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Summarizing discussion & future perspectives In Chapter 3 we evaluated the kinetics of MAPK p38 and JNK upon TRAIL exposure in two different NSCLC cell lines and examined the effect on apoptosis activation. These kinases are known to be activated directly by TRAIL through the formation of a secondary complex consisting of the serine/threonine kinase RIP1, caspase‐8 and FADD [7]. TRAIL treatment induced the phosphorylation of p38 and JNK1/2/3 only in sensitive H460 cells and not in the resistant A549 cell line. The use of chemical inhibitors and siRNAs indicated that JNK activation counteracted TRAIL‐induced apoptosis, whereas activation of p38 stimulated apoptosis. RIP1 was cleaved following TRAIL treatment concomitantly with detectable JNK phosphorylation. Further examination of the role of RIP1 by short hairpin (sh)RNA‐ dependent knockdown showed that p38 can be phosphorylated in both RIP1‐dependent and ‐independent manner, whereas JNK phosphorylation occurred independently of RIP1. The exact molecular mechanisms of p38 and JNK activation by TRAIL that occurs independently of RIP1 is yet unclear and remains to be unraveled. Immunoprecipitation experiments to determine the protein components that are responsible for the activation of these kinases will give us clues how to target these pathways more upstream. Inhibition of kinases that suppress apoptosis is expected to facilitate the elimination of cancer cells when combined with TRAIL receptor agonists. In addition, the activation of JNK by TRAIL occurred in a caspase‐8‐dependent manner, which was not the case for p38. Mcl‐1 was shown to be a downstream target of both p38 and JNK and silencing of Mcl‐1 with siRNA strongly augmented TRAIL‐induced apoptosis in H460 cells. Taken together, our results suggest a model in which TRAIL‐induced activation of p38 and JNK have counteracting effects on Mcl‐ 1 expression leading to pro‐ or anti‐apoptotic effects, respectively. The role of p38 and JNK in TRAIL signaling appears tumor‐type dependent. In prostate cancer cells, for example, activation of both kinases prevented TRAIL‐induced apoptosis [8]. JNK, on the other hand enhanced the apoptotic effect of TRAIL in human lymphoid cell lines [9]. This illustrates that caution is warranted when developing combination therapies with TRAIL receptor agonists and kinase inhibitors. Pre‐selection of suitable patients groups and tumor type‐dependent approaches will be required for optimal results in a clinical setting. Since Mcl‐1 appears to be the main target of p38 and JNK, the combined use of TRAIL with a Mcl‐1 inhibitor/antagonist, such as Gosypoll (AT‐101) and GX15‐070 could also provide an effective strategy. In colon, breast, and pancreatic cancer cells inhibition of Mcl‐1 with these inhibitors increased TRAIL‐induced apoptosis [10‐12]. In NSCLC cells this remains to be formally demonstrated, although our data has shown that Mcl‐1 knockdown sensitizes for TRAIL‐dependent apoptosis (Chapter 3). Gosypoll and GX15‐070 are both being evaluated in clinical trials (clinicaltrails.gov) and combined use with TRAIL might be interesting to explore in subsequent trials. ‐ 137 ‐
Chapter 8 Recently, various different TRAIL receptor complexes have been identified that trigger alternative forms of cell death, including the necroptosome and ripoptosome [13]. Currently, the more precise mechanisms that trigger the various kinases pathways are elusive. Further research is required to determine the molecular mechanisms triggering and regulating non‐canonical TRAIL signalling, which will also provide new clues for optimal use of TRAIL receptor agonists for the treatment of cancer. An unexpected feature of TRAIL is the induction of migration and invasion in tumor cells, which has been shown previously in colon, pancreatic ductal adenocarcinoma, and cholangiocarcinoma cancer cells [14‐16]. In Chapter 4 and as published [17], we describe TRAIL induced migration and invasion in NSCLC cells in a panel of NSCLC cell lines with different sensitivities for TRAIL. Migratory and invasive properties of TRAIL were observed in the resistant NSCLC cells, demonstrating possible pro‐tumorigenic activity of TRAIL in this cancer type for the first time. Using a kinomic approach with PepChip peptide arrays we were able to identify kinases that were activated in TRAIL resistant A549 cells and not in sensitive H460 cells. Following validation of the hits, the Src‐STAT3 pathway directly activated by TRAIL was found to be responsible for invasive behavior. Activation of Src could be linked with RIP1 since knockdown of RIP1 or chemical inhibition with necrostatin‐1 prevented activation. In literature, several other mechanisms have been described underlying TRAIL‐induced migration/invasion. In apoptosis resistant cholangiocarcinoma cancer cells migration was found to depend on NF‐κB [15], and in colon cancer cells K‐Ras, Raf‐1 and the ROCK/LIM kinase/cofilin pathway were involved [14]. It would be interesting to study if the RIP1‐Src‐STAT3 pathway is also involved in TRAIL‐induced migration in other tumor types as well. The migratory effect of TRAIL impedes its clinical value since unwanted pro‐tumorigenic effects may occur when administered in patients. We postulate that better knowledge of these mechanisms may provide markers for selecting patients that will likely benefit from TRAIL‐based therapy and will provide a rationalized basis for combination therapies with TRAIL agonists. Thus, kinases and their phosphorylated active forms may provide biomarkers to predict the sensitivity and/or consequences of TRAIL treatment. When required, TRAIL receptor agonists could be combined with small molecules that inhibit mediators of migration/invasion, such as Src, STAT3 or RIP1 inhibitors. In this context, markers for predicting TRAIL sensitivity in general would be of great therapeutic value. However, thus far only a few possible suitable biomarkers have been identified, which have not been tested in the clinic for upfront patient selection as yet. For example, TRAIL receptor O‐glycosylation was reported to be a good predictor for TRAIL sensitivity [18]. O‐ linked glycosylation involves the binding of glycosyl groups to threonine and serine side chains leading to improved ligand binding and receptor protein activation. In cancer tissue specific O‐glycosyltransferases were found to be overexpressed when compared to normal tissue and interestingly, were correlated with TRAIL sensitivity. In NSCLC, melanoma and pancreatic cancer cells, the expression of O‐glycosylation initiating enzyme GALNT14 was ‐ 138 ‐
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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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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 associated with TRAIL res
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Chapter 2 adhesion molecules [109].
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Chapter 2 melanoma cells from TRAIL
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Chapter 3 inhibitors and TRAIL rece
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Chapter 3 P38 and JNK have opposing
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Chapter 3 Figure 2 (continued). (e)
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Chapter 3 previously established H4
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Chapter 3 effect on TRAIL‐induced
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Chapter 3 induced apoptosis in H460
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Chapter 3 Reference List 1. Jemal A
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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 5 ABSTRACT TRAIL is an inte
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Page 101 and 102: Chapter 6 ABSTRACT TRAIL is a tumor
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Page 119 and 120: Chapter 7 ABSTRACT Thymidine phosph
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