towards improved death receptor targeted therapy for ... - TI Pharma

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INTRODUCTION TRAIL and Hsp90 inhibition Lung cancer is the most deadliest cancer type in both men and women. Non‐small cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancer cases. Even though, a lot of progress has been made in the treatment of NSCLC in recent years, the 5‐ years overall survival is still less than 15% [1]. Novel therapies for this type of cancer are therefore urgently needed. The TNF‐related apoptosis inducing ligand (TRAIL) apoptotic pathway is a promising avenue to eradicate tumor cells as it induces cell death specifically in tumor cells and not in normal cells [2;3]. TRAIL can bind as a homotrimer to five different TRAIL‐ or death receptors (DR), named TRAIL‐R1 (DR4) and TRAIL‐R2 (DR4), the decoy receptors, TRAIL‐R3 (DcR1) and TRAIL‐R4 (DcR2) and the soluble receptor, osteoprotegerin (OPG). When TRAIL binds to TRAIL‐R1 or TRAIL‐R2, a death inducing signaling complex (DISC) is formed activating the extrinsic pathway, which then results in apoptosis. The decoy receptors and OPG do not transduce death signals into cells, but instead are thought to reduce apoptosis induction via the functional receptors TRAIL‐R1 and –R2 by sequestering TRAIL [4]. The extrinsic apoptosis pathway can also crosstalk with the intrinsic apoptosis pathway. This occurs through Bid, which is cleaved by caspase‐8, producing truncated (t)Bid that results in activation of the pro‐apoptotic Bcl‐2 family members, BAX or BAK. These proteins cause induction of mitochondrial outer membrane permeabilization (MOMP) leading to cytosolic release of apoptogenic factors such as cytochrome c, which is a cofactor for caspase‐9 activation leading to effector caspases activation [4]. Currently, TRAIL is tested in several clinical trials in which different agonistic agents are used in addition to recombinant produced TRAIL, such as mapatumumab (anti‐TRAIL‐R1 antibody), AMG 655 (anti‐TRAIL‐R2 antibody) and CS‐1008 [5‐7] However, from preclinical studies it is known that around half of the tumor cell lines are refractory to TRAIL‐induced apoptosis that can be converted into a sensitive phenotype by combined use with other agents [8]. For example, various chemotherapeutics as well as novel targeted agents can enhance TRAIL‐induced apoptosis in NSCLC (see [8] and references therein). Further research is ongoing to identify other or additional mechanisms that affect TRAIL sensitivity and that may be exploited for therapeutic purposes. TRAIL apoptotic signaling can be suppressed as a result of activity of proteins such as Akt, EGFR, and NF‐κB that can modulate this apoptotic pathway in multiple ways [9‐12]. The heat shock proteins (Hsp’s) are a group of chaperone proteins that are crucial for the maintenance of the stability and function of their client proteins, allowing cells to survive in lethal conditions. Especially Hsp90, is upregulated in many tumors and has an essential role in maintaining the malignant transformation of cancer cells [13]. A role for Hsp90 has also been suggested in the tumorigenesis of NSCLC [14]. Hsp90 interacts with and stabilizes several key signaling proteins, such as Akt, ErbB2, c‐Met, and Raf‐1. Inhibition of Hsp90 prevents association with client proteins leading to ubiquitination of the unfolded ‐ 85 ‐
Chapter 5 targets and subsequent proteasomal degradation thus removing possible anti‐apoptotic proteins resulting in sensitization for apoptosis [15]. The small molecule, 17‐allylamino‐17‐ demethoxy‐geldanamycin (17‐AAG) is a potent Hsp90 inhibitor that induces cell cycle arrest and apoptosis in a variety of tumor cells, including NSCLC [16]. In this study we investigated whether 17‐AAG enhances the apoptosis‐inducing effect of TRAIL in NSCLC cells. Furthermore, the mode of action of the synergy of these two compounds was studied. MATERIALS & METHODS Cell lines and reagents The NSCLC cell lines, A549 and H460, were cultured in RPMI‐1640 supplemented with 10% Fetal Calf Serum (FCS). The cells were maintained in a humidified incubator at 37 °C and in a 5% CO2‐atmosphere. TRAIL (PeproTech EC Ltd, London, UK) was aliquoted in PBS at a final concentration of 500 ng/ml and stored at ‐20 °C. 17‐AAG was diluted in DMSO (stock solution of 1mM) and stored at ‐20 °C. The caspase inhibitors Z‐VAD‐fmk (general caspase inhibitor), Z‐IETD‐fmk (caspase‐8) and Z‐LEHD‐fmk (caspase‐9) were purchased from Bachem AG, dissolved in DMSO (10 mM or 20 mM) and stored at ‐20 °C. Drug cytotoxicity assays Drug cytotoxicity was determined using the methyl thiazole tetrazolium (MTT) assay. Cells were seeded in 96‐wells plates with a cell density of 9,000 cells/well. After 24 h, enabling attachment, cells were exposed (in triplicate) to increasing concentrations of the two drugs for 24 h. Next, the medium was removed and 50 µl MTT (10% diluted in HBSS buffer) per well was added and incubated for 1‐3 h at 37 °C. After the incubation, 150 μl DMSO per well was added and following 10‐15 min slowly shaking, the absorbance of the plate was measured at 540 nm with a TECAN plate reader by using the XFLUOR4 program. IC50 values were determined from the graphs. Synergy of TRAIL and 17‐AAG was determined by calculation of the combination index (CI) using Calcusyn (Calcusyn Biosoft, Cambridge, UK) as described earlier [17]. For calculation of the CI, only values above a fraction affected (FA) of 0.5 were used, equivalent to 50 ‐ 100% growth inhibition. A CI < 0.9 indicates synergism and >1.1 antagonism. Cell cycle and cell death measurements Cells were plated at a density of 200,000 cells/well in 6‐wells plates. After 24 h enabling attachment, the medium was replaced by medium containing the drug(s) as indicated. When indicated, caspase inhibitors were added to a final concentration of 20 µM, 2 h prior to drug treatment. After drug exposure cells were trypsinized, resuspended in medium collected from the matching sample and centrifuged for 5 min at 1200 rpm. After ‐ 86 ‐
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TRAIL-induced kinases activation an
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TRAIL‐induced kinases activation
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“In order to be irreplaceable one
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‐ 8 ‐
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Chapter 1 GENERAL INTRODUCTION Canc
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Chapter 1 OUTLINE OF THE THESIS As
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Chapter 1 Reference List 1. Jemal A
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Chapter 2 ABSTRACT Tumor necrosis f
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Chapter 2 INTRODUCTION The death li
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Chapter 2 In preclinical studies, a
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Chapter 2 Table 1| Summary of the k
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Chapter 2 proliferation could be pr
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Chapter 2 Figure 2. Canonical and n
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Chapter 2 associated with TRAIL res
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Chapter 2 adhesion molecules [109].
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Chapter 2 Reference List 1. Ashkena
Page 35 and 36: Chapter 2 37. Tolcher AW, Mita M, M
Page 37 and 38: Chapter 2 melanoma cells from TRAIL
Page 39 and 40: Chapter 2 and ERK pathways. Circula
Page 41 and 42: Chapter 3 ABSTRACT Tumor necrosis f
Page 43 and 44: Chapter 3 inhibitors and TRAIL rece
Page 45 and 46: Chapter 3 of amplification of the t
Page 47 and 48: Chapter 3 P38 and JNK have opposing
Page 49 and 50: Chapter 3 Figure 2 (continued). (e)
Page 51 and 52: Chapter 3 previously established H4
Page 53 and 54: Chapter 3 effect on TRAIL‐induced
Page 55 and 56: Chapter 3 induced apoptosis in H460
Page 57 and 58: Chapter 3 Reference List 1. Jemal A
Page 59 and 60: Chapter 3 40. Domina AM, Vrana JA,
Page 61 and 62: Chapter 4 ABSTRACT Tumor necrosis f
Page 63 and 64: Chapter 4 role in the activation of
Page 65 and 66: Chapter 4 the tip with a diameter o
Page 67 and 68: Chapter 4 RESULTS TRAIL induces a s
Page 69 and 70: Chapter 4 vehicle control or with 5
Page 71 and 72: Chapter 4 Non‐canonical TRAIL res
Page 73 and 74: Chapter 4 A549‐shRIP1 cells clear
Page 75 and 76: Chapter 4 Figure 7. Inhibition of S
Page 77 and 78: Chapter 4 DISCUSSION The TRAIL rece
Page 79 and 80: Chapter 4 Src‐independent mechani
Page 81 and 82: Chapter 4 variants. Cell Death Dis
Page 83 and 84: Chapter 4 ‐ 82 ‐
Page 85: Chapter 5 ABSTRACT TRAIL is an inte
Page 89 and 90: Chapter 5 RESULTS Synergistic activ
Page 91 and 92: Chapter 5 ng/ml TRAIL (Fig. 3B). Ap
Page 93 and 94: Chapter 5 by sub‐G1 levels in the
Page 95 and 96: Chapter 5 DISCUSSION In the present
Page 97 and 98: Chapter 5 Reference List 1. Jemal A
Page 101 and 102: Chapter 6 ABSTRACT TRAIL is a tumor
Page 103 and 104: Chapter 6 various stress stimuli [1
Page 105 and 106: Chapter 6 Subsequently, the membran
Page 107 and 108: Chapter 6 B H460 A549 Figure 1. Rep
Page 109 and 110: Chapter 6 TRAIL‐dependent cell de
Page 111 and 112: Chapter 6 B H460 A549 C Figure 4 (c
Page 113 and 114: Chapter 6 Figure 5 (continued). Mec
Page 115 and 116: Chapter 6 regulation and checkpoint
Page 117 and 118: Chapter 6 mechanism of immune evasi
Page 119 and 120: Chapter 7 ABSTRACT Thymidine phosph
Page 121 and 122: Chapter 7 Figure 1. Schematic overv
Page 123 and 124: Chapter 7 that was measured before
Page 125 and 126: Chapter 7 RESULTS TdR conversion to
Page 127 and 128: Chapter 7 dR is secreted from the c
Page 129 and 130: Chapter 7 Figure 4. Accumulation of
Page 131 and 132: Chapter 7 angiogenic properties. Ho
Page 133 and 134: Chapter 7 activity of enzymes. Natu
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Chapter 8 SUMMARIZING DISCUSSION AN
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Chapter 8 Recently, various differe
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Chapter 8 in phase II clinical tria
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Chapter 8 Reference List 1. Herbst
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Chapter 8 ‐ 144 ‐
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Chapter 9 NEDERLANDSE SAMENVATTING
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Chapter 9 waargenomen. Het gebruik
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Chapter 9 CONCLUSIE Het activeren v
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zelfs na werktijden (lees 23:45) ko
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Verder wil ik ook dr. Eric Ronken b
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