosphorylates the antiapoptotic Bcl-2 family members Bcl-xL, Bcl-2, and Mcl-1 to disrupt their interactions with Bax and Bak to promote apoptosis. However, the cellular consequence of activating JNK is not easily predicted owing to the number of JNK substrates and its seemingly contradictory roles in cell survival and cell death that are highly context dependent. 29,30 Within the Bcl-2 family, the net effect of JNK activation of Bcl-2 is not straightforward, because JNK can also phosphorylate Bad to suppress apoptosis 31 and Bcl-w can prevent activation of JNK. 32 To add to the complexity, prosurvival signaling induced by TRAIL can also directly activate JNK, although the functional consequence of this seems to be cell-type specific. However, this was not observed in HT29 and V9P cells used by El Fajoui et al 4 in this study. The observation that apoptosis induced by TRAIL and oxaliplatin is independent of caspase-8 and dependent on caspase-9 is interesting as caspase-8 plays an initiator role in both the type I and type II canonical signaling pathways of TRAIL. Type II cells are expected to be dependent on caspase-9, because the zymogen is activated by the apoptosome as a critical effector to 431

3 Figure 1. Oxaliplatin-induced JNK activation overlaps with the TRAIL-mediated intrinsic death pathway. TRAIL initiates cell death by binding proapoptotic death receptors DR4 or DR5 that colocalizes their intracellular death domains. This clustering recruits the Fas-associated death domain (FADD) and pro-caspase-8 that results in its activation through autocatalytic cleavage. In type II cells, active caspase-8 cleaves Bid to a truncated form, tbid, which subsequently interacts with proapoptotic Bcl-2 family members Bax and Bak. This interaction leads to permeabilization of the mitochondrial membrane and release of cytochrome c. Cytosolic cytochrome c then combines with Apaf-1 and ATP to form the apoptosome that activates caspase-9 to trigger apoptosis through the caspase cascade. Oxaliplatin complexes with DNA to form adducts that inhibit DNA replication and therefore induces cellular stress responses. As a consequence, JNK is activated and phosphorylates antiapoptotic Bcl-2 family members such as Bcl-xL at serine 62. This results in dissociation of these proteins with the proapoptotic Bcl-2 family members Bax and Bak. These previously sequestered proteins are now free to oligomerize and porate the mitochondrial membrane to induce apoptosis. initiate the caspase cascade. However, it has been previously noted that TRAIL-induced cell death can be triggered independently of caspase-8. We have previously found that caspase-9 may play a key role in the sensitivity of normal hepatocytes and esophageal epithelial cells to TRAIL, whereas cancer cells seem to depend on caspase-8. 37,38 The molecular details of caspase-8 independent cell death signaling by TRAIL and the determinants of the bifurcation of signaling events after caspase-8 activation between type I and type II cells remain unclear. Despite the biologically complex nature of JNK activation and its effects, this report highlights the importance of the intrinsic death pathway in the combination of TRAIL and oxaliplatin. These findings suggest that this combination may be effective specifically in type II cells that overexpress Bcl-xL. This has important clinical implications in stratifying patients who will benefit from this combination based on such tumor characteristics. The contribution of JNK-dependent Bcl-xL phosphorylation to overall TRAIL sensitivity in the background of high levels of other Bcl-2 targets of JNK such as Mcl-1 and Bcl-2 remain to be seen and will inform on the utility of this combination in such TRAIL-resistant tumors. Furthermore, exploring the robustness of oxaliplatin-induced JNK activation and its effects on Bcl-2 family members such as Bcl-xL in vivo will inform on the physiologic prevalence of this mechanism and the clinical utility of combining oxaliplatin with TRAIL. 432

4 JOSHUA E. ALLEN WAFIK S. EL-DEIRY Laboratory of Translational Oncology and Experimental Cancer Therapeutics Department of Medicine (Hematology/Oncology) Penn State Hershey Cancer Institute Penn State College of Medicine Hershey, Pennsylvania and Biochemistry and Molecular Biophysics Graduate Group University of Pennsylvania School of Medicine Philadelphia, Pennsylvania References 1. Falschlehner C, Schaefer U, Walczak H. Following TRAIL s path in the immune system. Immunology 2009;127: Abdulghani J, El-Deiry WS. TRAIL receptor signaling and therapeutics. Expert Opin Ther Targets 2010;14: Ricci MS, Kim SH, Ogi K, et al. Reduction of TRAIL-induced Mcl-1 and ciap2 by c-myc or sorafenib sensitizes resistant human cancer cells to TRAIL-induced death. Cancer Cell 2007;12: El Fajoui Z, Toscano F, Jacquemin G, et al. Oxaliplatin sensitizes human colon cancer cells to TRAIL through JNK-dependent phosphorylation of Bcl-xL. Gastroenterology 2011;141: Lovell JF, Billen LP, Bindner S, et al. Membrane binding by tbid initiates an ordered series of events culminating in membrane permeabilization by Bax. Cell 2008;135: Wolter KG, Hsu YT, Smith CL, et al. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 1997;139: Eskes R, Desagher S, Antonsson B, et al. Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol 2000;20: Hinz S, Trauzold A, Boenicke L, et al. Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis. Oncogene 2000;19: Burns TF, El-Deiry WS. Identification of inhibitors of TRAIL-induced death (ITIDs) in the TRAIL-sensitive colon carcinoma cell line SW480 using a genetic approach. J Biol Chem 2001;276: Fulda S, Meyer E, Debatin KM. Inhibition of TRAIL-induced apoptosis by Bcl-2 overexpression. Oncogene 2002;21: Walczak H, Bouchon A, Stahl H, et al. Tumor necrosis factorrelated apoptosis-inducing ligand retains its apoptosis-inducing capacity on Bcl-2- or Bcl-xL-overexpressing chemotherapy-resistant tumor cells. Cancer Res 2000;60: Ballestrero A, Nencioni A, Boy D, et al. Tumor necrosis factorrelated apoptosis-inducing ligand cooperates with anticancer drugs to overcome chemoresistance in antiapoptotic Bcl-2 family members expressing Jurkat cells. Clin Cancer Res 2004;10: Toscano F, Fajoui ZE, Gay F, et al. p53-mediated upregulation of DcR1 impairs oxaliplatin/trail-induced synergistic anti-tumour potential in colon cancer cells. Oncogene 2008;27: Meng RD, McDonald ER, Sheikh MS, et al. The TRAIL decoy receptor TRUNDD (DcR2, TRAIL-R4) is induced by adenovirusp53 overexpression and can delay TRAIL-, p53-, and KILLER/ DR5-dependent colon cancer apoptosis. Mol Ther 2000;1: Haldar S, Jena N, Croce CM. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A 1995;92: Haldar S, Chintapalli J, Croce CM. 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Signal transduction by the JNK group of MAP kinases. Cell 2000;103: Tournier C, Hess P, Yang DD, et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 2000;288: Fan M, Goodwin M, Vu T, et al. Vinblastine-induced Phosphorylation of Bcl-2 and Bcl-XL Is Mediated by JNK and Occurs in Parallel with Inactivation of the Raf-1/MEK/ERK Cascade. J Biol Chem 2000;275: Inoshita S, Takeda K, Hatai T, et al. Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to oxidative stress. J Biol Chem 2002;277: Nehme A, Baskaran R, Nebel S, et al. Induction of JNK and c-abl signalling by cisplatin and oxaliplatin in mismatch repair-proficient and -deficient cells. Br J Cancer 1999;79: Wang X, Li M, Wang J, et al. The BH3-only protein, PUMA, is involved in oxaliplatin-induced apoptosis in colon cancer cells. Biochem Pharmacol 2006;71: Woynarowski JM, Faivre S, Herzig MC, et al. Oxaliplatin-induced damage of cellular DNA. Mol Pharmacol 2000;58: Liu J, Lin A. Role of JNK activation in apoptosis: a double-edged sword. Cell Res 2005;15: Bogoyevitch MA, Kobe B. Uses for JNK: the many and varied substrates of the c-jun N-Terminal kinases. Microbiol Mol Biol Rev 2006;70: Yu C, Minemoto Y, Zhang J, et al. JNK suppresses apoptosis via phosphorylation of the proapoptotic Bcl-2 family protein BAD. Mol Cell 2004;13: Lee HW, Lee S-S, Lee SJ, et al. Bcl-w is expressed in a majority of infiltrative gastric adenocarcinomas and suppresses the cancer cell death by blocking stress-activated protein kinase/c-jun NH2- terminal kinase activation. Cancer Res 2003;63: Lin Y, Devin A, Cook A, et al. The death domain kinase RIP is essential for TRAIL (Apo2L)-induced activation of Ikappa B kinase and c-jun N-terminal kinase. Mol Cell Biol 2000;20: Muhlenbeck F, Haas E, Schwenzer R, et al. TRAIL/Apo2L activates c-jun NH2-terminal kinase (JNK) via caspase-dependent and caspase-independent pathways. 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5 36. Corazza N, Jakob S, Schaer C, et al. TRAIL receptor-mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality. J Clin Invest 2006;116: Ozoren N, Kim K, Burn TF, et al. The caspase 9 inhibitor Z-LEHD- FMK protects human liver cells while permitting death of cancer cells exposed to tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res 2000;60: Kim S-H, Kim K, Kwagh JG, et al. Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells. J Biol Chem 2004;279: Reprint requests Address requests for reprints to: Wafik S. El-Deiry, MD, PhD, 500 University Drive, Room T4423, Hematology/Oncology Division, Penn State Hershey Medical Center and Cancer Institute, Hershey, Pennsylvania wafik.eldeiry@gmail.com Conflicts of interest The authors disclose no conflicts by the AGA Institute /$36.00 doi: /j.gastro