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Introduction clinical Phase I/II studies in different tumor types. Phase I studies have shown that patients tolerate recombinant TRAIL (Apo2L/dulanerim) as well as agonistic antibodies quite well [9]. TRAIL can bind to five different receptors, the death receptors, TRAIL‐R1 (DR4) and TRAIL‐ R2 (DR5), the decoy receptors TRAIL‐R3 (DcR1) and TRAIL‐R4 (DcR2), and the soluble receptor osteoprotegerin (OPG) [10]. TRAIL activates the extrinsic pathway by binding to TRAIL‐R1 and TRAIL‐R2. The death inducing signaling complex (DISC) consisting of FADD and caspase‐8 is than formed, leading to caspase‐8 and caspase‐3 activation followed by apoptosis. Caspase‐8 can also cleave the pro‐apoptotic Bcl‐2 family member Bid into tBid, which in turn results in activation of other pro‐apoptotic family members, such as BAX or BAK. This leads to the induction of mitochondrial activator outer membrane permeabilization (MOMP) and release of cytochrome c and Smac/Diablo in the cytosol, resulting in the activation of apoptosis through the intrinsic pathway that involves caspases‐9 and ‐3 [11;12]. However, in addition to apoptosis induction that can be regarded as the canonical route of TRAIL signaling an increasing number of studies have shown that TRAIL can also activate diverse other intracellular non‐canonical signaling pathways in tumor cells. These include mitogen activated protein kinases (MAPKs), phosphoinositide 3‐kinase (PI3K) and Akt and nuclear factor κB (NF‐κB) that can enhance cell survival, cell proliferation and even migration/ invasion of tumor cells, thus possibly stimulating tumorigenesis [13;14]. Moreover, a large number of cancer cells, especially the highly malignant ones, are resistant to TRAIL‐induced apoptosis [15]. Some cancer cells that were initially sensitive to TRAIL can also develop resistance. TRAIL resistance is related to alterations at different points in the signaling pathway, mutations in the death receptors, inhibition of active DISC formation by cFLIP or more downstream by overexpression of anti‐apoptotic proteins amongst others XIAP and Bcl‐2 family members. The activation of the survival pathways aforementioned can also counteract the apoptotic effect of TRAIL. To circumvent TRAIL resistance and to increase the efficacy of this molecule, TRAIL can be combined with other anti‐cancer agents, such as kinase inhibitors, radiation and chemotherapy (see for extensive overview of TRAIL sensitizing strategies in lung cancer [16]). The aim of the research described in this thesis is to obtain more knowledge of the molecular mechanisms governing TRAIL resistance in NSCLC, in particular the role of kinases herein. In addition, we have explored several combination treatments for enhancing TRAIL efficacy. Ultimately, the work may lead to the identification of novel predictive markers and therapeutic approaches for optimizing TRAIL‐based therapies. ‐ 11 ‐
Chapter 1 OUTLINE OF THE THESIS As TRAIL‐induced apoptosis is frequently blocked in tumor cells and even TRAIL‐ dependent tumor promoting events may occur, it is important to obtain more knowledge of the underlying mechanisms causing these unwanted phenomena. In this thesis the focus is on examining whether kinases and which type of kinases are involved in TRAIL resistance. In Chapter 2 an overview is given of the different non‐canonical signaling cascades that have been found to be triggered by TRAIL in cancer cell models representing various tumor types. Some kinases have been identified that enhance TRAIL‐induced apoptosis in sensitive tumor cells such as Mitogen Activated Protein (MAP) kinases p38 and JNK. However, multiple other kinases have been found to contribute to non‐apoptotic signaling in TRAIL resistant tumor cells, including IĸB and PI3K/Akt. Yet other kinases such as the ROCK/ LIM kinase have been found to stimulate invasion. These different TRAIL inducible kinases are reviewed in detail in this chapter. In Chapter 3 we have studied the activation of p38 and JNK by TRAIL in sensitive and resistant NSCLC cells and the mechanism and consequences of activation have been evaluated. Previously, it has been shown that TRAIL activates these two kinases through the formation of the secondary complex, which consists among others of FADD, TRADD, Caspase‐8, FADD, TRAF2, and RIP1 [17]. In NSCLC cells the pro‐apoptotic or anti‐apoptotic effects of p38 and JNK activation by TRAIL were studied using selective chemical kinase inhibitors. The molecular mechanisms have been examined using siRNA‐dependent knockdown and ectopic overexpression strategies. In particular, the involvement of RIP1 and caspase‐8 in the activation of these pathways was investigated by silencing RIP1 expression with short hairpin (sh)RNA and using NSCLC H460 cells stably overexpressing the caspase‐8 inhibitor CrmA, respectively. In Chapter 4 we employed peptide arrays containing 1,024 different kinase pseudosubstrates as a kinomic approach to contrast kinase activation patterns in TRAIL apoptosis sensitive and resistant NSCLC cells. In this way, we also attempted to identify the kinases responsible for TRAIL‐induced migration and invasion that we observed in TRAIL resistant NSCLC cells. A novel non‐canonical TRAIL signaling route was revealed involving RIP1‐Src‐STAT3 signaling, which is preferentially activated by TRAIL‐R2. We propose that combined use of TRAIL with selective kinase inhibitors will either overcome TRAIL resistance and/ or prevent TRAIL‐induced cell migration. In addition to examining TRAIL‐induced protein kinase signaling and the application of protein kinase targeted inhibitors for enhancing TRAIL‐dependent apoptosis we also explored other combination strategies that indirectly affect kinases. An agent that targets heat shock proteins and a novel chemotherapeutic targeting thymidylate synthase (TS) that causes DNA damage through its incorporation into DNA were examined for potentiating the apoptosis‐inducing capacity of TRAIL. In Chapter 5 we examined the combined anti‐tumor effects of TRAIL together with a the novel anti‐cancer agent, 17‐ ‐ 12 ‐
Page 2 and 3: TRAIL-induced kinases activation an
Page 4 and 5: TRAIL‐induced kinases activation
Page 6: “In order to be irreplaceable one
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Page 11: Chapter 1 GENERAL INTRODUCTION Canc
Page 15 and 16: Chapter 1 Reference List 1. Jemal A
Page 17 and 18: Chapter 2 ABSTRACT Tumor necrosis f
Page 19 and 20: Chapter 2 INTRODUCTION The death li
Page 21 and 22: Chapter 2 In preclinical studies, a
Page 23 and 24: Chapter 2 Table 1| Summary of the k
Page 25 and 26: Chapter 2 proliferation could be pr
Page 27 and 28: Chapter 2 Figure 2. Canonical and n
Page 29 and 30: Chapter 2 associated with TRAIL res
Page 31 and 32: Chapter 2 adhesion molecules [109].
Page 33 and 34: 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 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,
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Chapter 4 role in the activation of
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Chapter 4 the tip with a diameter o
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Chapter 4 RESULTS TRAIL induces a s
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Chapter 4 vehicle control or with 5
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Chapter 4 Non‐canonical TRAIL res
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Chapter 4 A549‐shRIP1 cells clear
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Chapter 4 Figure 7. Inhibition of S
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Chapter 4 DISCUSSION The TRAIL rece
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Chapter 4 Src‐independent mechani
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Chapter 4 variants. Cell Death Dis
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Chapter 4 ‐ 82 ‐
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Chapter 5 ABSTRACT TRAIL is an inte
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Chapter 5 targets and subsequent pr
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Chapter 5 RESULTS Synergistic activ
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Chapter 5 ng/ml TRAIL (Fig. 3B). Ap
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Chapter 5 by sub‐G1 levels in the
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Chapter 5 DISCUSSION In the present
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Chapter 5 Reference List 1. Jemal A
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Chapter 6 ABSTRACT TRAIL is a tumor
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Chapter 6 various stress stimuli [1
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Chapter 6 Subsequently, the membran
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Chapter 6 B H460 A549 Figure 1. Rep
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Chapter 6 TRAIL‐dependent cell de
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Chapter 6 B H460 A549 C Figure 4 (c
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Chapter 6 Figure 5 (continued). Mec
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Chapter 6 regulation and checkpoint
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Chapter 6 mechanism of immune evasi
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Chapter 7 ABSTRACT Thymidine phosph
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Chapter 7 Figure 1. Schematic overv
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Chapter 7 that was measured before
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Chapter 7 RESULTS TdR conversion to
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Chapter 7 dR is secreted from the c
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Chapter 7 Figure 4. Accumulation of
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Chapter 7 angiogenic properties. Ho
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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 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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