Generation of tools to analyse the native TRAIL DISC ……………………………………...………...89 Establishment of protocols for the bacterial expression and purification of rhTRAIL……89 Characterisation of purified rhTRAIL regarding its potential for apoptosis induction, receptor binding and apoptotic cascade activation...………………………………...…91 Implementation of the native DISC analysis and cross--reactivity test of the DR5 antibody with DR4…………………………………………....…………………………………..…………...95 Interim summary and conclusions I…………………………………………....……………………….……..98 The stepwise tumourigenesis cells as model system to study the gain of TRAIL sensitivity through transformation …………………..……………………………...………………….…… 99 Normal and transformed cells of the stepwise transformation model differ in cell culture characteristics…………………………………………………………………………………………99 Transformed but not normal cells die upon rhTRAIL, even though DISC proteins are expressed at comparable levels and a DISC is formed in normal cells………………………...100 Interim summary and conclusion II…………………………………………....……………………...........106 Identification and validation of NMHCIIa, NMHCIIb, MLC2 and ßACT as novel DR5 and DR4 interactors……………………………………………………………………………......107 NMHCIIa, NMHCIIb, MLC2 and ßACT interact differentially with the DRs of normal and transformed cells…………………………………………………………………………………107 NMHCIIa, NMHCIIb and ßACT are expressed at similar protein levels in normal BJ and transformed BJELR cells whereas MLC2 is increased in transformed cells……….…110 Interim summary and conclusion III…………………………………..……………....…………………..111 Functional characterisation of the NMHCII/DR interaction with respect to TRAIL apoptotic response …………………………………………………………………………………………………..112 Chemical inhibition of NMII Mg2+--ATPase activity decreased NMHCIIa, NMHCIIb and MLC2 association with DR5 and DR4 and increased rhTRAIL--induced apoptosis in normal BJ cells…………………………………………………………………………………….112 Deregulated signalling pathways decreasing NMII Mg2+ ATPase activity in transformed cells……………………………………………………………..……………………….…………….116 NMHCIIa but not NMHCIIb protein depletion enhances rhTRAIL apoptosis in normal BJ cells by altering the DISC composition…....……………………………………….......…...117 Interim summary and conclusion IV…………………………………..…………….

TABLE OF FIGURES

PREAMBLE

Several main strategies to therapeutically approach cancer exist (Figure 1) and their variety, operation mode and effectiveness have been extensively reviewed, some with special regard to a certain type of cancer. Aiming to give a general overview about the difficulties of existing cancer therapies, the major problems shall be briefly summarised. Surgery as the first applied cancer therapy is suitable only for non-hematological tumours and if cancer has metastasised, the complete surgical excision is impossible. Radiation therapy and chemotherapy lack tumour cell--specificity. Whilst radiation therapy damages the genetic material of both normal and transformed cells, chemotherapy interferes with cell division of all rapidly dividing cells and thus affects amongst others cells of the intestinal lining. Since normal cells, unlike their transformed counterparts, usually are able to repair their DNA, normal cells recover after treatment.

However, this recovery phase for the patient is associated with inconceivable suffering from side effects or, if repair mechanisms in normal cells fail, secondary cancer can derive from the therapy itself. In targeted therapies, mutated, overexpressed or otherwise critical proteins in cancer cells are in the focus. If the cause for cancer is based on a single mutational event, like it is the case for acute promyelocytic leukemia (APL)

where a part of the promyelocytic leukemia (PML) gene has fused with the retinoic acid receptor alpha (RARa) gene and the maintenance of the leukemic phenotype depends on the presence of the PML--RARa fusion protein, addition of a single agent that induces the degradation of the PML--RARa protein (here: all--trans retinoic acid) can suffice to achieve Figure 1: Main strategies of cancer treatment and their method of action. Various strategies in the fight of cancer are being followed, but to date no single strategy has proven efficient and tumour cell--specific. The tumour mass in the middle was taken from [START_REF] Bode | Cancer prevention research --then and now[END_REF].

complete remission in human patients [as reviewed by de Thé and [START_REF] De The | Acute promyelocytic leukaemia: novel insights into the mechanisms of cure[END_REF].

Unfortunately, the vast majority of human cancers display multiple genetic changes, which even increase in number with tumour progression. Not always these changes are associated with alterations in the DNA sequence, but can be linked to inherited modifications in DNA methylation states of CpG dinucleotides that are referred to as 'epigenetic' alterations. Cancer cells show global genomic hypomethylation leading to chromosomal instability and gene--specific promoter hypermethylation resulting in the silencing of for instance tumour suppressor genes. Administration of epigenetic drugs such as inhibitors of DNA methlation can revert aberrant DNA methylation and histone modification, thus providing another approach in targeted cancer therapy [reviewed in [START_REF] Rodriguez--Paredes | Cancer epigenetics reaches mainstream oncology[END_REF]. In turn, other therapeutic tactics use monoclonal antibodies or peptides that bind to a cancer cell--specific surface protein, delivering coupled cytotoxic agents into the vicinity of the tumour cell. Major drawbacks using these approaches include the stability of the targeting protein as well as the circumstance that cancer cells constantly change the repertoir of expressed cell surface proteins and can downregulate or loose the targeted antigen. In immunotherapy, one of the major drawback is time, which is one one hand required to generate the vaccine or antibody and on the other hand is needed by the immune cells to react upon the boost, so that immunotherapy usually demands longer periods to observe effects on the tumour. Therefore, immunotherapy for most cancers is used as an adjuvant cancer treatment [reviewed by Sharma et al 2011, Gerritsen and[START_REF] Gerritsen | Current and emerging treatment options for castration--resistant prostate cancer: a focus on immunotherapy[END_REF]. Depending on the modality used (vaccine, antibody), strong or immediate passive immune responses can be observed, including flu--like symptoms. Usually only a limited number of tumour-associated antigens are present to be targeted and there is currently no experience with long--term effects of immunotherapy. Hormone therapy does not lead to cure, but keeps tumour growth under control until cancer cells become hormone resistant or refractory, which then will require a different set of therapy. The use of angiogenesis inhibitors to prevent blood vessel growth required for tumour nutrition is complicated by the fact that normal and transformed tissues require several, but the same angiogenic factors.

Usually, only one factor is targeted in therapy which is not sufficient for a full tumour remission or even could lead to the activation of compensatory pathways augmenting tumour invasion and metastasis [START_REF] Benest | Tension in the vasculature[END_REF]Augustin 2009, Ellis and[START_REF] Ellis | Cancer: The nuances of therapy[END_REF].

Moreover, the administration route of agents and the maintenance of stability and activity of agent are difficult.

The variety of possible targeted cancer therapies becomes evident, if pathological characteristics are considered that are common to all human malignancies [reviewed by Hanahan and[START_REF] Hanahan | The hallmarks of cancer[END_REF][START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF]. These include self-sufficiency in growth signalling, insensitivity to growth inhibitory signals, evasion from apoptosis, limitedless replicative potential, sustained angiogenesis and tissue invasion and metastasis (Figure 2). Recently, the addition of a seventh feature called 'inflammatory microenvironment' was postulated, since tumour cells are capable to increase cytokine production of surrounding matrix cells enabling metastasis [START_REF] Mantovani | Cancer: Inflaming metastasis[END_REF][START_REF] Cavallo | the immune hallmarks of cancer[END_REF]. Many novel therapies constantly arise immediately attacking these so--called 'hallmarks of cancer', some of which are approved by the US Food and Drug Administration (FDA) or are currently tested in clinical trials (Table 1) Whichever route might be taken, only tumour--specific therapies leading to cancer cell death--driven removal of all transformed cells from a patients body ultimately will lead to cure from cancer. Of note, agents of different origin are described selectively inducing tumour cell death. These comprise viruses that specifically replicate in cancer cells leading to their lysis, virus--derived proteins (E4ORF4, NS1, apoptin [reviewed by [START_REF] Noteborn | Proteins selectively killing tumor cells[END_REF]) that induce cytotoxicity in transformed but not normal cells if overexpressed, as well as human endogenous proteins with tumouricidal activity such as human alpha--lactalbumin made lethal to tumour cells (HAMLET), melanoma differentiation--associated gene 7 (MDA7)--encoded interleukin 24 (IL24) and tumour-necrosis factor alpha--related apoptosis inducing ligand (TRAIL). Whilst potential cancer therapies based on virus--derived tumouricidal proteins require an appropriate non-toxic vehicle allowing the inward transfer of the gene [START_REF] Peng | Inhibition of hepatocarcinoma by systemic delivery of Apoptin gene via the hepatic asialoglycoprotein receptor[END_REF] or protein [START_REF] Sun | PTD4--apoptin protein therapy inhibits tumor growth in vivo[END_REF] into target cells, HAMLET, IL24 and TRAIL mediate their cancer--cell specific and cytotoxic action from outside the cell, binding to their own receptors. However, in contrast to TRAIL, to date only little is known about the cell death mechanisms executed by HAMLET and IL24 and receptors have so far only been identified for latter. Secondly, whilst TRAIL can execute its tumouricidal function systemically with good tolerability, in vivo studies demonstrating HAMLET's therapeutic potential applied this agent only locally [START_REF] Gustafsson | Treatment of skin papillomas with topical alpha--lactalbumin--oleic acid[END_REF][START_REF] Mossberg | Bladder cancers respond to intravesical instillation of HAMLET (human alpha--lactalbumin made lethal to tumor cells)[END_REF]. Thus, future HAMLET--based cancer therapies might be restricted to a limited spectrum of cancer types. Finally, tumour-selective apoptosis observed for IL24 appears to result from IL24--mediated activation of the FAS ligand (FASL) and TRAIL pathways [START_REF] Ekmekcioglu | Killing of human melanoma cells induced by activation of class I interferon--regulated signaling pathways via MDA--7/IL--24[END_REF]. Whilst FASL or antibodies targeting its receptor displayed severe liver toxicity in mice eventually leading to the death of animals within a few hours [START_REF] Ogasawara | Lethal effect of the anti--Fas antibody in mice[END_REF][START_REF] Ni | Fas--mediated apoptosis in primary cultured mouse hepatocytes[END_REF],

TRAIL was proven safe in diverse animal models [START_REF] Kelley | Preclinical studies to predict the disposition of Apo2L/tumor necrosis factor--related apoptosis--inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety[END_REF]. Because of its practicability, systemic action and safety, our team focused its work on understanding the signaling of TRAIL and not of other endogenous proteins inducing tumour--selective apoptosis.

Indeed, the therapeutic promise of TRAIL is reflected in the large number of ongoing clinical trials [Yerbes et al 2011, http://clinicaltrials.gov] applying either the recombinant human protein (rhTRAIL), humanised antibodies targeting the TRAIL death receptors (DR5: lexatumumab=HGS--ETR2, AMG655, PRO95780; DR4: mapatumumab=HGS--ETR1), or adenoviral delivery of TRAIL coding sequences in monotherapy or in combination with chemotherapeutic drugs. Initial phase I clinical trials using rhTRAIL, HGS--ETR1, HGS--ETR2 or PRO95780 in monotherapy confirmed good tolerance for all agents and no dose--limiting toxicity in patients, partial response in 3% and stable disease in 53% of patients with advanced cancer for rhTRAIL [START_REF] Ashkenazi | Directing cancer cells to self--destruct with pro--apoptotic receptor agonists[END_REF], Herbst et al 2010[START_REF] Tolcher | Phase I pharmacokinetic and biologic correlative study of mapatumumab, a fully human monoclonal antibody with agonist activity to tumor necrosis factor--related apoptosis--inducing ligand receptor--1[END_REF][START_REF] Plummer | Phase 1 and pharmacokinetic study of lexatumumab in patients with advanced cancers[END_REF][START_REF] Camidge | A phase I safety and pharmacokinetic study of the death receptor 5 agonistic antibody PRO95780 in patients with advanced malignancies[END_REF].

Thereby, the most common adverse effects were nausea, fatigue, constipation and leucopenia in 10--20% of patients. Phase Ib clinical studies of rhTRAIL in combination with paclitaxel, carboplatin and bevacizumab revealed tumour progression--free survival of patients with advanced non--squamous non-small--cell lung cancer (NSCLC) [START_REF] Soria | Phase 1b study of dulanermin (recombinant human Apo2L/TRAIL) in combination with paclitaxel, carboplatin, and bevacizumab in patients with advanced non--squamous non--small--cell lung cancer[END_REF]. However, phase II clinical trials using rhTRAIL in combination with bevacizumab in patients with advanced NSCLC [START_REF] Soria | Randomized phase II study of dulanermin in combination with paclitaxel, carboplatin, and bevacizumab in advanced non-small--cell lung cancer[END_REF], HGS--ETR1 in patients with refractory colorectal cancer [START_REF] Trarbach | Phase II trial of mapatumumab, a fully human agonistic monoclonal antibody that targets and activates the tumour necrosis factor apoptosis--inducing ligand receptor--1 (TRAIL--R1), in patients with refractory colorectal cancer[END_REF] or HGS--ETR1 or PRO95780 in combination with paclitaxel or carboplatin in patients with advanced cancer [reviewed by Bellail and Cho 2012] did not reveal improvements of response rates. Thus, human cancers display resistance to TRAIL targeting agents in mono--and combination therapy, a difficulty that was known from early stage. Even before clinical trials were conducted,

INTRODUCTION

Tumour--specific cell--death is the ultimate goal of cancer therapy and eradication of all malignant cells from a human body is the only way for long--term cure. Endogenous mechanisms of programmed cell death exist [START_REF] Lockshin | Programmed cell death: early changes in metamorphosing cells[END_REF], distinguished

mainly by morphological criteria. One discriminates between apoptosis [Kerr et al 1072], autophagy and necrosis, but also other types and somehow intermediate forms have been described [reviewed by [START_REF] Kroemer | Classification of cell death: recommendations of the Nomenclature Committee on Cell Death[END_REF][START_REF] Galluzzi | Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012[END_REF].

Apoptosis

Morphologically, apoptosis features cell membrane blebbing, cell shrinkage, chromatin condensation, nucleosomal fragmentation and the breaking up of cells into fragments called apoptotic bodies. Contrary to the other forms of programmed cell death that do not involve membrane blebbing of dying cells, the dynamic plasma membrane alterations of apoptotic cells are thought to attract innate immune cells that facilitate their rapid removal from tissues before rupture and release of their cytoplasmic contents [START_REF] Savill | Corpse clearance defines the meaning of cell death[END_REF]]. In the body, apoptosis serves to remove irreparably damaged, surplus or infected cells. Apoptosis is accomplished via two different routes, the extrinsic and the intrinsic pathways (Figure 3), which are triggered by pro--apoptotic receptors and intracellular BCL2 proteins, respectively.

The extrinsic apoptosis pathway and type I versus type II signalling cells.

The extrinsic apoptosis pathway is activated by death domain (DD) containing cell surface receptors, the so--called death receptors [for activation by dependence receptors please see review [START_REF] Goldschneider | Dependence receptors: a new paradigm in cell signaling and cancer therapy[END_REF]] and formation of the death inducing signalling complex (DISC) (Figure 3) [START_REF] Kischkel | Cytotoxicity--dependent APO--1 (Fas/CD95)--associated proteins form a death--inducing signaling complex (DISC) with the receptor[END_REF]. The most extensively studied death receptors are TNF receptor 1 (TNFR1), FAS receptor (FAS), death receptor 5 (DR5) and death receptor 4 (DR4), whereby 2 DR5 isoforms, a long (DR5(L)) and a short one (DR5(S)) exist. Common to these receptors are highly homologous cystein--rich domains (CRD) in their extracellular part, defining their ligand specificity. The membrane--distal first CRD is also referred to as pre--ligand assembly domain (PLAD) enabling TNFR1, FAS, DR5 or DR4 interactions amongst each receptor type, independent of their ligands [START_REF] Chan | A domain in TNF receptors that mediates ligand--independent receptor assembly and signaling[END_REF][START_REF] Siegel | Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations[END_REF][START_REF] Clancy | Preligand assembly domain--mediated ligand-independent association between TRAIL receptor 4 (TR4) and TR2 regulates TRAIL--induced apoptosis[END_REF]. Extracellular ligand binding to death receptors oligomerises the receptors, which, at least in case of FAS, stabilises the intracellular receptor--DD in such a conformation that DD--containing adaptor molecules can bind (Figure 3) [START_REF] Scott | The Fas--FADD death domain complex structure unravels signalling by receptor clustering[END_REF]. While different DD--containing adaptor molecules exist, only FAS--associated death domain (FADD) additionally houses a death--effector domain (DED) [reviewed by Park et al 2007, Wu andLo 2009, deathdomain.org]. Via its DED, FADD interacts with tandem DED--comprising proteolytic enzymes, the initiator caspases procaspase--8a, --8b (CASP8) or procaspase--10 (CASP10)

or with the proteolytically inactive protein cellular FLICE--like inhibitory protein (cFLIP) [START_REF] Irmler | Inhibition of death receptor signals by cellular FLIP[END_REF]. The entity comprising at least the receptor, FADD, the initiator caspase(s) and/or cFLIP is called the death inducing signalling complex (DISC) (Figure 3). DISC formation results in the autocatalytic cleavage--stimulated activation of CASP8 and/or CASP10, which can be regulated by cFLIP. Out of 10 different cFLIP mRNA splice isoforms, only a long cFLIP (cFLIP(L)), a short cFLIP (cFLIP(S)), and a short isoform cloned from the Raji B--cell line (cFLIP(R)) are expressed at protein level [START_REF] Golks | c--FLIPR, a new regulator of death receptor-induced apoptosis[END_REF]. Moreover, 2 cleavage products of cFLIP(L) exist, cFLIP(L)p43 resulting from FASL--stimulated CASP cleavage at residue D376 inside the DISC [START_REF] Scaffidi | The role of c--FLIP in modulation of CD95--induced apoptosis[END_REF] and cFLIP(L)p22 resulting from ligand--independent CASP cleavage at amino acid D196 within the cytosol of malignant B and T lymphoblastoma cell lines [START_REF] Golks | The c--FLIP--NH2 terminus (p22--FLIP) induces NF--kappaB activation[END_REF].

Whilst cFLIP(S) blocks CASP activation at the DISC, probably competing with CASP for FADD binding, and cFLIP(R) due to its structural similarity with cFLIP(S) might act similarly, the role of cFLIP(L) at the FAS--DISC is controversial [START_REF] Chang | c--FLIP(L) is a dual function regulator for caspase--8 activation and CD95--mediated apoptosis[END_REF]. Some reports stated an anti--apoptotic cFLIP(L) function at the FAS--DISC due to its ability to block CASP8 activation [START_REF] Irmler | Inhibition of death receptor signals by cellular FLIP[END_REF][START_REF] Rasper | Cell death attenuation by 'Usurpin', a mammalian DED--caspase homologue that precludes caspase--8 recruitment and activation by the CD--95 (Fas, APO--1) receptor complex[END_REF], or to activate pro--survival pathways via adaptor protein recruitment to the TNFR1-- and FAS--DISC [START_REF] Hu | Activation of NF--kappaB by FADD, Casper, and caspase--8[END_REF][START_REF] Kataoka | The caspase--8 inhibitor FLIP promotes activation of NF--kappaB and Erk signaling pathways[END_REF]. Studies on the cFLIP(L) cleavage products demonstrated that upon its generation, cFLIP(L)p43 stays bound to the FAS--DISC, blocking CASP8 processing [START_REF] Scaffidi | The role of c--FLIP in modulation of CD95--induced apoptosis[END_REF]. Or, after FASL treatment, cFLIP(L)p43 forms a complex with CASP8

and the E3 ubiquitin--protein ligase TNF receptor--associated factor 2 (TRAF2) to activate the transcription factor nuclear factor kappa B (NFKB) [START_REF] Kataoka | N--terminal fragment of c--FLIP(L) processed by caspase 8 specifically interacts with TRAF2 and induces activation of the NF--kappaB signaling pathway[END_REF].

Contrary, cFLIP(L)p22 found in nonapoptotic malignant cells, primary phytohaemaglutinin--activated T and B cells, and mature lipopolysaccharide (LPS)-activated dendritic cells induced NFKB activity, which prevented these cells from FASL-and TRAIL--induced apoptosis [Golks et al 2006, reviewed by Lavrik and[START_REF] Lavrik | Regulation of CD95/Fas signaling at the DISC[END_REF]. Other studies described a pro--apoptotic cFLIP(L) activity based on its ability to activate CASP8 cleavage at the FAS--DISC [START_REF] Micheau | The long form of FLIP is an activator of caspase--8 at the Fas death--inducing signaling complex[END_REF][START_REF] Dohrman | Cellular FLIP long form augments caspase activity and death of T cells through heterodimerization with and activation of caspase--8[END_REF].

Apparently, at the DR5/DR4--DISC, cFLIP(L) causes a likewise double--edged effect, as certain Jurkat clones expressing cFLIP(L) display reduced cell viability upon TRAIL treatment [START_REF] Irmler | Inhibition of death receptor signals by cellular FLIP[END_REF]. A recent report expressing different cFLIP variants in human HACAT keratinocytes demonstrated that all forms protected from FASL--and TRAIL--apoptosis, but the CASP8 cleavage pattern was changed in such a way that cFLIP(L) induced CASP8 processing to its active CASP8 p43/41 fragments irrespective of cFLIP cleavage and that cFLIP(S) or cFLIP(L)p43 blocked CASP8 cleavage [START_REF] Kavuri | Cellular FLICE--inhibitory protein (cFLIP) isoforms block CD95--and TRAIL death receptor--induced gene induction irrespective of processing of caspase--8 or cFLIP in the death--inducing signaling complex[END_REF]. Importantly, cFLIP knockout in mice causes the same embryonic lethality than FADD--or CASP8--deficient mice and cFLIP--depleted mouse fibroblasts are highly sensitive to TNFα--and FAS--apoptosis [START_REF] Yeh | Requirement for Casper (c--FLIP) in regulation of death receptor--induced apoptosis and embryonic development[END_REF]. Remarkably, the substrate--range of initiator CASP8 depends on its state of maturity. Using recombinant proteins in in vitro assays, the CASP8 substrate--range was shown to be limited to CASP8 itself and cFLIP [START_REF] Chang | Interdimer processing mechanism of procaspase--8 activation[END_REF][START_REF] Hughes | Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival[END_REF]. In contrast, cleaved CASP8 does not process inactive CASP8 molecules, but efficiently processes effector caspases (like 3, 6, 7) thus triggering the apoptotic signal [START_REF] Lavrik | The active caspase--8 heterotetramer is formed at the CD95 DISC[END_REF].

Downstream of DISC formation and signalling events, the pro--apoptotic signal is propagated via 2 different pathways, dependent on the cell type (Figure 3). In type I cells the activation of effector procaspases--3 (CASP3), --6 (CASP6) and --7 (CASP7) by CASP8 and/or CASP10 suffices to induce apoptosis [START_REF] Scaffidi | Two CD95 (APO--1/Fas) signaling pathways[END_REF], and no cleavage of BH3--interacting domain death agonist (BID) to activate the intrinsic apoptosis pathway is required (Figure 3) [START_REF] Yin | Bid--deficient mice are resistant to Fas--induced hepatocellular apoptosis[END_REF][START_REF] Ozoren | Defining characteristics of Types I and II apoptotic cells in response to TRAIL[END_REF][START_REF] Kandasamy | Involvement of proapoptotic molecules Bax and Bak in tumor necrosis factor--related apoptosis--inducing ligand (TRAIL)--induced mitochondrial disruption and apoptosis: differential regulation of cytochrome c and Smac/DIABLO release[END_REF][START_REF] Rathmell | Bak--/--mouse: a model for apoptosis[END_REF]. In type II cells, apoptosis requires the release of apoptogenic factors from the mitochondria, which is mediated by CASP8 and/or CASP10 cleaving BID [START_REF] Luo | Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors[END_REF], Milhas et al 2005, Fischer et binding (Figure 3) [START_REF] Du | Smac, a mitochondrial protein that promotes cytochrome c--dependent caspase activation by eliminating IAP inhibition[END_REF][START_REF] Verhagen | Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins[END_REF][START_REF] Suzuki | A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death[END_REF]. This inhibtion has biological significance as during FASL--and TRAIL--apoptosis, depletion of IAP family members such as XIAP renders cells independent to BID cleavage and hence directs them from type--II to type--I signalling cells [START_REF] Jost | XIAP discriminates between type I and type II FAS--induced apoptosis[END_REF][START_REF] Varfolomeev | X chromosome--linked inhibitor of apoptosis regulates cell death induction by proapoptotic receptor agonists[END_REF].

The medical relevance of apoptosis in cancer therapy. Control over apoptosis is of immense therapeutic importance for many human health conditions, be it the inhibition of cell death in heart cells in cardiac ischaemia [START_REF] Hayakawa | Sensitivity to apoptosis signal, clearance rate, and ultrastructure of fas ligand--induced apoptosis in in vivo adult cardiac cells[END_REF], of neurons in Alzheimer's [START_REF] Rohn | Activation of caspase--8 in the Alzheimer's disease brain[END_REF] During this arrest, repair mechanisms can act in the cell. However, if the repair fails, p53

stimulates the expression of pro--apoptotic factors (PUMA, NOXA, BAX, APAF1) and inhibits anti--apoptotic protein expression (BCL2, BCLXL) (Figure 3) [reviewed by [START_REF] Brosh | When mutants gain new powers: news from the mutant p53 field[END_REF]. In tumours that escape apoptosis, either p53 itself is inactivated, or p53--regulating proteins are uncontrolled such as the hyperactivation of the E3 ubiquitin protein--ligase MDM2, which promotes p53 degradation [reviewed by [START_REF] Hahn | Rules for making human tumor cells[END_REF]. Alternatively, anti--apoptotic proteins are frequently upregulated in many cancers, such as BCL2 and IAP family members [Beroukhim et al 2010, Smolewski and[START_REF] Smolewski | Inhibitors of apoptosis proteins (IAPs) as potential molecular targets for therapy of hematological malignancies[END_REF]. Many tumour cells rely on the deregulation of these proteins to survive, so that cells transformed with the oncogenic transcription factor MYC highly proliferate, but undergo apoptosis if survival factors like BCL2 are withdrawn [START_REF] Askew | Constitutive c--myc expression in an IL--3--dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis[END_REF][START_REF] Shi | Role for c--myc in activation--induced apoptotic cell death in T cell hybridomas[END_REF][START_REF] Evan | Induction of apoptosis in fibroblasts by c--myc protein[END_REF]. This way, tumour cells are more prone to apoptosis than normal cells, but can counterbalance detriments increasing pro--survival factors. Certain cancer cells master the balancing act between apoptosis and survival so well that they survive although displaying elevated levels of active effector caspases [START_REF] Yang | Coexistence of high levels of apoptotic signaling and inhibitor of apoptosis proteins in human tumor cells: implication for cancer specific therapy[END_REF]. Generally, evasion from apoptosis is thought to promote and enable metastasis [reviewed by [START_REF] Mehlen | Metastasis: a question of life or death[END_REF]. Potential future cancer therapies currently tested in clinical trials target both the extrinsic and intrinsic apoptosis pathways, whereby pro--apoptotic receptor agonists (PARA) and antagonists of the anti-apoptotic BCL2 family are in focus [reviewed by [START_REF] Ashkenazi | Directing cancer cells to self--destruct with pro--apoptotic receptor agonists[END_REF][START_REF] Pavet | Towards novel paradigms for cancer therapy[END_REF].

Contrary to the intrinsic pathway, targeting the extrinsic pathway has the advantage that it triggers tumour cell apoptosis independent of p53 and that mutations inactivating the extrinsic pathway occur less frequent [reviewed by [START_REF] Ashkenazi | Directing cancer cells to self--destruct with pro--apoptotic receptor agonists[END_REF].

Most importantly, DR5-- and/or DR4--activating PARA, unlike other pro--apoptotic agents can discriminate between malignant and normal cells and thus carry a significant lower risk of side effects.

The pro-apoptotic subset of TNF cytokines and its most prominent member(s)

The extrinsic apoptotis pathway is elicited by endogenous proteins [reviewed by [START_REF] Noteborn | Proteins selectively killing tumor cells[END_REF][START_REF] Pavet | Towards novel paradigms for cancer therapy[END_REF] such as cytokines of the tumour necrosis factor (TNF)

superfamily. Cytokines are immunomodulatory proteins that alter the interaction between cells, the communication between cells, or the behavior of cells. The TNF superfamily of cytokines for example can induce inflammation, apoptosis, proliferation, invasion, angiogenesis, metastasis and morphogenesis [reviewed by [START_REF] Aggarwal | Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey[END_REF]. Whilst all TNF cytokines are pro--inflammatory, TRAIL, the ligand of DR5 and DR4, is additionally classified into the pro--morphogenic and pro--apoptotic subsets of TNF cytokines (Figure 4, A) [reviewed by [START_REF] Gonzalvez | New insights into apoptosis signaling by Apo2L/TRAIL[END_REF]Ashkenazi 2010, Aggarwal et al 2012], even though TRAIL's affiliation to the pro--apoptotic subclass is most renown and hitherto characterised best. Of all other pro--apoptotic TNF members, TRAIL shares highest protein sequence homology with FAS ligand (FASL), which binds and activates the FAS death receptor (Figure 4, B). The most well--known TNF superfamily member however is the activator of death receptor TNFR1, tumour necrosis factor alpha (TNFα), which presumably was the first representative of this class of proteins to be identified [START_REF] Aggarwal | Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey[END_REF]. Today, most of what is accepted in TRAIL signalling was originally identified in TNFα--or FASL--signalling and only later proven to account also for TRAIL. However, it shall be stated clearly that TNFα, FASL and TRAIL are different signalling systems and many aspects that have been substantiated for TNFα or FASL lack demonstration for TRAIL. The variety of human TNF cytokine genes (18 in total) arose from duplication events [reviewed by Ware 2003, Wiens and[START_REF] Wiens | Origin and evolution of TNF and TNF receptor superfamilies[END_REF]. When duplicated genes are preserved in evolution, this is because a higher dosage of the gene product is advantageous, because the copies have distinct functions or the copies share the same function [as reviewed by [START_REF] Innan | Population genetic models of duplicated genes[END_REF]]. As TRAIL is unique for its tumour cell-specific induction of apoptosis, it clearly differs in its functionality as compared to TNFα or FASL. Thus, major differences in signalling pathways must exist. and FASL are shed from cell membranes by metalloproteases in vivo [START_REF] Moss | Cloning of a disintegrin metalloproteinase that processes precursor tumour--necrosis factor--alpha[END_REF][START_REF] Tanaka | Expression and localization of membrane type matrix metalloproteinase--1 (MT1--MMP) in trophoblast cells of cultured mouse blastocysts and ectoplacental cones[END_REF][START_REF] Schneider | Conversion of membrane--bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity[END_REF], metalloprotease inhibitors did not increase TRAIL surface levels in any cell--type tested [START_REF] Mariani | Differential regulation of TRAIL and CD95 ligand in transformed cells of the T and B lymphocyte lineage[END_REF]]. Instead, sTRAIL in vitro was generated by cysteine proteases [START_REF] Mariani | Differential regulation of TRAIL and CD95 ligand in transformed cells of the T and B lymphocyte lineage[END_REF]], but the identity of the critical enzyme(s) is still unclear. It has been demonstrated that the membrane--bound and not the soluble form of FASL is critical for apoptosis [START_REF] Schneider | Conversion of membrane--bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity[END_REF][START_REF] O'reilly | Membrane--bound Fas ligand only is essential for Fas-induced apoptosis[END_REF] and likewise, a higher pro--apototic activity of mTRAIL than of sTRAIL was observed [START_REF] Schneider | Conversion of membrane--bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity[END_REF][START_REF] Wajant | Differential activation of TRAIL--R1 and --2 by soluble and membrane TRAIL allows selective surface antigen--directed activation of TRAIL--R2 by a soluble TRAIL derivative[END_REF]. However, whilst Schneider et al do not obtain apoptosis induction at 5µg mL --1 (!) sTRAIL in otherwise highly TRAIL--sensitive BJAB cells and they do not demonstrate that single M2--antibody treatment does not induce apoptosis, one cannot exclude that the sTRAIL preparation utilised in that study was biologically inactive. Moreover, reports by Schneider and

Wajant investigated TRAIL's cytotoxic action only on transformed cells, so that a higher apoptosis--inducing potential, procured by crosslinking, might be detrimental for TRAIL's tumour--cell specificity, like it was concluded for FASL [START_REF] Schneider | Conversion of membrane--bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity[END_REF].

Compared to other TNF members, TRAIL is unique as it contains an unpaired cysteine residue (Cys 230 ) in its extracellular TNF homology domain, which coordinates a zinc (Zn 2+ )--ion that stabilises TRAIL's homotrimeric, bell--shaped structure [START_REF] Hymowitz | A unique zinc--binding site revealed by a high--resolution X--ray structure of homotrimeric Apo2L/TRAIL[END_REF], Bodmer et al 2000]. It was the first example of metal binding--mediated trimerisation of a cytokine [START_REF] Hymowitz | A unique zinc--binding site revealed by a high--resolution X--ray structure of homotrimeric Apo2L/TRAIL[END_REF] and mutation of this residue or interchain disulfide bond formation on Cys 230 lead to TRAIL's dissociation into dimeric and/or monomeric subunits, a decline in TRAIL's binding affinity to its receptor and hence a decrease in its pro--apoptotic activity [START_REF] Bodmer | Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL[END_REF]. At physiologic pH TRAIL specifically exerts its pro--apoptotic action by binding to its receptors, whilst at pH--values smaller than 4.5 TRAIL changes its conformation, which increases its binding ability with membranes, induces pore formation and membrane permeabilisation and thus enforces TRAIL's unspecific cytotoxicity to cells [START_REF] Nam | Association of human tumor necrosis factor--related apoptosis inducing ligand with membrane upon acidification[END_REF]. Moreover, at a pH--value of 6.5 TRAIL--induced cell death in tumour cells adopted necrotic and apoptotic features [START_REF] Meurette | TRAIL (TNF--related apoptosis--inducing ligand) induces necrosis--like cell death in tumor cells at acidic extracellular pH[END_REF]. Thus, the extracellular pH seems critical for the tumour--specificity and mode of cell death induction by TRAIL. The importance of TRAIL's protein structure for its specific action became evident when initial indications for a certain susceptibility of human normal cells towards TRAIL--apoptosis in vitro [START_REF] Jo | Apoptosis induced in normal human hepatocytes by tumor necrosis factor--related apoptosis--inducing ligand[END_REF][START_REF] Leverkus | Regulation of tumor necrosis factor--related apoptosis--inducing ligand sensitivity in primary and transformed human keratinocytes[END_REF][START_REF] Nitsch | Human brain--cell death induced by tumour--necrosis--factor--related apoptosis--inducing ligand (TRAIL)[END_REF][START_REF] Nesterov | Tumor necrosis factor--related apoptosis--inducing ligand (TRAIL) triggers apoptosis in normal prostate epithelial cells[END_REF][START_REF] Lane | Differential induction of apoptosis by tumor necrosis factor--related apoptosis--inducing ligand in human ovarian carcinoma cells[END_REF][START_REF] Steele | Differential susceptibility to TRAIL of normal versus malignant human urothelial cells[END_REF] were associated to amine (polyhistidine, FLAG, leucine zippers) tags attached to the TRAIL sequence [START_REF] Lawrence | Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions[END_REF] TRAIL expression in health and disease. In non--pathologic conditions TRAIL is expressed on human immune effector cells, such as interleukin 12--stimulated natural killer (NK) cells [Zhang et al 2008], peripheral blood T and B lymphocytes [START_REF] Kayagaki | Type I interferons (IFNs) regulate tumor necrosis factor--related apoptosis--inducing ligand (TRAIL) expression on human T cells: A novel mechanism for the antitumor effects of type I IFNs[END_REF][START_REF] Ehrlich | Regulation of soluble and surface-bound TRAIL in human T cells, B cells, and monocytes[END_REF], interferon--activated monocytes [START_REF] Griffith | Monocyte--mediated tumoricidal activity via the tumor necrosis factor--related cytokine, TRAIL[END_REF], LPS-induced monocytes and macrophages [START_REF] Halaas | Lipopolysaccharide induces expression of APO2 ligand/TRAIL in human monocytes and macrophages[END_REF], neutrophils [START_REF] Tecchio | IFNalpha--stimulated neutrophils and monocytes release a soluble form of TNF--related apoptosis--inducing ligand (TRAIL/Apo--2 ligand) displaying apoptotic activity on leukemic cells[END_REF] and dendritic cells. Even if its pioneering and several following reports denoted that TRAIL mRNA in contrast to TNFα and FASL mRNA is expressed in many normal human tissues [START_REF] Wiley | Identification and characterization of a new member of the TNF family that induces apoptosis[END_REF][START_REF] Pitti | Induction of apoptosis by Apo--2 ligand, a new member of the tumor necrosis factor cytokine family[END_REF], one should consider that TRAIL detection in tissues might originate from immune cells that permanently reside in the different tissues analysed [START_REF] Lohmann--Matthes | Pulmonary macrophages[END_REF][START_REF] Berent--Maoz | Human mast cells express intracellular TRAIL[END_REF].

Elevated mTRAIL expression on immune effector cells, increased sTRAIL levels in body fluids and the enhanced TRAIL expression on normal cells was reported in connection with many human diseases leading to tissue--damage and cell death.

Examples are autoimmune disorders (multiple sclerosis [START_REF] Wandinger | TNF--related apoptosis inducing ligand (TRAIL) as a potential response marker for interferon--beta treatment in multiple sclerosis[END_REF],

rheumatoid arthritis [START_REF] Ichikawa | TRAIL--R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis[END_REF][START_REF] Audo | Mechanisms and clinical relevance of TRAIL--triggered responses in the synovial fibroblasts of patients with rheumatoid arthritis[END_REF], systemic lupus erythematosus [START_REF] Lub | Soluble TRAIL concentrations are raised in patients with systemic lupus erythematosus[END_REF], systemic sclerosis [START_REF] Azab | Elevated serum TRAIL levels in scleroderma patients and its possible association with pulmonary involvement[END_REF]), cardiovascular diseases (myocardial infarction [START_REF] Nakajima | Enhanced expression of the apoptosis inducing ligand TRAIL in mononuclear cells after myocardial infarction[END_REF]), viral infections (hepatitis C [START_REF] Mundt | Involvement of TRAIL and its receptors in viral hepatitis[END_REF], HIV--1 [START_REF] Herbeuval | TNF--related apoptosis--inducing ligand (TRAIL) in HIV--1--infected patients and its in vitro production by antigen-presenting cells[END_REF]), neurologic diseases (Alzheimer's disease [START_REF] Cantarella | Neutralization of TRAIL death pathway protects human neuronal cell line from beta--amyloid toxicity[END_REF][START_REF] Uberti | TRAIL is expressed in the brain cells of Alzheimer's disease patients[END_REF]), metabolic diseases (obesity [START_REF] Bernardi | TRAIL as Biomarker and Potential Therapeutic Tool for Cardiovascular Diseases[END_REF], type I diabetes [Lamhamedi--Cherradi et al 2003a]), pulmonary diseases (asthma [START_REF] Collison | Emerging role of tumour necrosis factor-related apoptosis--inducing ligand (TRAIL) as a key regulator of inflammatory responses[END_REF]), bacterial infections (periodontal disease [START_REF] Mori | Osteoblast apoptosis in periodontal disease: role of TNF--related apoptosis--inducing ligand[END_REF]), hematologic diseases (myelodysplastic syndromes [START_REF] Campioni | Evidence for a role of TNF--related apoptosis--inducing ligand (TRAIL) in the anemia of myelodysplastic syndromes[END_REF]) and further degenerating diseases like intervertebral disk degeneration in scoliosis or vertebrae fracture [START_REF] Bertram | Expression of TRAIL and the death receptors DR4 and DR5 correlates with progression of degeneration in human intervertebral disks[END_REF]. Since TRAIL--stimulated apoptosis requires a functional DR5 and/or DR4--mediated extrinsic apoptosis pathway within the target cell, the origins for each of these conditions are apparently potent sensitisers of (normal) cells to TRAIL--apoptosis.

Contrary to above mentioned conditions, cancer does not cause tissue--damage by dying but through hyperproliferating cells. TRAIL expression in certain transformed cells has therefore been related to their resistance to TRAIL--apoptosis [START_REF] Frank | Expression of TRAIL and its receptors in human brain tumors[END_REF][START_REF] Rieger | Human astrocytic brain tumors express AP02L/TRAIL[END_REF][START_REF] Zhao | Functional expression of TRAIL by lymphoid and myeloid tumour cells[END_REF][START_REF] Inoue | Functional expression of tumor necrosis factor--related apoptosis--inducing ligand in human colonic adenocarcinoma cells[END_REF][START_REF] Koyama | Expression of TNF--related apoptosis--inducing ligand (TRAIL) and its receptors in gastric carcinoma and tumor--infiltrating lymphocytes: a possible mechanism of immune evasion of the tumor[END_REF]. Although TRAIL induces apoptosis in transformed cells in vitro [START_REF] Wiley | Identification and characterization of a new member of the TNF family that induces apoptosis[END_REF] and in vivo [START_REF] Wiley | Identification and characterization of a new member of the TNF family that induces apoptosis[END_REF][START_REF] Walczak | Tumoricidal activity of tumor necrosis factor--related apoptosis--inducing ligand in vivo[END_REF], some cancer cell lines and many patient--derived primary cultures in vitro are resistant to TRAIL treatment [START_REF] Nguyen | Relative resistance of fresh isolates of melanoma to tumor necrosis factor--related apoptosis--inducing ligand (TRAIL)-induced apoptosis[END_REF][START_REF] Macfarlane | Mechanisms of resistance to TRAIL--induced apoptosis in primary B cell chronic lymphocytic leukaemia[END_REF][START_REF] Koschny | Bortezomib sensitizes primary human astrocytoma cells of WHO grades I to IV for tumor necrosis factor--related apoptosis--inducing ligand--induced apoptosis[END_REF][START_REF] Todaro | Apoptosis resistance in epithelial tumors is mediated by tumor--cell--derived interleukin--4[END_REF]. Cancer cells thus might have to escape from TRAIL--apoptosis to be able to grow at all in vivo.

TRAIL function in vivo.

Based on its expression on immune effector cells and its feature to induce apoptosis selectively in tumour cells in vitro and in human tumour xenografts implanted in SCID mice [START_REF] Wiley | Identification and characterization of a new member of the TNF family that induces apoptosis[END_REF][START_REF] Walczak | Tumoricidal activity of tumor necrosis factor--related apoptosis--inducing ligand in vivo[END_REF], TRAIL is thought to function in tumour surveillance. In support of this assumption, treatment of wildtype mice with TRAIL--neutralising antibodies promoted tumour initiation if mice were challenged with the chemical carcinogen methylcholanthrene (MCA) [Takeda et al 2002] and increased liver metastases if mice were inoculated with murine cancer cells [START_REF] Takeda | Involvement of tumor necrosis factor-related apoptosis--inducing ligand in surveillance of tumor metastasis by liver natural killer cells[END_REF][START_REF] Seki | Tumor necrosis factor--related apoptosis--inducing ligand--mediated apoptosis is an important endogenous mechanism for resistance to liver metastases in murine renal cancer[END_REF]. Likewise, TRAIL knockout increased tumourigenesis in response to MCA [START_REF] Cretney | Increased susceptibility to tumor initiation and metastasis in TNF--related apoptosis--inducing ligand--deficient mice[END_REF] and augmented metastases if mice were inoculated with murine lymphoma cells [START_REF] Sedger | Characterization of the in vivo function of TNF--alpha--related apoptosis--inducing ligand, TRAIL/Apo2L, using TRAIL/Apo2L gene--deficient mice[END_REF], renal carcinoma or mammary carcinoma cells [START_REF] Cretney | Increased susceptibility to tumor initiation and metastasis in TNF--related apoptosis--inducing ligand--deficient mice[END_REF]. Overexpression of TRAIL in skin keratinocytes prevented from experimentally induced skin carcinogenesis [Kedinger et al 2011]. Contradictorily, mice deficient in TRAIL or their single TRAIL death receptor and DR5 homologue (TRAIL--R) [START_REF] Wu | Molecular cloning and functional analysis of the mouse homologue of the KILLER/DR5 tumor necrosis factor--related apoptosis--inducing ligand (TRAIL) death receptor[END_REF] did not spontaneously develop tumours [START_REF] Yue | Loss of TRAIL--R does not affect thymic or intestinal tumor development in p53 and adenomatous polyposis coli mutant mice[END_REF]. Moreover, although detachment of tumour cells from the extracellular matrix was suggested to increase sensitivity to exogenously added TRAIL [reviewed by

Grosse--Wilde and [START_REF] Grosse--Wilde | Metastasis suppressor function of tumor necrosis factor--related apoptosis--inducing ligand--R in mice: implications for TRAIL--based therapy in humans[END_REF], TRAIL did not inhibit metastasis of TRAIL--sensitive cancer cells to all organs equally well [START_REF] Seki | Tumor necrosis factor--related apoptosis--inducing ligand--mediated apoptosis is an important endogenous mechanism for resistance to liver metastases in murine renal cancer[END_REF]. Finally, TRAIL--deficient mice developed hematologic malignancies only at late stage of life [START_REF] Zerafa | Cutting edge: TRAIL deficiency accelerates hematological malignancies[END_REF], if then to a larger extend than control cells. The role of TRAIL in tumour surveillance therefore has not been fully resolved.

Even more controversial are results from studies investigating TRAILs putative function in the negative selection of premature T lymphocytes in the thymus and the development of autoimmunity, which was suggested based on TRAIL expression on dendritic and epithelial cells. Whilst some studies reported more severe and accelerated experimentally induced autoimmune diseases in TRAIL deficient or TRAIL inhibited mice [START_REF] Hilliard | Roles of TNF--related apoptosis--inducing ligand in experimental autoimmune encephalomyelitis[END_REF], Lamhamedi--Cherradi et al 2003a, Lamhamedi--Cherradi et al 2003b[START_REF] Mi | Blockade of tumor necrosis factor--related apoptosis--inducing ligand exacerbates type 1 diabetes in NOD mice[END_REF], Cretney et al 2005], others could not experimentally elicit accelerated diseases at all [START_REF] Simon | Tumor necrosis factor--related apoptosis--inducing ligand in T cell development: sensitivity of human thymocytes[END_REF], Cretney et al 2003]. Moreover, TRAIL-- or TRAIL--R knockout mice did not spontaneously develop autoimmunity [Cretney et al 2003], even not with prolonged age [Cretney et al 2003, reviewed in Cretney et al 2006] and TRAIL--R knockout mice demonstrated normal negative selection [START_REF] Diehl | TRAIL--R as a negative regulator of innate immune cell responses[END_REF].

Thus, TRAIL may not have a direct role in negative selection and prevention of spontaneous autoimmunity. Instead, it is currently postulated that TRAIL may act on autoreactive T cells by inhibiting cytokine and antibody production and in this way inhibit proliferation of autoreactive T lymphocytes causing inflammation [reviewed by [START_REF] Shepard | The Biology of TRAIL and the Role of TRAIL--Based Therapeutics in Infectious Diseases[END_REF].

Finally, adapted from the finding that many cells of the innate immune system increase their TRAIL expression upon their activation by a variety of pro--inflammatory cytokines (IFNα, IFNβ, IFNγ, IL2, TNFα) [START_REF] Kayagaki | Type I interferons (IFNs) regulate tumor necrosis factor--related apoptosis--inducing ligand (TRAIL) expression on human T cells: A novel mechanism for the antitumor effects of type I IFNs[END_REF][START_REF] Ehrlich | Regulation of soluble and surface-bound TRAIL in human T cells, B cells, and monocytes[END_REF][START_REF] Kemp | Plasmacytoid dendritic cell--derived IFN-alpha induces TNF--related apoptosis--inducing ligand/Apo--2L--mediated antitumor activity by human monocytes following CpG oligodeoxynucleotide stimulation[END_REF][START_REF] Liu | The involvement of TNF--alpha--related apoptosis-inducing ligand in the enhanced cytotoxicity of IFN--beta--stimulated human dendritic cells to tumor cells[END_REF][START_REF] Takeda | Involvement of tumor necrosis factor-related apoptosis--inducing ligand in surveillance of tumor metastasis by liver natural killer cells[END_REF][START_REF] Johnsen | Regulation of APO--2 ligand/trail expression in NK cells--involvement in NK cell--mediated cytotoxicity[END_REF][START_REF] Kashii | Constitutive expression and role of the TNF family ligands in apoptotic killing of tumor cells by human NK cells[END_REF] and that the TRAIL gene promoter is induced by interferons [START_REF] Gong | Genomic organization and transcriptional regulation of human Apo2/TRAIL gene[END_REF][START_REF] Sato | Antiviral response by natural killer cells through TRAIL gene induction by IFN--alpha/beta[END_REF][START_REF] Clarke | Tumor suppressor IRF--1 mediates retinoid and interferon anticancer signaling to death ligand TRAIL[END_REF], TRAIL--apoptosis is suggested to function in the clearance of virus-infected cells [reviewed by [START_REF] Shepard | The Biology of TRAIL and the Role of TRAIL--Based Therapeutics in Infectious Diseases[END_REF][START_REF] Cummins | The TRAIL to viral pathogenesis: the good, the bad and the ugly[END_REF]Badley 2009]. It appears that on one hand, TRAIL--mediated apoptosis of virus--infected cells is protective to the host. On the other hand, some viruses may activate TRAIL--apoptosis and use the TRAIL system to release and disseminate the virus. Furthermore, TRAIL--apoptosis can negatively contribute to virus pathogenesis as certain virus proteins induce TRAIL-expression on uninfected macrophages, which subsequently facilitate TRAIL--apoptosis in uninfected T lymphocytes, a process referred to as 'bystander killing' [START_REF] Miura | Critical contribution of tumor necrosis factor--related apoptosis--inducing ligand (TRAIL) to apoptosis of human CD4+ T cells in HIV--1--infected hu--PBL--NOD--SCID mice[END_REF], Zhang et al 2001].

In conclusion, even though further studies are required to tailor TRAIL's exact function in immune surveillance, its potential to induce apoptosis in transformed, virus-infected or diseased cells in vivo is doubtless. Especially in light of TRAIL's anti--tumour function it must not be forgotten that spontaneous tumour--development usually requires the accumulation of several genetic alterations over time. If instead tumour formation is accelerated challenging a system with mutagenic substances, TRAIL's tumour--surveillance activity became evident. Since not all metastasising cells are more susceptible to TRAIL--apoptosis than their primary tumour cells [START_REF] Vigneswaran | Differential susceptibility of metastatic and primary oral cancer cells to TRAIL--induced apoptosis[END_REF] and most probably not all tissues display equal abundances of resident immune effector cells, clarification of general TRAIL sensitivities of metastatic cells and as well as tissue--distribution of resident immune effector cells may allow us to understand current gaps and inconsistencies in TRAIL's in vivo anti--tumour function.

Potential therapeutic advantages of TRAIL

Several features make TRAIL a promising cancer therapeutic agent. First and foremost, in contrast to conventional therapies that lead to systemic toxicity, TRAIL induces apoptosis in human cancer cells, whilst sparing healthy normal ones. This tumour--selectivity is the key therapeutic trait of TRAIL, which separates TRAIL from other pro--apoptotic TNF cytokines. In support of this, whilst TNFα proved unsuccessful in preclinical studies causing deadly inflammatory response, liver--and kidney toxicity [START_REF] Leist | The 55--kD tumor necrosis factor receptor and CD95 independently signal murine hepatocyte apoptosis and subsequent liver failure[END_REF] and intravenous injection of FASL into severe combined immunodeficiency (SCID) mice caused apoptosis of xenografted human hepatocytes [START_REF] Hao | TRAIL inhibits tumor growth but is nontoxic to human hepatocytes in chimeric mice[END_REF], preclinical and finally clinical studies have shown that rhTRAIL was well tolerated. Particularly, rhTRAIL did not cause undesired accumulation of the recombinant protein or immune response in patients [reviewed by Ashkenazi et al 2008]. Apart from its safety, further therapeutic advantages of TRAIL are its activation of the extrinsic apoptosis pathway independent of the p53 status in tumour cells [START_REF] Galligan | Chemotherapy and TRAIL--mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c--FLIP[END_REF] and the circumstance that common oncogenes like MYC and RAS sensitise cells to TRAIL (see 'TRAIL--apoptosis and the stepwise transformation model') [START_REF] Nesterov | Oncogenic Ras sensitizes normal human cells to tumor necrosis factor--alpha--related apoptosis--inducing ligand-induced apoptosis[END_REF], Wang et al 2005]. In preclinical trials rhTRAIL demonstrated growth inhibitory and cytotoxic activity against a wide variety of tumours originating from solid and hematologic human malignancies xenografted into SCID or athymic mice.

Thereby, rhTRAIL showed anti--tumour activity as single agent or in combination with several chemotherapeutics [reviewed by Ashkenazi et al 2008]

Trail apoptotic signalling and DISC formation

Generally, TRAIL--induced apoptosis follows the extrinsic pathway common to the pro--apoptotic TNF cytokines described above. However, compared to other TNF members, there are considerable differences and uncertainties in the membrane-localisation of DISC proteins, the proteomic composition of the native DISC and the subsequent intracellular fate of the DISC components (Figure 5), which all impact on the signalling outcome and therefore are important to highlight. Contrary to the TNFα receptor TNFR1, FADD binding to DR5 or DR4 does not require the adaptor molecule TNFR1--associated death domain protein (TRADD). Via its DED, FADD co--recruits CASP8 and/or CASP10 to DR5 and DR4 and thus enables their activation, a process that was shown to require both proximity--induced dimerisation and proteolytic cleavage in FASL and TNFα signalling [START_REF] Boatright | A unified model for apical caspase activation[END_REF][START_REF] Donepudi | Insights into the regulatory mechanism for caspase--8 activation[END_REF][START_REF] Hughes | Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival[END_REF], Oberst et al 2010[START_REF] Lavrik | Regulation of CD95/Fas signaling at the DISC[END_REF] and that generally got accepted for TRAIL--signalling as well. Whilst CASP8 is essential for TRAIL-apoptosis, the role of CASP10 in the DISC is controversial [START_REF] Kischkel | Death receptor recruitment of endogenous caspase--10 and apoptosis initiation in the absence of caspase--8[END_REF][START_REF] Sprick | Caspase--10 is recruited to and activated at the native TRAIL and CD95 death--inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase--8[END_REF].

Notably, PLAD--mediated and ligand--independent heterotypic receptor interactions between TRAIL receptors (Figure 5) [START_REF] Clancy | Preligand assembly domain--mediated ligand-independent association between TRAIL receptor 4 (TR4) and TR2 regulates TRAIL--induced apoptosis[END_REF] 5). Thereby, O--glycosyltransferases were found overexpressed in cancer tissues and correlated with TRAIL sensitivity [START_REF] Wagner | Death--receptor O--glycosylation controls tumor--cell sensitivity to the proapoptotic ligand Apo2L/TRAIL[END_REF]. Furthermore, S--nitrosylation (covalent binding of a nitrogen monoxide to a reactive thiol cysteine) of DR4 but not DR5 stimulated CASP8 cleavage via a not further defined mechanism [START_REF] Tang | Nitrosylcobalamin promotes cell death via S nitrosylation of Apo2L/TRAIL receptor DR4[END_REF]. Finally, S--palmitoylation (the reversible attachment of palmitate to a cysteine residue) of DR4 and not DR5 promoted receptor oligomerisation, DISC formation and TRAIL--apoptosis localising receptors to lipid rafts (Figure 5) [START_REF] Rossin | Palmitoylation of the TRAIL receptor DR4 confers an efficient TRAIL--induced cell death signalling[END_REF], which are detergent--resistant membrane domains rich in sphingomyelin, sphingolipids and cholesterol. However, whilst S--nitrosylation was only observed upon nitrosylcobalamin treatment and endogenous S--nitrosylation could not be determined in 3 cancer cell lines [START_REF] Tang | Nitrosylcobalamin promotes cell death via S nitrosylation of Apo2L/TRAIL receptor DR4[END_REF], oligomerisation--promoting effects of S--palmitoylation was only demonstrated in an overexpression system [START_REF] Rossin | Palmitoylation of the TRAIL receptor DR4 confers an efficient TRAIL--induced cell death signalling[END_REF].

The biological significance of S--nitrosylation and S--palmitoylation thus remain unclear.

The requirement of death receptor recruitment into lipid rafts for effective apoptosis signalling originally has been reported for FAS signalling [START_REF] Gajate | Cytoskeleton--mediated death receptor and ligand concentration in lipid rafts forms apoptosis--promoting clusters in cancer chemotherapy[END_REF] and was suggested for DR5 and DR4 by 2 studies working with NSCLC and HeLa cells, respectively (Figure 5) [START_REF] Song | Lipid rafts and nonrafts mediate tumor necrosis factor related apoptosis--inducing ligand induced apoptotic and nonapoptotic signals in non small cell lung carcinoma cells[END_REF][START_REF] Rossin | Palmitoylation of the TRAIL receptor DR4 confers an efficient TRAIL--induced cell death signalling[END_REF]. This notion recently got challenged by findings where no association of the full, native TRAIL DISC with lipid rafts was found in hematopoietic (BJAB, Jurkat, Z138), nor in HeLa cells [START_REF] Dickens | A Death Effector Domain Chain DISC Model Reveals a Crucial Role for Caspase--8 Chain Assembly in Mediating Apoptotic Cell Death[END_REF]. Whilst the HeLa cell study [START_REF] Rossin | Palmitoylation of the TRAIL receptor DR4 confers an efficient TRAIL--induced cell death signalling[END_REF] did not demonstrate a co--localisation of FADD and CASP8 with lipid rafts, the NSCLC study [START_REF] Song | Lipid rafts and nonrafts mediate tumor necrosis factor related apoptosis--inducing ligand induced apoptotic and nonapoptotic signals in non small cell lung carcinoma cells[END_REF] did, so that it was suggested that raft structures might play a greater role in in epithelial (NSCLC) tumour cells [START_REF] Dickens | A Death Effector Domain Chain DISC Model Reveals a Crucial Role for Caspase--8 Chain Assembly in Mediating Apoptotic Cell Death[END_REF]. The biological sinificance of lipid rafts in TRAIL apoptosis signalling thus appears to be limited to only few cell types and requires clarification.

One of the most striking differences between TRAIL--induced and TNFα--or FASL-stimulated apoptosis is the requirement of ligand--induced receptor internalisation for apoptosis to occur (Figure 5). Whilst receptor internalisation is crucial for TNFR1-- and Another level of complexity is added to the regulation DISC formation by the fact that next to DR5 and DR4, also the activity of CASP8 and cFLIP can be modulated by posttranslational modifications. For instance, CASP8 phosphorylation at tyrosine 380 by the focal adhesion kinase and proto--oncogene tyrosine protein kinase (SRC) was reported, which prevented CASP8 cleavage at the DISC. Expression of a non--phosphorylatable CASP8 mutant or SRC inhibition sensitised TRAIL--resistant human colon cancer DLD1 cells to TRAIL--apoptosis [START_REF] Cursi | Src kinase phosphorylates Caspase--8 on Tyr380: a novel mechanism of apoptosis suppression[END_REF][START_REF] De Toni | P60--c--src suppresses apoptosis through inhibition of caspase 8 activation in hepatoma cells, but not in primary hepatocytes[END_REF].

Increased phosphorylation of cFLIP(L) and cFLIP(S) was observed upon bile acid GCDCA treatment of hepatocellular cancer cells, which lowered their affinity for FADD and thus lead to an increased recruitment of CASP8 and CASP10 to the TRAIL--DISC. Bile acid effects could be prevented by PKC inhibition (chelerythrine) [START_REF] Higuchi | Bile acids stimulate cFLIP phosphorylation enhancing TRAIL--mediated apoptosis[END_REF].

Notably, endogenous cFLIP phosphorylation has not been reported yet. Finally, ubiquitinylation of DR5 [START_REF] Johnson | The proteasome inhibitor PS--341 overcomes TRAIL resistance in Bax and caspase 9--negative or Bcl--xL overexpressing cells[END_REF], CASP8 and CASP10 [START_REF] Mcdonald | Suppression of caspase--8--and --10-associated RING proteins results in sensitization to death ligands and inhibition of tumor cell growth[END_REF] and cFLIP [Poukkula et al 2005, Chen et where CASP8 cleavage was promoted. Overexpression of an additionally identified CASP8--bound deubiquitinase called A20 prevented CUL3--mediated effects (Figure 5) [START_REF] Jin | Cullin3--based polyubiquitination and p62--dependent aggregation of caspase--8 mediate extrinsic apoptosis signaling[END_REF].

Previous FAS and consequently DR5-- and DR4--signalling models suggested that the receptor, FADD and CASP8, CASP10 or cFLIP are assembled in a 3:3:3 ratio, meaning TRAIL-DISC and non-apoptotic forms of programmed cell death. Recently, the death receptors of TNFα and FASL were shown to induce necrotic cell death, a process dependent on the kinase activity of receptor interacting serine/threonine protein kinase 1 and 3 (RIPK1, RIPK3) [START_REF] Cho | Phosphorylation--driven assembly of the RIP1--RIP3 complex regulates programmed necrosis and virus--induced inflammation[END_REF][START_REF] He | Receptor interacting protein kinase--3 determines cellular necrotic response to TNF--alpha[END_REF], Zhang et al 2009, summarised by Vandenabeele and Melino 2012]. A similar necrotic activity has been proposed for TRAIL [Holler et al 2000], but the interaction of DR5 and DR4 with RIPK1 has been questioned early on [START_REF] Kischkel | Apo2L/TRAIL--dependent recruitment of endogenous FADD and caspase--8 to death receptors 4 and 5[END_REF]. In fact, only 1 publication so far has demonstrated an interaction between DR5 or DR4 with RIPK1 by co-immunoprecipitation in an endogenous system [START_REF] Harper | Modulation of tumor necrosis factor apoptosis--inducing ligand-- induced NF--kappa B activation by inhibition of apical caspases[END_REF]. This finding recently was challenged by a study coming from the same team, which failed to detect RIPK1 in mass spectrometric analyses of the native DISC immunoprecipited targeting TRAIL from BJAB, Jurkat, Z138 and HeLa cells [START_REF] Dickens | A Death Effector Domain Chain DISC Model Reveals a Crucial Role for Caspase--8 Chain Assembly in Mediating Apoptotic Cell Death[END_REF]. It could however be possible that the detection of RIPK1 requires the immunoprecipitation of proteins downstream the receptors, such as FADD or CASP8. Compared to other TNF family members, TRAIL has the lowest affinity to OPG at physiological temperature [START_REF] Truneh | Temperature--sensitive differential affinity of TRAIL for its receptors. DR5 is the highest affinity receptor[END_REF]. Therefore, even though binding of TNF family members to OPG contributes to bone remodelling [START_REF] Emery | Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL[END_REF], the physiologic role if TRAIL/OPG binding remains unclear.

Anti

Probably the most potent inhibitors of CASP8 activation in the DISC are the DISC-binding cFLIP proteins cFLIP(L), cFLIP(L)p43 and cFLIP(S). Contrary to the FAS--DISC [START_REF] Chang | c--FLIP(L) is a dual function regulator for caspase--8 activation and CD95--mediated apoptosis[END_REF], Micheau et al 2002, Dohrman et activation upon TRAIL--challenge [START_REF] Sharp | Selective knockdown of the long variant of cellular FLICE inhibitory protein augments death receptor--mediated caspase--8 activation and apoptosis[END_REF]. In line with this, expression of cFLIP(L), cFLIP(L)p43 and cFLIP(S) in FLAG--TRAIL sensitive normal human HACAT keratinocytes, blocked CASP8 cleavage [START_REF] Kavuri | Cellular FLICE--inhibitory protein (cFLIP) isoforms block CD95--and TRAIL death receptor--induced gene induction irrespective of processing of caspase--8 or cFLIP in the death--inducing signaling complex[END_REF]. Unfortunately, most studies describing the (anti--)apoptotic cFLIP function of cFLIP proteins at the DISC were conducted focusing on the FAS--DISC, and the outcomes might not be automatically translatable to the TRAIL--DISC.

Further non--canonical DISC--binding proteins have been identified in cancer cells to negatively impact on initiator CASP activation in the TRAIL--DISC (PED/PEA15, the DDX3/GSK3/cIAP1 capping complex, PRMT5) [reviewed by [START_REF] Pennarun | Playing the DISC: turning on TRAIL death receptor--mediated apoptosis in cancer[END_REF][START_REF] Shirley | Regulating TRAIL receptor--induced cell death at the membrane : a deadly discussion[END_REF]. For reasons of space these will not be further described.

TRAIL non-apoptotic signalling and complex II formation

Even though much less efficient than TNFα, TRAIL can activate pro--survival signalling pathways such as nuclear factor B (NFKB), the mitogen--activated protein kinases (MAPK) stress--activated protein kinase (JNK), p38 and extracellular--signal regulated kinase (ERK), as well as RAC--alpha serine/threonine--protein kinase (AKT) (Figure 5). This is thought to depend on the formation of spatially and temporally distinct receptor/adaptor complexes, as described in the following.

TRAIL-induced NFKB, MAPK (JNK, p38, ERK) and AKT activation via complex II formation. Studies utilising overexpression systems [START_REF] Schneider | TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD--dependent apoptosis and activate NF--kappaB[END_REF], Degli--Esposti et al 1997a] revealed that DR5, DR4 and DcR2 activated NFKB, a transcription factor involved in pro--survival signalling activating genes like cFLIP, cIAP1, cIAP2, XIAP and BCLXL. Subsequently, TRAIL--induced NFKB activation was observed in TRAIL--resistant human embryonic kidney 293 (HEK293) cells, pan--CASP inhibited HeLa cells [START_REF] Harper | Modulation of tumor necrosis factor apoptosis--inducing ligand-- induced NF--kappa B activation by inhibition of apical caspases[END_REF], TRAIL--resistant Jurkat [START_REF] Ehrhardt | TRAIL induced survival and proliferation in cancer cells resistant towards TRAIL--induced apoptosis mediated by NF--kappaB[END_REF] and cholangiocarcinoma cell lines [START_REF] Ishimura | Trail induces cell migration and invasion in apoptosis--resistant cholangiocarcinoma cells[END_REF], in which TRAIL--stimulated NFKB activation promoted cell survival, proliferation [START_REF] Ehrhardt | TRAIL induced survival and proliferation in cancer cells resistant towards TRAIL--induced apoptosis mediated by NF--kappaB[END_REF] or migration [START_REF] Ishimura | Trail induces cell migration and invasion in apoptosis--resistant cholangiocarcinoma cells[END_REF]. Generally, NFKB is activated via the I kappa B kinase (IKK) complex consisting of 3 kinases IKKα, IKKβ and IKKγ whose activation leads to phosphorylation and degradation of the inhibitor of kappa B (IKB). This degradation in turn permits NFKB translocation to the nucleus where it stimulates transcription [reviewed by [START_REF] Li | Positive and negative signaling components involved in TNFalpha-induced NF--kappaB activation[END_REF]. Since death receptor signalling appears to be influenced by the formation of spatially and temporally distinct receptor/adaptor complexes, in this context the formation of a secondary complex (complex II) has been proposed

(Figure 5). Complex II is suggested to form subsequent to DISC formation and requires recruitment of the DD--containing RIPK1 for IKK activation [START_REF] Lin | The death domain kinase RIP is essential for TRAIL (Apo2L)--induced activation of IkappaB kinase and c--Jun N--terminal kinase[END_REF]. Via a yet unspecified mechanism FADD and activated CASP8 are described to dissociate from DR5 and DR4 to then bind multiple proteins (the adaptor TRADD, RIPK1, IKKγ and the E3 ubiquitin--protein ligase (TRAF2)) forming complex II [Varfolomeev et al 2005, Jin and[START_REF] Jin | Distinct signaling pathways in TRAIL--versus tumor necrosis factor--induced apoptosis[END_REF]]. Besides activating NFKB, complex II was reported to signal to JNK and p38 in TRAIL--resistant HT1080 fibrosarcoma cells [START_REF] Varfolomeev | Molecular determinants of kinase pathway activation by Apo2 ligand/tumor necrosis factor--related apoptosis-inducing ligand[END_REF], as well as to ERK in normal human vascular smooth muscle cells fostering survival and migration [Secchiero et al 2004a], and in normal human endothelial cells promoting migration, invasion, proliferation and differentiation [Secchiero et al 2004b]. Finally, complex II also activated AKT in normal human endothelial cells (Figure 5) [START_REF] Secchiero | TRAIL promotes the survival and proliferation of primary human vascular endothelial cells by activating the Akt and ERK pathways[END_REF].

Complex II--mediated activation of NFKB depended on the presence of FADD, RIPK1 and IKKγ, whilst activation of JNK demanded TRAF2 [START_REF] Lin | The death domain kinase RIP is essential for TRAIL (Apo2L)--induced activation of IkappaB kinase and c--Jun N--terminal kinase[END_REF] and triggering of p38 required RIPK1 and TRAF2 [START_REF] Varfolomeev | Molecular determinants of kinase pathway activation by Apo2 ligand/tumor necrosis factor--related apoptosis-inducing ligand[END_REF]. The detailed mechanisms for how this DR5 and/or DR4--derived complex II activates either NFKB, MAPK or AKT signalling pathway is not known and intermediate signalling components are not yet defined, but studies of the TNFR1--derived complex II reported that complex II signalling is altered in dependence of the ubiquitinylation and/or phosphorylation state of RIPK1. Intriguingly, a recent study could not detect RIPK1 being associated with the TRAIL DISC in several hematopoetic tumor cell lines [START_REF] Dickens | A Death Effector Domain Chain DISC Model Reveals a Crucial Role for Caspase--8 Chain Assembly in Mediating Apoptotic Cell Death[END_REF], which challenges their own previous findings [START_REF] Harper | Modulation of tumor necrosis factor apoptosis--inducing ligand-- induced NF--kappa B activation by inhibition of apical caspases[END_REF]. Regardless of whether a DR5/DR4--derived complex II exists, cFLIP(L)p22 was capable to interact with IKKγ and activate NFKB in TRAIL-resistant tumour cells in absence of a ligand [START_REF] Golks | The c--FLIP--NH2 terminus (p22--FLIP) induces NF--kappaB activation[END_REF]. The molecular pathway for a complex II--mediated activation of ERK has only been outlined in its basic features for FASL signalling and was proposed to involve FASL--induced and cFLIP(L)p43-dependent recruitment of TRAF2, RIPK1 and the proto--oncogene serine/threonine-protein kinase RAF1 [START_REF] Kataoka | The caspase--8 inhibitor FLIP promotes activation of NF--kappaB and Erk signaling pathways[END_REF]. About the mechanism of DR5/DR4--derived AKT activation even less is known, so far only its dependence on the phosphoinositol--3-kinase (PI3K) has been described [START_REF] Secchiero | TRAIL promotes the survival and proliferation of primary human vascular endothelial cells by activating the Akt and ERK pathways[END_REF]. Although the molecular players for the activation of TRAIL's non--apoptotic signalling cascades have not been elucidated yet, evidence for the regulation of TRAIL's pro--survival signalling via events such as exclusion from lipid rafts [START_REF] Song | Lipid rafts and nonrafts mediate tumor necrosis factor related apoptosis--inducing ligand induced apoptotic and nonapoptotic signals in non small cell lung carcinoma cells[END_REF] already exist. Possibly, the TRAIL--induced activation of pro--survival pathways occurs independent of a complex II, but involves yet unidentified adaptor and effector molecules recruited to the TRAIL--receptors. Evidence supporting this hypothesis comes, for instance, from overexpression studies suggesting a ligand--independent activation of AKT through DcR2 [START_REF] Lalaoui | TRAIL--R4 promotes tumor growth and resistance to apoptosis in cervical carcinoma HeLa cells through AKT[END_REF], or from studies on endogenous systems indicating that AKT--phosphorylated MAP kinase-activating death domain protein (MADD) binds to DR4 [START_REF] Li | Akt--phosphorylated mitogen--activated kinase--activating death domain protein (MADD) inhibits TRAIL--induced apoptosis by blocking Fas--associated death domain (FADD) association with death receptor 4[END_REF]. Remarkably, in case of TNFα--signalling endogenous MADD was required to recruit the adaptor protein growth factor receptor--bound protein 2 (GRB2) and son of sevenless homolog 1 (SOS1)

to TNFR1 and to subsequently activate RAS and the MAPK/ERK kinase kinases 1 and 2 (MEKK1/2) [START_REF] Kurada | MADD, a splice variant of IG20, is indispensable for MAPK activation and protection against apoptosis upon tumor necrosis factor--alpha treatment[END_REF]. Thus, at least JNK and ERK might be activated by TRAIL via additional and/or other routes than a TRAIL receptor--derived complex II.

Enlightenment about how TRAIL and its receptors activate pro--survival signalling pathways is essential, since TRAIL--induced CASP activation occurs rapidly, and complex II generation takes place subsequent to the formation of the DISC. Therefore it was hypothesised that (apart from CASP activation itself) the level of pro--survival signal activity prior to ligand stimulation sets the threshold of cells to undergo TRAIL--apoptosis and not pro--survival pathways activated downstream of DISC formation [as suggested for NFKB by [START_REF] Gonzalvez | New insights into apoptosis signaling by Apo2L/TRAIL[END_REF]]. However, this assumption has to be redefined if TRAIL would activate pro--survival pathways via faster routes than complex II, especially if these pathways negatively feedback on CASP8 activation in the DISC.

The stepwise system cells and transformation-associated changes in the actin cytoskeleton

Cancer development in humans involves a sequence of events. As cancer incidence increases with age, these events appear to occur over many decades. Since patient--derived cancer cell lines conserve the status of a tumour the time the biopsy was taken, these cell lines contain already a multitude of alterations. The decisive and minimally required events that lead to neoplastic transformation thus are hard to trace back. A huge step towards understanding the cancer progress was achieved, when Hahn and Weinberg succeeded to systematically transform human cells in vitro (Figure 7) [START_REF] Hahn | Creation of human tumour cells with defined genetic elements[END_REF]. The generation of their stepwise transformation model aided to limit the pathogenesis of human cancers to a set of genetic and biochemical rules, as summarised in the following [and reviewed by [START_REF] Hahn | Rules for making human tumor cells[END_REF]. The stepwise transformation model. In vitro, neoplastic transformation of normal human fibroblasts (BJ), embryonic kidney cells (HEK) or mammary epithelial cells (HMEC) is achieved by the introduction of 3 genetic elements, the telomerase catalytic subunit (HTERT), the Simian virus early region (SV40ER) and a constitutively active mutant of HRAS (HRASG12V) (Figure 7) [START_REF] Hahn | Creation of human tumour cells with defined genetic elements[END_REF]. Thereby, cell transfection with HTERT serves to immortalise the otherwise growth--limited normal cells via maintenance of the telomeres. Interestingly, a reactivation of telomerases is often observed in cancers. Although SV40ER encodes large T (LT), small T (ST) and 17KT proteins, subsequent experiments utilising only the LT and ST viral oncoproteins and oncogenic RAS demonstrated that the disruption of intracellular pathways regulated by LT, ST and RAS is sufficient for the carcinogenic conversion of human cells [START_REF] Hahn | Creation of human tumour cells with defined genetic elements[END_REF], Yu et al 2001, Hahn et al 2002[START_REF] Rangarajan | Species--and cell type--specific requirements for cellular transformation[END_REF]. Whilst LT localises to the cells nucleus, it inhibits the transcription factor retinoblastoma protein (pRB), which determines whether or not a cell proceeds through G1 phase. Indeed, the pRB is commonly inactivated in cancers. In addition, LT blocks the pRB--related p107 and p130 proteins, which are repressors of E2F transcription factors that, in turn, are promoters of cell cycle entry and progression genes. Most importantly, LT restrains p53, the promoter of cell cycle arrest or apoptosis inducing genes that is inactivated in 50% of all tumours [reviewed by [START_REF] Cheng | Cellular transformation by Simian Virus 40 and Murine Polyoma Virus T antigens[END_REF]. Through its inhibition of pRB and p53, LT prevents HTERT--immortalised cells to undergo senescence.

Contrary to LT, the ST protein is localised in the cytoplasm where it binds and inhibits one or more isoforms of the tumour suppressor protein phosphatase 2A (PP2A), which dephosphorylates a wide variety of substrates. The introduction of an oncogene such as hyperactive RAS serves to generate a constitutive mitogenic signal, which reduces the dependence of transformed cells on external mitogenic stimulation.

Importantly, fully transformed stepwise system cells (BJELR, HA1ER, HMLER) display cancer cell--characteristic features defined as anchorage--independent growth, loss of contact inhibition and tumourigenicity in vivo, which were assessed by colony formation/growth in soft agar, BrdU--incorporation upon confluency and tumour formation capacity in immunocompromised mice, respectively. Tumours obtained from injecting fully transformed cells in mice are anaplastic, strongly angiogenic and non-metastatic. The principal benefit that the stepwise transformation system holds, is that it provides tumourigenic cells for which their normal counterparts of isogenic background are available. This milestone cannot be achieved with patient--derived cancer cells. Secondly, contrary to cancer cells originating from tumour explants, the genetic alterations that induced neoplastic transformation are clearly defined in the in vitro transformed tumour cells. Therefore, a targeted dissection of the cellular pathways that are modulated by LT, ST and HRAS is enabled, which will help to understand the etiology of transformation or transformation--associated features.

TRAIL-apoptosis and the stepwise transformation model. One feature connected to transformation is the gain of sensitivity to TRAIL--apoptosis. Whilst normal and HTERT--immortalised cells of the stepwise tumourigenesis model are resistant to rhTRAIL--apoptosis, HTERT/SV40ER--pretransformed cells are weakly sensitive and HTERT/SV40ER/HRASG12V--transformed cells are highly sensitive (unpublished observations from our laboratory). Hitherto, 2 studies used the stepwise tumourigenesis model to explore the causal mechanism(s) for TRAIL sensitivity of fully transformed cells (BJELR, HA1ER). These reported that constitutively active RAS, via the RAF1/MEK1/ERK pathway, stabilises the MYC protein (Figure 8). In turn, MYC transcriptionally upregulates DR4 and DR5, thus leading to a sensitisation of BJELR and HA1ER cells to TRAIL--apoptosis [START_REF] Nesterov | Oncogenic Ras sensitizes normal human cells to tumor necrosis factor--alpha--related apoptosis--inducing ligand-induced apoptosis[END_REF], Wang et al 2004, Wang et al 2005]. Indeed, like pharmacological MEK1 inhibition [START_REF] Nesterov | Oncogenic Ras sensitizes normal human cells to tumor necrosis factor--alpha--related apoptosis--inducing ligand-induced apoptosis[END_REF], siRNA-mediated MYC depletion diminishes TRAIL--apoptosis in BJELR (unpublished observations from our laboratory), which implies that MYC is one of the main drivers of TRAIL sensitivity in these cells. The importance of RAF1/MEK1/ERK pathway and MYC for TRAIL sensitivity has also been reported in other transformed cells, where the dependence of TRAIL apoptosis on MEK1 activity [START_REF] Drosopoulos | Transformation by oncogenic RAS sensitizes human colon cells to TRAIL--induced apoptosis by up--regulating death receptor 4 and death receptor 5 through a MEK--dependent pathway[END_REF] or MYC's ability to repress cFLIP was shown [START_REF] Ricci | Direct repression of FLIP expression by c--myc is a major determinant of TRAIL sensitivity[END_REF][START_REF] Amanullah | Deregulated c--Myc prematurely recruits both Type I and II CD95/Fas apoptotic pathways associated with terminal myeloid differentiation[END_REF]. Contrary to these studies, APC/ß--catenin mediated activation of MYC in colorectal cancer cells was associated with a downregulation of cFLIP(L), but left cells resistant to TRAIL. Only upon retinoic acid (RA) treatment of MYC--activated cells, an upregulation of DR5 and DR4 and a sensitisation to TRAIL was observed [START_REF] Zhang | Chemoprevention of colorectal cancer by targeting APC--deficient cells for apoptosis[END_REF]. Thus, MYC upregulation in malignant cells appears to have different effects on TRAIL sensitivity.

Regarding MYC effects in normal cells, findings of Wang et al confirmed previous reports, which showed that ectopic MYC expression in normal fibroblasts sensitises to many different apoptotic stimuli [reviewed by [START_REF] Pelengaris | c--MYC: more than just a matter of life and death[END_REF] such as FASL-- [START_REF] Hueber | Requirement for the CD95 receptor--ligand pathway in c--Myc--induced apoptosis[END_REF] or TNFα--induced apoptosis [START_REF] Klefstrom | Induction of TNF--sensitive cellular phenotype by c--Myc involves p53 and impaired NF--kappaB activation[END_REF]. Not only the death receptors, but also CASP8 and CASP9 might be MYC target genes, as indicated by a large--scale analyses of MYC binding to E--box consensus element CACGTG--containing promoters in live human cells [START_REF] Fernandez | Genomic targets of the human c--Myc protein[END_REF]. Indeed, an increase of full--length CASP8 protein levels can be seen upon exogenous MYC expression in BJ cells [START_REF] Wang | Synthetic lethal targeting of MYC by activation of the DR5 death receptor pathway[END_REF]. Since overexpression of MYC occurs in a variety of human cancers, sensitivity to (TRAIL--) apoptosis is a physiological consequence of transformation. Whilst the mechanism(s) eliciting elevated MYC expression levels has (have) not been established in human cancer, in normal human epithelial cells MYC expression is activated upon cell adhesion to fibronectin and requires cytoskeletal rearrangements [START_REF] Benaud | Regulation of the expression of c--Myc by beta1 integrins in epithelial cells[END_REF]. Interestingly, advanced differentiation and epithelial cell organisation represses endogenous MYC expression [START_REF] Simpson | Epithelial cell organization suppresses Myc function by attenuating Myc expression[END_REF].

Transformation-associated changes in the actin cytoskeleton. Morphological alterations linked to transformation are reminiscent to cytoskeletal changes of cells at the onset of mitosis. Cells round up, matrix adhesion is lost, a contractile actin-myosin ring is formed and cortical rigidity increases. Moreover, transformed cells display motile blebs and bulging pseudopodia, loose contact inhibition and stress fibers. All these events involve actin remodelling mediated by signalling pathways that implicate RHO family GTPases. Primarily the RHO--GTPases CDC42, RAC1 and RHOA have been associated with actin cytoskeleton rearrangements, whereby CDC42 induces filopodia [Nobes andHall 1995, Kozma et al 1995], RAC1 regulates lamellipodia formation and membrane ruffling [Ridley et al 1992] and RHOA regulates stress fiber and focal adhesion assembly (Figure 9) [START_REF] Ridley | The small GTP--binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors[END_REF]. Adelstein 2000] and is mainly regulated by RHOA. In its entirety the actin cytoskeleton is a complex, highly dynamic and adaptive structure, which assembles in response to environmental cues. Thereby, the assembly of actin into higher--order structures like networks, fibers or protrusions in normal and transformed cells is regulated by the RHO--GTPases RHOA, CDC42 and RAC1, whereby actin fiber--dependent processes such as adhesion and motility are primarily regulated by RHOA [reviewed by [START_REF] Hall | Rho GTPases and the actin cytoskeleton[END_REF][START_REF] Schoenwaelder | Bidirectional signaling between the cytoskeleton and integrins[END_REF][START_REF] Hernandez--Alcoceba | The Ras family of GTPases in cancer cell invasion[END_REF][START_REF] Evers | Rho family proteins in cell adhesion and cell migration[END_REF][START_REF] Parsons | Cell adhesion: integrating cytoskeletal dynamics and cellular tension[END_REF][START_REF] Rottner | Actin dynamics and turnover in cell motility[END_REF][START_REF] Spiering | Dynamics of the Rho--family small GTPases in actin regulation and motility[END_REF].

LT and ST-induced actin cytoskeleton changes. More than 2 decades ago it was demonstrated that the actin cytoskeleton is rearranged rapidly when normal fibroblasts or epithelial cells are transformed with simian virus 40 (SV40) [START_REF] Weber | Localization and Distribution of Actin Fibers in Normal, Transformed and Revertant Cells[END_REF]. Obvious morphological alterations of SV40--transformed cells included a disorganised actin cytoskeleton and an increased filopodial and lamellipodial production enhancing cellular motility and contributing to the loss of contact inhibition.

In the following, the expression level of several actin cytoskeleton proteins was found deregulated upon SV40--transformation. For instance, SV40--transformed cells exhibited decreased levels of a subset of the tropomyosin family of proteins [START_REF] Hendricks | Tropomyosin is decreased in transformed cells[END_REF][START_REF] Matsumura | Isolation and characterization of tropomyosin--containing microfilaments from cultured cells[END_REF], which stabilise and protect stress fibers from disassembly by binding to F--actin [Bernstein et al 1982, summarised Likewise, ST mutants that could not bind PP2A also failed to transform human cells expressing LT, hTERT and HRAS [START_REF] Mungre | Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small--t antigen in vitro decrease viral transformation[END_REF][START_REF] Porras | A novel simian virus 40 early--region domain mediates transactivation of the cyclin A promoter by small--t antigen and is required for transformation in small--t antigen--dependent assays[END_REF], Yu et al 2001, Hahn et al 2002[START_REF] Chen | Identification of specific PP2A complexes involved in human cell transformation[END_REF], reviewed by Westermarck and Hahn 2008]. Thus, the ST/PP2 interaction is required for rearrangements of the actin cytoskeleton as well as for ST--mediated transformation. Summarising the role of PP2A inhibition in actin disassembly, decrease of cell adhesion and anchorage--independent growth would extend the scope of this paragraph and has been extensively reviewed elsewhere transfection with oncogenic HRAS [START_REF] Kumar | Cytoskeletal changes in human transformed cells: studies on HOS cells[END_REF][START_REF] Janssen | Ras--and Raf--induced down-modulation of non--muscle tropomyosin are MEK--independent[END_REF] or RAS effectors [START_REF] Coller | Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion[END_REF] provoked repression of cytoskeleton protein expression such as tropomyosin [START_REF] Janssen | Ras--and Raf--induced down-modulation of non--muscle tropomyosin are MEK--independent[END_REF][START_REF] Coller | Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion[END_REF] and regulatory myosin light chain 2 (MLC2) [START_REF] Kumar | Cytoskeletal changes in human transformed cells: studies on HOS cells[END_REF][START_REF] Gerrits | Gene duplication and conversion events shaped three homologous, differentially expressed myosin regulatory light chain (MLC2) genes[END_REF], or it leads to inactivation of actin cytoskeleton regulating kinases such as ROCK1 [START_REF] Izawa | Possible involvement of the inactivation of the Rho--Rho--kinase pathway in oncogenic Ras--induced transformation[END_REF]. Moreover, in cancers, RAS mutations lead to overexpression and/or hyperactivation of p21--activated kinase 1 (PAK1), which dissolves stress fibers and reorganises focal adhesions (Figure 12)

[reviewed by [START_REF] Dummler | Pak protein kinases and their role in cancer[END_REF]. Enforced expression of cytoskeleton proteins can revert the oncogenic HRAS--induced tumourigenic phenotype back to normal [reviewed by [START_REF] Pawlak | Post--transcriptional down--regulation of ROCKI/Rho-kinase through an MEK--dependent pathway leads to cytoskeleton disruption in Ras--transformed fibroblasts[END_REF]. Unfortunately, most of what is known about transformation--induced cytoskeleton changes originates from studies looking at either SV40--or oncogenic HRAS--transformation, but not at the alterations that occur if transforming cells with both oncogenes. One could argue that only cytoskeleton alterations induced by HRASG12V are relevant, as it is required to convey all cancer cell-specific features and to fully transform cells such as BJELR or HA1ER. However, one has to consider, that alterations observed with single HRASG12V--transformation might necessitate a normal cellular backbone. These alterations may not fully develop, or could differentially develop if the normal backbone has been disrupted by a previous transformation step with SV40ER. For instance, as ST--transformation is associated with an increase of RAC1 and CDC42 and a decline in RHOA expression levels, HRAS most probably will not impact on stress fiber contraction and focal adhesion formation via the PI3K/RAC1/RHOA pathway in ST--transformed cells (Figure 12). Moreover, as transformation with ST upregulates MYC levels, probably other signalling pathways than MYC--stabilisation by HRASG12V might be of importance. Therefore, studying HRASG12V--induced effects in an SV40ER--pretransformed backbone is essential to manifest differences in the cytoskeleton structure that are critical for features displayed by fully transformed cells.

Actin cytoskeleton and cancer cell apoptosis

For a while it has become clear that the actin cytoskeleton is not only a support giving structure that tents up cells, but that it actively influences incoming signalling pathways such as growth signals and that it controls cell adhesion and migration. In the past 20 years evidence accumulated that the actin cytoskeleton initiates or inhibits apoptosis, which is critical with regard to the disrupted cytoskeletal architecture of transformed cells. In addition to its apparent 'reservoir' function for pro--apoptotic proteins, nuclear translocation of actin upon treatment of mast cells with actin depolymerising latrunculin A was observed, but did not induce apoptosis [START_REF] Pendleton | AAA releases results of first anatomy salary/faculty survey[END_REF]. Similarly, it was shown that caspase--generated actin fragments were targeted to mitochondria after they have undergone N--myristoylation [START_REF] Utsumi | C--terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N--myristoylated upon caspase--mediated cleavage and targeted to mitochondria[END_REF]. Moreover, these fragments induced morphological changes resembling apoptotic cells if ectopically expressed in normal 3T3 fibroblasts [START_REF] Mashima | Caspase--mediated cleavage of cytoskeletal actin plays a positive role in the process of morphological apoptosis[END_REF]. However, this did not cause caspase activation or cleavage of the caspase substrate and apoptosis marker poly ADP--ribose polymerase (PARP) [START_REF] Mashima | Caspase--mediated cleavage of cytoskeletal actin plays a positive role in the process of morphological apoptosis[END_REF]. Thus, nuclear or mitochondrially translocated actin does not seem to have apoptosis--inducing function, but might regulate the progression of apoptosis in a way that has yet to be defined.

The actin cytoskeleton regulates

In Thus, cofilin phosphorylation status regulates a 'flee or die' situation and provides one explanation for how ST--transformed cells might be protected from apoptosis.

Thymosin ß, which prevents actin polymerisation, disrupted stress fibers and induced apoptosis if overexpressed in fibroblasts [START_REF] Hall | Thymosin beta--10 accelerates apoptosis[END_REF], or ovarian cancer cell lines [START_REF] Lee | Overexpression of the thymosin beta--10 gene in human ovarian cancer cells disrupts F--actin stress fiber and leads to apoptosis[END_REF]. Strikingly, overexpression of F--actin stabilising tropomodulin 1 inhibited thymosin ß--apoptosis competing for actin binding [START_REF] Rho | The interaction between E--tropomodulin and thymosin beta--10 rescues tumor cells from thymosin beta--10 mediated apoptosis by restoring actin architecture[END_REF]. Whilst thymosin ß proteins appear to be upregulated in HRASG12V--transformed cells [START_REF] Nummela | Thymosin beta4 is a determinant of the transformed phenotype and invasiveness of S--adenosylmethionine decarboxylase--transfected fibroblasts[END_REF], reviewed by Sribenja et al 2009], no direct information about tropomodulin 1 expression in SV40 or oncogenic HRAS transformation exists. However, recent studies indicate that certain tropomodulins are downregulated in tissues of oncogenic KRAS--transgenic mice [START_REF] Lee | Profiling of transcripts and proteins modulated by K--ras oncogene in the lung tissues of K--ras transgenic mice by omics approaches[END_REF]. These data suggest that HRASG12V-transformed cells might be sensitised to apoptosis via a deregulation of tropomodulin and thymosin ß proteins. If so, neither the mode of protein deregulation, nor the mechanism of pre--sensitisation to apoptosis is known.

Another actin--depolymerising protein that was shown to be downregulated in cells harboring activating KRAS--mutations in colorectal cancer cell lines and primary tumours is gelsolin. Interfering with gelsolin expression using siRNA enhanced CASP9 and CASP7 activation upon butyrate--induced apoptosis [START_REF] Klampfer | Oncogenic Ras promotes butyrate-induced apoptosis through inhibition of gelsolin expression[END_REF]. Moreover, overexpression of full--length gelsolin protected Jurkat cells from apoptosis induced via the mitochondrial pathway (ceramide, dexamethasone, staurosporine, thapsigargin and protoporphyrin IX) [START_REF] Ohtsu | Inhibition of apoptosis by the actin--regulatory protein gelsolin[END_REF], Koya et al 2000] and prevented apoptosis in neuronal cells [START_REF] Harms | Neuronal gelsolin prevents apoptosis by enhancing actin depolymerization[END_REF]. Gelsolins protective role has been associtated to its ability to control the closure of mitochondrial ion channels to prevent cytochrome c efflux [START_REF] Kusano | Human gelsolin prevents apoptosis by inhibiting apoptotic mitochondrial changes via closing VDAC[END_REF]. At the same time, downstream of mitochondrial activation, CASP3--generated gelsolin fragments with high actin severing capacity [START_REF] Kothakota | Caspase--3--generated fragment of gelsolin: effector of morphological change in apoptosis[END_REF] represents an important mediator of anoikis, whereby the role of NMII in this process in undetermined.

Generally, involvement of myosin II has mainly been described for TNF--apoptosis and therefore will be introduced in the next paragraph. In essence, actin and actin-binding proteins take a central role in apoptosis, whereby both actin fiber dynamics as well as the intracellular localization of the actin cytoskeleton proteins are of apparent importance in the regulation of BCL2 protein storage, mitochondrial membrane permeabilisation and the intracellular localization of pro--apoptotic factors.

The actin cytoskeleton regulates the extrinsic apoptosis pathway. In addition to their impact on the intrinsic apoptosis pathway described in the previous section, actin cytoskeleton proteins also regulate apoptosis induced by cytokines of the TNF family. Especially the role of actin and ezrin has been analysed, since an interaction of actin mediated by ezrin [START_REF] Lozupone | Identification and relevance of the CD95--binding domain in the N--terminal region of ezrin[END_REF] and moesin [START_REF] Hebert | Rho--ROCK--dependent ezrin--radixin-moesin phosphorylation regulates Fas--mediated apoptosis in Jurkat cells[END_REF] was demonstrated for FAS. The current FAS signalling model proposes that the interaction of actin and ezrin with FAS is required to localise FAS into lipid rafts and to mediate ligand-stimulated FAS internalisation, which is required for DISC formation and thus FAS-apoptosis. However, regarding the contradictory data in literature, the actual role of actin or ezrin in FAS--apoptosis is far from being understood, as it will be outlined in this paragraph.

Initially observed prior to FASL stimulation [START_REF] Parlato | CD95 (APO--1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway[END_REF]. Again, both studies did not exclude that actin cytoskeleton rupture was not associated with a decline in FAS or DISC protein expression levels. Finally, in type II cells actin filament disruption using cytochalasin increased TRAIL--apoptosis, which was not associated with an enhancement of CASP8 activation [Gasparian et al 2008]. As this study did not demonstrate that CASP8 expression levels upon cytochalasin D were unaltered, it remains unclear whether an intact actin cytoskeleton is required for the expression of FAS and DISC components, and/or whether it is required for the DISC formation process itself.

Ezrin originally was found to colocalise with FAS in FAS--sensitive, activated CD4+

T lymphocytes, as evidenced in cell fractionation assays and immunocytochemistry, latter demonstrating a polarised ezrin/FAS distribution. Ezrin depletion resulted in reduced FAS--stimulated percentages of annexin V positive cells and a scattered FAS distribution [START_REF] Parlato | CD95 (APO--1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway[END_REF], which implied that the ezrin--mediated polarisation of FAS in activated CD4+ T lymphocytes is required to sensitise cells to FAS--apoptosis.

Congruently, ezrin was co--immunoprecipitated with FAS from FAS--sensitive, but not from FAS--resistant T lymphocytes [START_REF] Luciani | CD95/phosphorylated ezrin association underlies HIV--1 GP120/IL--2--induced susceptibility to CD95(APO--1/Fas)--mediated apoptosis of human resting CD4(+)T lymphocytes[END_REF]. However, one should note that this study neither proved comparable FAS expression levels in the investigated cells nor comparable FAS immunoprecipitation levels. Regarding these missing but essential informations it therefore does not seem surprising that contradictory data for the role of the ezrin/FAS interaction in FAS--apoptosis exist. Namely, in type I signalling H9 T lymphoblastoma cells ezrin knockdown increased the percentages of FASL--or TRAIL-induced annexin V--positive cells. Moreover, ezrin knockdown in activated peripheral blood T lymphocytes or expression of dominant negative ezrin mutants in type I H9 lymphoblastoma cells increased percentages of FASL--induced annexin V--positivity.

Finally, ezrin depletion enforced the FASL--stimulated activation of the CASP cascade in whole cell lysates [START_REF] Kuo | Ezrin is a negative regulator of death receptor--induced apoptosis[END_REF]. However, the proapoptotic effect of ezrin loss regarding DISC formation remains unclear, as apparently more FAS was immunopurified from ezrin knockdown H9 lymphoblastoma cells, which puts the reported increased DISC formation into question [START_REF] Kuo | Ezrin is a negative regulator of death receptor--induced apoptosis[END_REF]. Contrary to a report that indicated actin as an important constitutent of the endocytotic machinery that internalises FAS after induction in type I signalling SKW6.4 and H9 lymphoblastoma cells [START_REF] Algeciras--Schimnich | Actin dependent CD95 internalization is specific for Type I cells[END_REF], this study denoted that FASL--induced internalization of FAS was unaffected by ezrin knockdown in exactly the same cell type (H9 lymphocytes) [START_REF] Kuo | Ezrin is a negative regulator of death receptor--induced apoptosis[END_REF]. Thus, either actin does not play a role in FAS internalisation, or ezrin alone does not mediate binding of actin to FAS. Apparently, latter situation is the case, as also moesin (but not radixin) was reported to bind FAS

AIMS OF THE THESIS

The overall aim of the thesis was to contribute to our understanding of the molecular features that account for the tumour--selective apoptosis induction by TRAIL. apoptosis the 7A6 antigen flips on the outside of the mitochondrial membrane and marks one of the earliest events in the apoptosis process (Figure 19) [START_REF] Zhang | A mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells[END_REF][START_REF] Koester | Monitoring early cellular responses in apoptosis is aided by the mitochondrial membrane protein--specific monoclonal antibody APO2.7[END_REF], Büssing et al 1999]. Unless otherwise indicated, all following volumes and cell numbers refer to 1 cm 2 growth surface of cell culture vessel. Cells treated in apoptosis assays were plated 1d prior to rhTRAIL treatment at a density of 10x10 4 cells in case of BJAB cells, 10x10 3 cells if BJ cells were assayed or 50x10 3 cells if BJELR cells were investigated. Cells were plated in 250µL medium. In knockdown experiments, which required prolonged cell culture prior to apoptosis assay, the cell culture medium was renewed 1d ahead of rhTRAIL addition. Treatment with rhTRAIL itself was performed in 175 µL conditioned medium. Pre--dilutions of rhTRAIL were generated at appropriate concentrations so that 2.5µL of the cytokine dilution could be added per well. After 16h incubation cell supernatants were collected in FACS tubes and cells were rinsed once with 250µL phosphate buffered saline (PBS) that also was collected in the FACS tube. Adherent cells were trypsinised with 125µL 0.5% trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco). To ensure the generation of single cell suspensions, cells were monitored under the microscope and if necessary cells were carefully triturated with pipettes. Detached cells were transferred to the FACS tubes and the cell culture well was washed with 500µL PBS. Thereafter viable and dead cells were pelleted at 240xg and 4°C for 5 min. The supernatants were removed and cells were permeabilised adding 100µL of an ice--cold solution of digitonin, dissolved at a final concentration of 0.1µg mL--1 in 0.25% fetal calf serum (FCS) and PBS. To ensure proper permeabilisation, cells were again carefully pipetted up and down to obtain single cells.

After incubating the cells for 10min at 4°C, permeabilisation was checked with trypan blue stain under the microscope and was then stopped with 2mL ice--cold 2.5% FCS in PBS. Cells were centrifuged at 240xg and 4°C for 5 min, supernatants were removed and cells were stained with 5µL Apo2.7 antibody diluted in 25µL 2.5% FCS and PBS in the dark at room temperature (RT) for 25min. After staining, cells were resuspended in 250--500µL 2.5% FCS and PBS and immediately analysed by FACS. Importantly, BJELR cells should never exceed 80% confluency, as this would impact on TRAIL sensitivity. We recommend to rather split BJELR cells on 2 consecutive days than letting them overgrow. For selection, medium of BJELR cells was freshly supplemented with 100µg mL --1 hygromicine, 400µg mL --1 G418 and 0.5µg mL --1 puromicin. Same cell culture rules than for BJELR cells applied for transformed HA1ER cells, except the basic cell culture medium differed [DMEM 1g L --1 glucose, 10% FCS #97]. All cells were grown at 37°C, 5% carbon dioxide (CO2) and constant humidity.

Bacterial transformation. For bacterial transformations competent

Chemical NMII inhibition. For chemical inhibition of NMII function blebbistatin

(Sigma) dissolved in dimethyl sulfoxide (DMSO) or ML7 (Sigma) resuspended in a 1:1 mixture of ethanol and water were used. Prior to challenging of cells with rhTRAIL, cells were incubated for 30min with the respective agent or solvent and then co--incubated with rhTRAIL.

DISC immunoprecipitation. The basic protocol for immunoprecipitating the DISC was described by [START_REF] Walczak | Biochemical analysis of the native TRAIL Death--Inducing Signaling Complex[END_REF] and originated from the book Apoptosis and Cancer, but shall below be described in brief. Per immunoprecipitation 30x10 6 BJAB cells [targeted total protein amount per IP: 0.5--1.5mg] were stimulated for 1h in fresh medium supplemented with 1μg mL --1 rhTRAIL. Lysing equal numbers of BJAB and stepwise system cells yielded 3--fold higher protein concentrations for Thereafter 2mL of pre--cleared supernatants (corresponding to 2mg of total protein) were mixed with 50µL of beads coupled to one of the IP--AB. A fraction of the pre--cleared supernatant was kept as 'input' controls. After over night (ON) incubation shaking head-to--head at 4°C, IP beads were pelleted at 4°C and 106x for 2 min and washed in 1mL ice-cold lysis buffer containing 1% Triton--X100, protease--and phosphatase inhibitors.

Importantly, flicking the tube was avoided, instead the tubes were reversed 3--5 times, until all beads were removed from the tip of the tube. All tubes in one round of washing were removed an equal number of times. In total, the washing and centrifugation steps were repeated 5 times. To elute the DISC proteins from the beads 50µL of 2--fold concentrated Laemmli buffer [125mM Tris--HCl pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 10% glycerol] was added, tubes were carefully flicked to bathe all beads and samples were incubated at 75°C for 10 min. In the following the samples were centrifuged at 4460xg for 2 min and the supernatants were recovered as 'IP'. Collected Plasmids. For the bacterial expression of rhTRAIL a pET9A vector was used encoding amino acid V114--G281 of human TRAIL [START_REF] Pitti | Induction of apoptosis by Apo--2 ligand, a new member of the tumor necrosis factor cytokine family[END_REF].

Plasmid

RESULTS

We hypothesised that the differential sensitivity of normal and transformed cells towards TRAIL apoptosis arises at the DISC. Aiming to perform comparative DISC analyses in normal and transformed cells, several basic technologies needed to be inaugurated in the laboratory. Therefore the following first results section will provide protocols for the bacterial expression and purification of untagged recombinant human TRAIL. The purified ligand subsequently was characterised concerning its capacity to induce apoptosis, to bind to its specific and un--specific TNF family receptors and to activate the apoptotic cascade. Moreover, the protocol and pitfalls of the native DISC analysis as well as the specificity of the utilised IP--antibodies will be described.

In the second part of the results section the cells of the stepwise tumourigenesis model will be characterised regarding their cell culture characteristics, TRAIL sensitivity, DISC protein expression levels, DR5 surface expression, activation of the apoptotic cascade and DISC formation.

Since comparative DISC analyses did not reveal a complete explanation for the differential response of normal and transformed cells to TRAIL--apoptosis, we suspected the presence of unknown DISC--interacting proteins that regulate observed differential sensitivity. We therefore applied mass spectrometry--based analyses of DR5-immunoprecipitates and identified NMHCIIa, NMHCIIb, MLC2 and ßACT as novel DR5

and DR4 interactors that differentially co--purified with the DR of normal and transformed cells, as described in the third segment of the results section.

In contrast to actin contributing to FAS and TNFR1--mediated apoptosis, the co-purification of NMII with DR was entirely novel and by the time we identified NMII there was only one paper available linking NMII activity to TNFR1 cell surface transport.

Therefore we focused our analyses on understanding the functional role of NMHCIIa and NMHCIIb in TRAIL--apoptosis, which will be presented in the fourth part of the results section. In brief, the fourth chapter will point out that chemical inhibition of NMII is

Generation of tools to analyse the native TRAIL DISC

DISC immunoprecipitation experiments require considerable amounts (15--45µg) of sTRAIL, therefore the primary goal was to establish the cytokine production and purification in the laboratory. Undesirable cytotoxicity of certain TRAIL variants towards normal hepatocytes has been associated to the hyper--oligomerisation capacity of protein (FLAG)--or histidine (His)--tags [START_REF] Lawrence | Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions[END_REF][START_REF] Ganten | Preclinical differentiation between apparently safe and potentially hepatotoxic applications of TRAIL either alone or in combination with chemotherapeutic drugs[END_REF].

Therefore we decided to utilise a native, untagged, biologically active [START_REF] Hymowitz | A unique zinc--binding site revealed by a high--resolution X--ray structure of homotrimeric Apo2L/TRAIL[END_REF] rhTRAIL variant comprising amino acid 114 to 281, which has been proven as non--toxic to isolated [START_REF] Lawrence | Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions[END_REF] or mouse--xenografted normal human hepatocytes [START_REF] Hao | TRAIL inhibits tumor growth but is nontoxic to human hepatocytes in chimeric mice[END_REF].

Establishment of protocols for the bacterial expression and purification of rhTRAIL. The rhTRAIL purification from bacterial cultures --conducted in accordance to described protocols [START_REF] Kim | Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells[END_REF]-- is accomplished via resin--immobilised Ni 2+ metal ions that are able to bind electron--rich molecules, such as histidine. Importantly, also non--His--tagged rhTRAIL can be purified via Ni 2+ --binding methods as the herein expressed native rhTRAIL sequence contains 6 histidines (Figure 20, A) that are sufficient for Ni 2+ binding [START_REF] Ashkenazi | Safety and antitumor activity of recombinant soluble Apo2 ligand[END_REF][START_REF] Kim | Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells[END_REF].

Although standard protocols [START_REF] Kim | Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells[END_REF] immediately allowed the purification of native rhTRAIL purified from Escherichia coli strain BL21DE3 (Figure 20, B--C), the ligand lacked pro--apoptotic activity when applied on transformed cells (not shown). Since this inactivation could be due to protein overexpression leading to protein aggregation and formation of inclusion bodies [Qiaexpressionist 2003], three major changes were incorporated in the purification protocol. Firstly, the growth temperature was decreased from 30°C to 18°C to reduce expression levels leading to a higher amount of soluble protein [Qiaexpressionist 2003]. Secondly, as the biologically active trimeric TRAIL structure [START_REF] Bodmer | Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL[END_REF][START_REF] Trabzuni | Functional analysis of tumour necrosis factor--alpha--related apoptosis--inducing ligand (TRAIL): cysteine--230 plays a critical role in the homotrimerization and biological activity of this novel tumoricidal cytokine[END_REF][START_REF] Seol | A caspase--9 variant missing the catalytic site is an endogenous inhibitor of apoptosis[END_REF] is maintained by an unpaired cysteine residue (Cys 230 ) that coordinates one Zn 2+ -ion per TRAIL trimer [START_REF] Hymowitz | A unique zinc--binding site revealed by a high--resolution X--ray structure of homotrimeric Apo2L/TRAIL[END_REF], the culture medium was supplemented with 100µM ZnSO4. Thirdly, to enhance oligomerisation of native, untagged rhTRAIL and to decrease the yields of presumably biologically inactive monomers [START_REF] Kim | Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells[END_REF],

the reducing agent contained in the purification buffer and the dialysis buffer was exchanged from 2--mercaptoethanol to 5mM DTT. Due to the new reducing agent rhTRAIL prufication was performed as batch purification using Ni 2+ --NTA beads, because

Ni 2+ ions of commercially available HiTrap columns chelated at 5mM DTT. Finally, to increase yields of rhTRAIL through efficient breaking up of the bacterial cell wall, the sonication time was extended from 1 to 2min. In this way, the biological, pro--apoptotic activity of rhTRAIL could be conserved. The putative biologically active homotrimeric subunit structure of rhTRAIL can be seen in (Figure 20,D).

Characterisation of purified rhTRAIL regarding its potential for apoptosis induction, receptor binding and apoptotic cascade activation.

Although TRAIL is expressed as a monomer, high--resolution X--ray structures have shown that it exists as a homotrimer [START_REF] Hymowitz | A unique zinc--binding site revealed by a high--resolution X--ray structure of homotrimeric Apo2L/TRAIL[END_REF]. Therefore one assumes that TRAIL spontaneously assembles into a homotrimeric structure in solution. Crystal structure studies have shown that the binding of TRAIL to DR5 takes place via the interface of TRAIL monomers, so that one homotrimeric sTRAIL molecule binds and clusters up to 3 extracellular DR5 domains [START_REF] Hymowitz | Triggering cell death: the crystal structure of Apo2L/TRAIL in a complex with death receptor 5[END_REF]. Therefore an intact homotrimeric TRAIL structure is essential for TRAIL binding to its receptors and for its biological activity.

To examine if purified rhTRAIL properly binds to its receptors without cross-- After the binding of rhTRAIL to its specific receptors was confirmed in vitro, we tested whether rhTRAIL can execute its apoptotic function and if so, whether rhTRAIL induces apoptosis specifically via its DRs and not via unwanted cytotoxic side effects.

activating
Therefore we used the TRAIL--sensitive malignant B--cells, the Burkitt's lymphoma cells (BJAB), which express DR5, DR4, DcR1 and DcR2 on their cell surface and whose major apoptosis mediator is DR5 [START_REF] Thomas | Direct binding of Fas--associated death domain (FADD) to the tumor necrosis factor--related apoptosis--inducing ligand receptor DR5 is regulated by the death effector domain of FADD[END_REF]. BJAB cells are monoallelic for their major TRAIL--apoptosis signalling receptor DR5 (BJAB DR5--/+ ), allowing the depletion of the DR5 gene by chemical mutagenesis (BJAB DR5--/--) [START_REF] Thomas | Direct binding of Fas--associated death domain (FADD) to the tumor necrosis factor--related apoptosis--inducing ligand receptor DR5 is regulated by the death effector domain of FADD[END_REF]. To date, BJAB DR5--/--cells are currently the only patient--derived cancer cell system for which a constitutive DR5 knockout (BJAB DR5--/--) is available. BJAB DR5--/+ and BJAB DR5--/--cells were treated with different concentrations of rhTRAIL for 16h and the percentage of apoptotic cells were quantified. For that, we detected cell populations positive for 7A6, a protein that translocates from the inner to the outer mitochondrial membrane if the cell undergoes apoptosis. Importantly, rhTRAIL induced dose--dependent apoptosis in BJAB DR5--/+ cells, whilst no substantial (< 10%) cell death was evidenced in BJAB DR5--/-- (Figure 21,B). Therefore, the purified ligand specifically induces apoptosis via its receptors and does not display unspecific cytotoxicity.

In the chronological sequence of apoptosis 7A6 positivity is posterior to the formation of the DISC with cleavage--mediated CASP8 activation as its key signature. The Once the biological activity and the specificity of the purified rhTRAIL to its receptors were confirmed, the next goal was to assess the stability of the recombinant protein upon storage. Repeated freeze--thaw cycles usually degrade a protein [Deutscher 2009]. Therefore we applied different concentrations of single rhTRAIL eluates immediately after purification and dialysis (no freeze), as well as after the first (1 st freeze) and second (2 nd freeze) freezing at --20°C for 1 day each on BJAB DR5--/+ cells and measured apoptosis 16 h after start of treatment. Even at very low rhTRAIL concentrations like 0.1 or 0.5ng mL --1 no significant decline in the pro--apoptotic activity could be observed for rhTRAIL eluates that were used either before or after freezing.

Therefore purified rhTRAIL is stable in the selected buffer composition for at least 2 consecutive freeze--thaw cycles. ) and in BJAB cells whose DR5 receptor was knocked out (BJAB DR5--/--). Apoptosis was quantified as 7A6 positivity in FACS analysis after 16h of treatment. Commer--cially available TRAIL (cTRAIL) was included as positive control. Values derive from 3--4 independent experiments. (C) Representative Western blot for caspase--8 (CASP8) cleavage kinetics in BJAB DR5--/+ and BJAB DR5--/--cells upon administration of 1µg mL --1 rhTRAIL for the indicated times.

The antibody reveals the full--length CASP8 and its active p43/p41 and p18 fragments. The blot for transcription intermediary factor 1--alpha (TIF1a) served as loading control. Depicted is 1 of 2 independent experiments. (D) Western blots for the apoptotic cascade activation in BJAB DR5--/+ and BJAB DR5--/--cells after 1h incubation with 1µg mL --1 purified rhTRAIL. The antibodies against the initiator caspases CASP8 and caspase--9 (CASP9) detect both the full--length and the cleaved fragments. The antibodies against effector caspase--3 (CASP3) and --7 (CASP7) specifically react with the active p19/p17 CASP3, respectively with the active p20 CASP7 cleavage products. The BID antibody exclusively reacts with the full--length protein. Whole cell lysates originating from cells treated for 1h with 25µM etoposide were included as positive control. Indicated are the blots for 1 of 2 independent experiments. (E) Dosage-response curves for different rhTRAIL eluates that were applied at indicated concentrations on BJAB DR5--/+ cells either immediately after purification (no freeze) or after the first and second freezing. Apoptosis was assessed as in (B). Values of the upper 3 diagrams derive from one single experiment and are fused in the diagram at the bottom. The abbrevation 'N.d.' is used for 'not determined'.

detected (Figure 22,B). Thus the DR5--IP AB does not significantly cross--react with DR4

and the DR4--IP AB does not substantially cross--react with DR5.

A second DISC--IP challenge originates from the mode of IP--ABs immobilisation to the beads that allow DISC isolation by sedimentation. Usually, beads are covalently coupled to immunoglobulin--binding proteins to which the IP--AB binds. Unfortunately these immunoglobulin--binding proteins can hamper Western blot analyses of IPs as they cross--react with detection--ABs, leading to an unspecific signal at the molecular weight (MW) of the immunoglobulin--binding protein (here: protein G ≈ 30KDa, e.g. signal above FADD and in between CASP8 p43/p41 and p18 in DR5--IP, Figure 25,F). Depending on the host organism in which the secondary AB was generated and the type of immunoglobulin--binding protein employed in the experiment, this cross--reactivity is weaker or stronger [Frank 1997]. Therefore we tested different sets of detection antibodies with different types of immunoglobulin--binding proteins. While optimising the DISC--IP and Western blot analysis protocol, we found that the use of protein G-coupled sepharose beads in combination with chicken--derived secondary antibodies give lowest unspecific signals (not shown).

Thirdly, Western blot detection of the single members comprising the DISC can be hampered by a cross--reaction of detection--AB with IP--AB, which still can occur although crossing species [START_REF] Lal | Clean Western blot signals from immunoprecipitated samples[END_REF]. Due to this cross--reaction that gives rise to unspecific signals at the MWs of the IP--AB's heavy and light chain (50 and 25KDa), certain members that linger in the DISC cannot be doubtlessly identified. This is the case for cFLIP(L) and cFLIP(S) (MW=55KDa, 25 KDa respectively) when co-immunoprecipitated with DR5, DR4 or CASP8 (Supplementary, Figure 31). The degree of cross reaction and AB--leakage from the beads depends on the applied IP--AB. Due to the unspecific cross--reactions between detection ABs and immunoglobulin--binding proteins or IP--ABs, we think it is indispensable to load eluates of IP--AB--coupled beads next to the respective DISC immunoprecipitates. In this way a clear identification of specific signals is ensured.

Finally, the abundance of the single DISC members within the DISC varies drastically depending on the cellular response upon TRAIL and certain AB affinities are weak so that the detection becomes difficult as it is the case with FADD. In the course of DISC--IP protocol optimisation we found that loading of 15µL IP--eluate suffices for detection of all DISC members studied here.

Interim summary and conclusions I

The expression and purification of untagged rhTRAIL was successfully established and the alleged biologically active homotrimeric structure was detected on non--reducing SDS--PAGE gels. As shown by SPR experiments purified rhTRAIL binds with high affinity specifically to its receptors, but not non--specific receptors from the TNF family. FACS--assisted 7A6 immunogenicity and Western blot analyses demonstrated the biological (here: pro--apoptotic) activity of purified rhTRAIL in patient--derived BJAB lymphoma cells and that rhTRAIL activates the extrinsic and intrinsic apoptotic pathway in a DR5--dependent manner. Moreover, apoptosis assays with 7A6 confirmed that purified rhTRAIL maintains its pro--apoptotic activity for at least 2 freeze--thaw cycles without myjor activity loss.

DISC analyses revealed that purified rhTRAIL induces DISC formation in accordance to prevalent models in BJAB cells. Furthermore, no PLAD--mediated heterotypic receptor interactions were detected under our experimental conditions, indicating that either ligand--independent DR5/DR4 clustering does not occur in this cell model or that this interaction is lost under the given IP conditions. Importantly, the DR5--IP AB does not cross--react with DR4 and the DR4--IP AB does not cross--react with DR5.

In summary the basic tools required for the isolation and analysis of the native TRAIL DISC have been established and validated. The action of rhTRAIL on cells of the stepwise transformation model and TRAIL DISC formation in that cell system are investigated in the following chapter. [START_REF] Mills | Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation[END_REF], cytokinesis [Fishkind et al 1991, Burton and[START_REF] Burton | Traction forces of cytokinesis measured with optically modified elastic substrata[END_REF]] and cell movement [Trinkaus 1973, Friedl and[START_REF] Friedl | Tumour--cell invasion and migration: diversity and escape mechanisms[END_REF]]. In addition, membrane blebbing was observed on diverse viable cancer cells [START_REF] Cunningham | Actin polymerization and intracellular solvent flow in cell surface blebbing[END_REF][START_REF] Sahai | Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis[END_REF][START_REF] Charras | Non--equilibration of hydrostatic pressure in blebbing cells[END_REF][START_REF] Charras | Reassembly of contractile actin cortex in cell blebs[END_REF]. Although BJERL appear much smaller than BJ cells (Figure 23), lysis of the same cell numbers yielded equal amounts of total protein (not shown). We therefore employed equal cell numbers of BJ and BJELR cells in DISC--IP experiments. In addition, we assumed that BJELR cells appear much smaller than BJ cells either because of their slow and loose attachment to the growth surface or because of their high proliferation rate that might not leave enough time for a full de--novo synthesis of cytoplasm. In fact, whilst BJ cells duplicate once every 48h, BJELR divide on average every 24h.

The stepwise tumourigenesis cells as model system to study the gain of TRAIL sensitivity through transformation

Transformed but not normal cells die upon rhTRAIL, even though DISC

proteins are expressed at comparable levels and a DISC is formed in normal cells.

After the apoptotic activity of purified rhTRAIL has been confirmed on BJAB lymphoma cells (Figure 21, B--D), we tested the ligand on normal BJ and transformed BJELR cells of the stepwise transformation model. Thereby we applied high dosages (1µg mL --1 ) of purified rhTRAIL to ensure receptor saturation with ligand (Figure 24,A). Following 16h of treatment with 1µg mL --1 rhTRAIL, significantly more BJELR than BJ cells died (44% (± 10%) versus 11% (±4), p=0.002) (Figure 24,B). The analyses leads us to conclude that purified rhTRAIL acts in a tumor--specific manner inducing apoptosis in transformed but not normal cells, likewise it has been reported for other recombinant TRAIL preparations [START_REF] Ashkenazi | Safety and antitumor activity of recombinant soluble Apo2 ligand[END_REF][START_REF] Walczak | Tumoricidal activity of tumor necrosis factor--related apoptosis--inducing ligand in vivo[END_REF].

The observation that purified rhTRAIL specifically induces apoptosis in cancer cells adds meaning for addressing the central question of this work: how does a cell which is initially TRAIL--resistant become sensitive to TRAIL--apoptosis via the process of transformation?

Earlier publications comparing HTERT and SV40ER--pre--transformed (BJEL, HA1E) cells with fully transformed (BJELR, HA1ER) cells [START_REF] Nesterov | Oncogenic Ras sensitizes normal human cells to tumor necrosis factor--alpha--related apoptosis--inducing ligand-induced apoptosis[END_REF][START_REF] Lund | Transformation--dependent silencing of tumor--selective apoptosis--inducing TRAIL by DNA hypermethylation is antagonized by decitabine[END_REF] or overexpressing oncogenes in normal BJ [START_REF] Wang | Synthetic lethal targeting of MYC by activation of the DR5 death receptor pathway[END_REF] and cancer cells [START_REF] Drosopoulos | Transformation by oncogenic RAS sensitizes human colon cells to TRAIL--induced apoptosis by up--regulating death receptor 4 and death receptor 5 through a MEK--dependent pathway[END_REF], respectively, related the gain of TRAIL--sensitivity upon oncogenic transformation to an increase in DR5 total protein and surface expression levels. Therefore we tested the possibility that rhTRAIL--resistance in normal BJ cells could arise from absent or aberrant protein expression of key DISC members. Analysing We detected rapid cleavage of CASP8, but not CASP10 upon rhTRAIL addition.

Therefore CASP8 seems to be the initiator CASP of rhTRAIL signalling and CASP10 appears to play (if at all) only a minor role during apoptosis--induction in stepwise-system cells, a finding supported by others [START_REF] Dickens | A Death Effector Domain Chain DISC Model Reveals a Crucial Role for Caspase--8 Chain Assembly in Mediating Apoptotic Cell Death[END_REF].

Moreover, for the investigated time--points no decline in full--length BID levels and no appearance of cleaved CASP9 p35 fragments was observed (Figure 24, F, right panel), which is in line with previous observations [START_REF] Nesterov | Oncogenic Ras sensitizes normal human cells to tumor necrosis factor--alpha--related apoptosis--inducing ligand-induced apoptosis[END_REF]. Since BID knockdown prevents rhTRAIL--apoptosis in BJELR cells [START_REF] Lund | Transformation--dependent silencing of tumor--selective apoptosis--inducing TRAIL by DNA hypermethylation is antagonized by decitabine[END_REF] or other effector caspases [START_REF] Li | Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[END_REF][START_REF] Slee | Cleavage of BID during cytotoxic drug and UV radiation--induced apoptosis occurs downstream of the point of Bcl--2 action and is catalysed by caspase--3: a potential feedback loop for amplification of apoptosis--associated mitochondrial cytochrome c release[END_REF][START_REF] Hayakawa | Bid truncation mediated by caspases--3 and --9 in vinorelbine--induced apoptosis[END_REF].

We detected cleavage of effector CASP7 into its active p20 fragment only 4h after rhTRAIL additon (Figure 24, F). Considering that CASP7 is a substrate of granzyme B, CASP10 and CASP3 [START_REF] Gu | Processing and activation of CMH--1 by granzyme B[END_REF][START_REF] Fernandes--Alnemri | In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD--like domains[END_REF][START_REF] Fernandes--Alnemri | Mch3, a novel human apoptotic cysteine protease highly related to CPP32[END_REF] and that no CASP10 processing but early CASP3 cleavage was observed (Figure 24, E), CASP7 presumably is processed by CASP3 in transformed BJELR cells.

Low total CASP8 cleavage levels in the whole entity of analysed normal cell Similar DISC compositions as described for DR5--IP, were obtained when DR4 or CASP8 was immunoprecipitated in normal BJ and transformed BJELR cells (Supplementary, Figure 31). Contrary to reports demonstrating TRADD co--immunoprecipitation with GST--tagged rhTRAIL from murine embryonic fibroblasts [START_REF] Cao | The role of TRADD in TRAIL-induced apoptosis and signaling[END_REF] or with FADD upon HIS--TRAIL--stimulation of H460 lung carcinoma cells [START_REF] Jin | Distinct signaling pathways in TRAIL--versus tumor necrosis factor--induced apoptosis[END_REF], we could not detect the co--purification of TRADD with CASP8 in normal or transformed cells (Supplementary, Figure 31). Although we cannot exclude that in stepwise system cells TRADD interacts with DISC components at a different timepoint than at 1h of rhTRAIL stimulation, the chosen analysis time laid in the middle of the reported TRADD recruitment time which was between 15min [START_REF] Cao | The role of TRADD in TRAIL-induced apoptosis and signaling[END_REF] and 1.5 and 3h [START_REF] Jin | Distinct signaling pathways in TRAIL--versus tumor necrosis factor--induced apoptosis[END_REF]] of TRAIL stimulation. Although there is currently no indication for complex II formation in stepwise--system cells, analyses of the co--immunoprecipitation of other pro--survival proteins constituting complex II await completion.

Observed differences in the composition of the DISC in normal BJ and transformed BJELR cells and simultaneous absence of DcR2 and TRADD supported our initial hypothesis that there could be unknown DISC interactors in normal and transformed cells regulating DISC formation, composition and TRAIL signalling.

Interim summary and conclusion II

As evidenced on cells of the BJ stepwise transformation system, purified rhTRAIL acts in a tumor--specific manner, killing transformed but not normal cells. WB analyses and FACS--assisted readouts of DR5 surface stainings revealed that the varying rhTRAIL response of normal BJ and transformed BJELR cells is not due to differential DISC protein expression or DR5 surface levels. Instead, WB analyses unveiled that rhTRAIL-resistance in BJ cells originates from poor initial CASP8 cleavage and/or recruitment, which is not compensated at prolonged rhTRAIL incubation. Apparently CASP8 is the apical caspase of rhTRAIL signalling in the stepwise--system cells, as rapid CASP8 but no CASP10 cleavage was observed in BJELR cells.

Interestingly, DISC analyses showed that normal BJ cells are capable of forming a partial DISC. Even though rhTRAIL--stimulated DR5/DR4 heterocomplex formation was similar in normal and transformed cells, FADD recruitment, CASP8 processing and/or recruitment was inefficient in normal cells. Reduced FADD recruitment, CASP8 cleavage and/or recruitment at the DISC was not due to translocation of anti--apoptotic DcR2 into the DISC. So far, no indications for complex II formation was observed, as there was no TRADD co--recruited.

In summary, rhTRAIL--resistance in normal cells is associated with poor CASP8 activation at the DISC that cannot be fully explained by previously described resistance mechanisms. The identification of novel DISC interactors that could regulate the divergent DISC formation is described in the next chapter. In the following, we intended to confirm the identified NMHCIIa/DR5 and ßACT/DR5 interaction. We therefore expanded regular Western blot--based DISC analysis on the detection of NMHCIIa and ßACT, which affirmed their interaction with DR5 and DR4. Both MS and Western blot analyses showed higher levels of NMHCIIa and ßACT interacting with DR5 in normal cells in the unstimulated state. Upon rhTRAIL stimulus, NMHCIIa and ßACT were disassembled from DR5 in transformed but not in normal cells (Figure 25, D; for the full DISC analysis please see Supplementary, Figure 31). Fibroblasts, like a variety of cultured cells express 2 (IIa, IIb) of 3 existing (IIa, IIb, IIc) NMHC isoforms [START_REF] Lo | Nonmuscle myosin IIb is involved in the guidance of fibroblast migration[END_REF]. We therefore controled DR--IPs for the interaction with NMHCIIb and found that also NMHCIIb was co--purified with DR5, following the same pattern than NMHCIIa (Figure 25 Importantly, analysis of the DISC immunoprecipitating DR4 gave comparable interaction patterns of cytoskeleton proteins with DR4 than with DR5 (Figure 25, D and Supplementary, Figure 31).

Questioning the biological significance of the NMII/DR and ßACT/DR interaction in cells other than mesenchymal fibroblasts, we analysed HTERT, SV40ER and HRASG12V--transformed HA1ER cells and confirmed the interaction of NMHCIIa, NMHCIIb and ßACT with DR5 in those cells (Supplementary, Figure 32). Analysis of normal HEK cells as their normal counterparts so far was hampered by poor cell growth, not yielding sufficient sample material. Whilst the co--purification of NMHCIIa, NMHCIIb and ßACT was confirmed in transformed HA1ER cells, no association of NMHCIIa or NMHCIIb with the DR5 or DR4 of highly rhTRAIL--sensitive BJAB cell was found (data not shown). This suggests a difference in cells growing in adhesion or suspension.

In a secondary MS approach where whole DR5--IP eluates immediately were given for in--solution digestion and analysis (Supplementary, Figure 33,A), again

NMHCIIa and ßACT and in addition NMHCIIb was identified (Supplementary, Figure 33,B). In sample conditions where DR5 was identified in DR5--IP, parallel control IgG--IP purified neither DR5, nor co--precipitated NMHCIIa (Supplementary, Figure 33,C). NMHCIIa, NMHCIIb and ßACT are expressed at similar protein levels in normal BJ and transformed BJELR cells whereas MLC2 is increased in transformed cells. Cellular transformation is often associated with the down--regulation of actin-associated proteins such as TM, CALD, MLC2, calponin, transgelin, ABP280, alpha-actinin, MARCKS, schwannomin, thymosin ß4, gelsolin [START_REF] Button | Actin, its associated proteins and metastasis[END_REF], Pawlak and Helfman 2001[START_REF] Iida | Overexpression of Plk3 causes morphological change and cell growth suppression in Ras pathway--activated cells[END_REF]. Given the reduced interaction of NMHCIIa, NMHCIIb, MLC2 and ßACT with the DR of BJERL cells (Figure 25, D, Supplementary, Figure 31), we carried out Western blot analysis of BJ and BJELR whole cell lysates and found that whilst NMHCIIa, NMHCIIb and ßACT were expressed at comparable levels, MLC2 protein levels are increased in transformed cells (Figure 26). Importantly, the applied anti--MLC2

antibody detects all three existent MLC2 isoforms, namely smMLC2, nmMLC2 and nmlMLC2. Due to their sequence homology no antibody can be generated capable to distinguish between these MLC2 isoforms. An oncogenic HRAS--transformation associated decrease of only the smMLC2 isoform mRNA and protein levels has been reported, whilst same studies describe no transformation--associated alterations of nmMLC2 and nmlMLC2 protein levels [START_REF] Kumar | Cytoskeletal changes in human transformed cells: studies on HOS cells[END_REF][START_REF] Kumar | Regulation of smooth muscle alpha--actin promoter in ras--transformed cells: usefulness for setting up reporter gene--based assay system for drug screening[END_REF][START_REF] Gerrits | Gene duplication and conversion events shaped three homologous, differentially expressed myosin regulatory light chain (MLC2) genes[END_REF]. This apparent discrepancy to our results might be due to the fact that oncogenic HRAS--transformation was investigated in fibroblasts [START_REF] Kumar | Cytoskeletal changes in human transformed cells: studies on HOS cells[END_REF][START_REF] Kumar | Regulation of smooth muscle alpha--actin promoter in ras--transformed cells: usefulness for setting up reporter gene--based assay system for drug screening[END_REF] and immortalised mouse embryonic fibroblasts [START_REF] Gerrits | Gene duplication and conversion events shaped three homologous, differentially expressed myosin regulatory light chain (MLC2) genes[END_REF], but not in SV40ER--pretransformed cells, and therefore could depend on the genetic background.

Indeed, Gerrits et al demonstrate Western blots with constantly higher nmMLC2 (and seemingly also smMLC2 and nmlMLC2) protein levels in tumourigenic human cancer cells in comparison to nontumourigenic cells, but unfortunately do not discuss this result.

Interim summary and conclusion III

Mass spectrometry probing of either on--membrane digested differential bands or whole DR5--IP eluates of unstimulated and rhTRAIL--treated normal BJ and transformed BJELR cells identified NMHCIIa, NMHCIIb and ßACT as novel DR5--interactors.

Subsequent Western blot analysis confirmed the interaction and revealed that MLC2 Even though a weaker interaction of NMHCIIa, NMHCIIb, MLC2 and ßACT with DR5 and DR4 of transformed BJELR than normal BJ cells was found, Western blots indicated similar protein expression levels for all proteins except MLC2 that was increased in transformed cells.

In summary, rhTRAIL--resistance in normal cells is associated with robust and sustained NMHCII/DR interaction, which is weaker and lost in TRAIL--sensitive transformed cells. Explorations of whether the newly identified interaction is of functional meaning for TRAIL apoptosis are communicated in the subsequent chapter.

IV -Functional characterisation of the NMHCII/DR interaction with respect to TRAIL apoptotic response

Arrangement of the actomyosin cytoskeleton differs in normal and cancer cells, being disrupted in the latter [START_REF] Weber | Localization and Distribution of Actin Fibers in Normal, Transformed and Revertant Cells[END_REF][START_REF] Bar--Sagi | Induction of membrane ruffling and fluid--phase pinocytosis in quiescent fibroblasts by ras proteins[END_REF] For that we used ML7, an MLCK--specific inhibitor [START_REF] Saitoh | Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase[END_REF][START_REF] Bain | The specificities of protein kinase inhibitors: an update[END_REF] that competes with ATP binding to MLCK. Of note, 30 min after ML7 addition at concentrations ≤50µM normal cells began to display retraction of cell protrusions and cell rounding, which was then followed by a recovery phase. At 2.5h after addition of ≤50µM ML7, cells fully recovered normal morphology.

Normal BJ cells that were incubated for 30 min with 50µM ML7 showed a reduction in ±4)) (Supplementary, Figure 34).

As the observed overall sensitising effect of normal BJ cells to TRAIL--apoptosis was stronger with blebbistatin than with ML7 (52% ±3 versus 29% ±0.3), both agents reduced the NMII/DR interaction, blebbistatin did not affect the ßACT/DR interaction whilst ML7 abolished ßACT from DR, we concluded that TRAIL--DISC mediated apoptosis may require the interaction with ßACT. upon high ML7 dosages, which revealed that BJELR cells did not survive ML7 levels as high as BJ cells (Supplementary, Figure 34). These data suggest that NMII activity in transformed BJELR cells is hindered (at least) by deregulation of MLCK and PAK1.

Work on further inherent mechanisms blocking NMII ATPase activity were not concluded at the time this manuscript was generated, and therefore can be read in the section 'Work in Progress'. Although NMHCIIa and NMHCIIb are highly homologous [START_REF] Bresnick | Molecular mechanisms of nonmuscle myosin--II regulation[END_REF], siRNA-mediated knockdown is specific (Figure 29,A). Depletion of NMHCIIb but not NMHCIIa impaired cell proliferation in normal cells as compared to scramble (SCR) control cells (NMHCIIb=2.18x10 5 ±4.4x10 4 cells, p=0.03; NMHCIIa=4.3x10 5 ±1.3x10 5 cells; SCR=3.6x10 5 ±5.3x10 4 cells) (Figure 29,B). We noted a differential influence of NMHCIIa or NMHCIIb protein loss on the adhesive properties of normal BJ cells, since KD of NMHCIIa but not NMHCIIb decreased attachment of BJ cells to the cell culture plate, which is in line with reported loss of focal adhesions in NMHCIIa KD cells [START_REF] Li | Positive and negative signaling components involved in TNFalpha-induced NF--kappaB activation[END_REF].

NMHCIIa--KD cells detached much faster during trypsinisation than SCR--transfected control, mock--transfected control or NMHCIIb--KD BJ cells. Significantly (p=6.9x10 --6 ) more NMHCIIa--KD cells underwent rhTRAIL--induced apoptosis (63% ±7) than Thus, NMHCIIa is a novel DR interactor that regulates the response of normal cells to TRAIL.

Identification of TRAIL DISC binding proteins that putatively are influenced by or that influence the NMHCIIa/DR5 interaction

Interfering with cell adhesion and spreading is a way to sensitise to TRAIL-apoptosis and adherent cells were reported as more sensitive to TRAIL if they were treated in suspension [START_REF] Goldberg | Global effects of anchorage on gene expression during mammary carcinoma cell growth reveal role of tumor necrosis factor--related apoptosis--inducing ligand in anoikis[END_REF][START_REF] Algeciras--Schimnich | Molecular ordering of the initial signaling events of CD95[END_REF][START_REF] Kurenova | Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor--interacting protein[END_REF][START_REF] Laguinge | DR5 receptor mediates anoikis in human colorectal carcinoma cell lines[END_REF][START_REF] Phipps | Targeting cell spreading: a method of sensitizing metastatic tumor cells to TRAIL--induced apoptosis[END_REF]. Recently, a study analysing the proteome of focal adhesions in presence and absence of blebbistatin reported the recruitment of signalling proteins regulating adhesion, spreading, migration, proliferation and survival to focal adhesions was either promoted or prevented by NMII contractility [START_REF] Kuo | Analysis of the myosin--II--responsive focal adhesion proteome reveals a role for beta--Pix in negative regulation of focal adhesion maturation[END_REF]. We observed a strong NMII/DR interaction and resistance to rhTRAIL--apoptosis in normal cells and a weak NMII/DR interaction and sensitivity to rhTRAIL--apoptosis in transformed BJELR cells. Hence, we hypothesised that NMII could bring pro--survival proteins into the DISC of normal cells.

Pro-survival adapter protein GRB2 and serine/threonine kinase RAF1 constitutively bind to DR5 and their binding is not dependent on NMHCIIa.

Interestingly, the proliferation signalling adapter and SH3 protein GRB2 was indicated to interact with NMHCIIa [START_REF] Blagoev | A proteomics strategy to elucidate functional protein--protein interactions applied to EGF signaling[END_REF] and proven to directly bind to TNFR1 via a PLAP motif in the receptor sequence [START_REF] Hildt | Identification of Grb2 as a novel binding partner of tumor necrosis factor (TNF) receptor I[END_REF]. Generally, SH3 proteins recognise PXXP and RXXK motifs [START_REF] Cohen | Modular binding domains in signal transduction proteins[END_REF][START_REF] Berry | A high--affinity Arg--X--X--Lys SH3 binding motif confers specificity for the interaction between Gads and SLP--76 in T cell signaling[END_REF] that are found in both DR5 long and short isoforms (Figure 30,A). Therefore we investigated DR5--and DR4--immunoprecipitates for the co--purification of GRB2. Like it is the case for TNFR1 [START_REF] Hildt | Identification of Grb2 as a novel binding partner of tumor necrosis factor (TNF) receptor I[END_REF], GRB2 interacts constitutively with DR5 (Figure 30,B). Even though the cytoplamic sequence of DR4 does not contain a PXXP or RXXK motif (Figure 30, A), GRB2 co--immunoprecipitated when DR4 was targeted (not shown). This could on one hand be due to the diversity of SH3 domain--binding motifs of which there are new ones constantly identified [START_REF] Kaneko | The SH3 domain----a family of versatile peptide--and protein--recognition module[END_REF]. On the other hand the GRB2/DR4 interaction could be mediated via GRB2's SH2 domain, just as it is the case for tyrosine kinase receptors such as EGFR [START_REF] Batzer | Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor[END_REF][START_REF] Okutani | Grb2/Ash binds directly to tyrosines 1068 and 1086 and indirectly to tyrosine 1148 of activated human epidermal growth factor receptors in intact cells[END_REF][START_REF] Chook | The Grb2--mSos1 complex binds phosphopeptides with higher affinity than Grb2[END_REF][START_REF] Jones | A quantitative protein interaction network for the ErbB receptors using protein microarrays[END_REF]. Of note, no co--purification of TNFR1 or EGFR occurred in DR5--IPs (not shown), indicating that co--purification of GRB2 with DR5--IPs is due to GRB2 binding to RESULTS

123

DR5, not to an interaction of DR5 with TNFR1 or EGFR that consequently would bring GRB2. To test the hypothesis that NMII could bring GRB2 to the receptor, we analysed DR5--IP eluates originating from previous NMHCIIa KD experiments in normal BJ cells for differential GRB2 recruitment and found no alterations (Figure 30,C). Thus, GRB2 binding to DR5 in normal BJ cells does not depend on NMHCIIa.

Binding of GRB2 to TNFR1 is essential for TNFα--induced activation of RAF1 [START_REF] Hildt | Identification of Grb2 as a novel binding partner of tumor necrosis factor (TNF) receptor I[END_REF]. In TNFα--and FAS--signalling RAF1 is recruited into and activated by phosphorylation [START_REF] Broustas | Phosphorylation of the myosin-binding subunit of myosin phosphatase by Raf--1 and inhibition of phosphatase activity[END_REF]. Of note, RAF1 gene knockout in mice lead to apoptosis and embryonic lethality, a phenotype that could be rescued using RAF1 mutants without kinase activity towards MEK [START_REF] Mikula | Embryonic lethality and fetal liver apoptosis in mice lacking the c--raf--1 gene[END_REF], Hüser et al 2001[START_REF] Matallanas | Raf family kinases: old dogs have learned new tricks[END_REF]. Previous experiments demonstrated that siRNA--mediated NMHCIIa depletion amongst others resulted in reduced cFLIP(L) recruitment and/or processing at the DR5--DISC (Figure 29,E). We reasoned that if cFLIP(L)p43 would recruit RAF1 into the DISC and if lower cFLIP(L)p43 levels are observable in NMHCIIa KD cells, one would be able to detect less RAF1 recruitment. Therefore we investigated DR5--IP eluates originating from previous NMHCIIa KD experiments for the presence of RAF1. Surprisingly, RAF1 constitutively co--purified with DR5 to comparable levels in scrambled transfected and NMHCIIa KD normal BJ cells (Figure 30,C). Thus, like with GRB2, RAF1 interaction with the DR5 DISC appears to be independent of the NMHCIIa/DR5 interaction.

Depletion of GRB2 does not enhance the DISC-mediated rhTRAIL apoptosis

in normal BJ cells. In contrast to NMHCIIa KD, GRB2 ablation in normal BJ cells increased cell adhesion and critically decreased cell proliferation as assessed by total cell counts 4d after start of siRNA--mediated KD (GRB2=1.54x10 5 ±2.4x10 4 cells, p=0.03; SCR=4.3x10 5 ±1.4x10 4 cells) (Figure 30,D). Loss of GRB2 protein was accompanied by a decline in phosphorylated RAF1, phosphorylated MEK1/2 and total MEK1/2 levels as evidenced by Western blot analyses of normal BJ cell WCL 4d after transfection (Figure 30,E). To explore effects of GRB2 deficiency on TRAIL sensitivity, we left lipofectamine-transfected (mock), scramble siRNA--transfected (SCR), and GRB2 siRNA--transfected cells 4d after start of transfection untreated or treated them with 1µg mL --1 rhTRAIL for 16h and assessed the percentage of apoptotic cells. Results yielded a mild but significant difference in rhTRAIL--induced apoptosis between GRB2 depleted and SCR control cells (GRB2=27% ±1.3, p=0.03; SCR=20% ±1). However, solely the reduction of GRB2 protein levels without rhTRAIL stimulation was sufficient to decrease cell viability of normal BJ cells (GRB2=7% ±2, p=0.06; SCR=3% ±1.6) (Figure 30, F). Thus, GRB2 deficiency in normal BJ cells did not drastically enhance the apoptosis response to TRAIL. Instead, it reduced cell proliferation and basal cell viability. Investigating the effects on TRAIL sensitivity therefore was difficult to assess, as cell densities and secreted factors at the moment of rhTRAIL addition are critical for the cell response to TRAIL.

Aiming to compensate cell density differences at the start of rhTRAIL treatment, we reduced the KD duration of GRB2 from 4 to 2 days and assessed the rhTRAIL apoptosis response by analysing CASP8 and CASP3 cleavage in normal BJ cells in Western blots. We found that 2d KD was sufficient to reduce GRB2 protein levels.

However, after 30min or after 17h of 1µg mL --1 rhTRAIL we did not observe enhanced signals for CASP8 or CASP3 cleavage, indicating that loss of GRB2 has no impact on the DISC--mediated rhTRAIL apoptotic response in normal BJ cells (Figure 30,G).

Interim summary and conclusion V

GRB2 and RAF1 constitutively bind to DR5 and in case of GRB2 also to DR4. Their binding to DR5 is not dependent on NMIIa activity, since NMHCIIa siRNA--mediated protein depletion diminished NMHCIIa/DR5 interaction, but had no influence on the levels of co--immunoprecipitated GRB2 and RAF1 upon immunoprecipitation of DR5.

The finding that RAF1 constitutively co--purifies with DR5 implies that RAF1 recruitment into the TRAIL DISC cannot be mediated by cFLIP(L)p43, denoting an apparent difference to TNFα and FAS signalling.

Reduction of GRB2 protein levels using siRNA KD in normal BJ cells decreased cell proliferation as assessed by total cell counts. This was associated with an observed decline in levels of phosphorylated RAF1, phosphorylated MEK1/2 and total MEK1/2 protein in Western blot. Thus the RAF1/MEK1/2 pathway that is known to activate the growth--stimulating extracellular signal--regulated kinase 1/2 (ERK1/2) is impaired upon GRB2 loss in BJ cells. Diminished GRB2 protein levels lead to a decline in basal BJ cell viability and moderate enhancement of rhTRAIL--induced apoptosis. However, even at shorter GRB2 KD duration we did not find evidence that GRB2 sensitises normal BJ cells to TRAIL via a DISC--mediated mechanism, since 2days of GRB2 KD did not reveal enforced CASP8 cleavage signals in Western blot analyses of whole cell lysates.

Depletion of pro--survival molecule GRB2 from DR5 hence does not tune rhTRAIL signalling to apoptosis in normal cells.

Figure 30 continued: were loaded onto SDS--PAGE gels and analysed by regular Western blot for the presence of DISC components and growth factor receptor--bound protein 2 (GRB2).* (C) Inputs and DR5--IP eluates originating from experiments described in Figure 29 and analysed in Figure 29,E were explored for co--purification of GRB2 and RAF proto--oncogene serine/threonine--protein kinase (RAF1).* (D) BJ cells were transfected with 15nM siRNA against GRB2 (G), scramble control siRNA (S) or mock-transfected with lipofectamine (M) for 4d. Cell culture medium was renewed 1d and 3d after start of transfection. Total cell numbers were counted 4d after start of transfection. Values are means of 3 independent experiments. (E) Whole cell lysates (WCL) of BJ cells transfected as described in (D) were analysed for expression, respectively phosphorylation levels of GRB2, RAF1 and MEK1/2 4d after start of knockdown (KD).* (F) TRAIL--induced apoptosis upon GRB2 siRNA KD in BJ cells. Four days after start of KD BJ cells were left untreated (--) or were treated with 1µg mL --1 rhTRAIL (+). Apoptosis was assessed as 7A6 positivity 16 hours after the addition of rhTRAIL. Values represent mean outcome of 3 independent experiments. (G) TRAIL--induced caspase cleavage after 2d of GRB2 KD in BJ cells. BJ cells were transfected as described in (D) and cell culture medium was renewed 1d after start of transfection. At day 2 of transfection cells were left untreated (--) or were treated with 1µg mL --1 rhTRAIL for 30 min or for 17h.

After that whole cell lysates (WCL) were analysed for GRB2 expression and caspase 8 and 3 (CASP8, CASP3) cleavage. Depicted are Western blots for 1 out of *2 independent experiments. Significances were calculated using unpaired, 2--tailed Student's t--test.

WORK IN PROGRESS

By the time this thesis manuscript was prepared, certain aspects regarding (I) the DISC definition in normal and transformed cells, (II) the mechanism of the NMII/DR interaction, (III) the molecular basis for the reduced NMII activity in transformed cells, (IV) the implication of NMII in DR transport and (V) the NMII/DR interaction in BJEL cells were not fully resolved. Since they are, in parts, essential to the previously shown results, these aspects shall be introduced herein.

I -Optimisation of DISC analyses to enable the distinction between divergence in protein recruitment or processing

As described in the 'Results' section, DISC analyses of normal BJ and transformed BJELR cells revealed lower rhTRAIL--induced levels of the CASP8 p18 cleavage product at the DR of normal cells (Results, Figure 24, G, Supplementary, Figure 31). Since processing of full--length CASP8 within the DISC appears to be accomplished rapidly, we currently cannot specify whether the reduction in CASP8 p18 fragments in normal BJ cells is due to reduced CASP8 processivity or due to reduced recruitment of CASP8 to the DR. DISC--analyses of normal and transformed cells that will be incubated with a pan--CASP inhibitor (zVAD) prior to challenge with rhTRAIL will allow us to answer whether the reduced sensitivity of normal cells to rhTRAIL--apoptosis solely is due to reduced CASP8 processing or additionally involves diminished recruitment of the full--length CASP8 protein.

Likewise described for CASP8, comparative DISC analyses of normal BJ and transformed BJELR cells and of scrambled--and NMHCIIa--siRNA transfected normal BJ cells, respectively, revealed alterations in the signal levels of the cFLIP(L) p43 cleavage product. As the full--length cFLIP(L) signal always is covered by unspecific signals originating from cross--reaction of the primary cFLIP(L) antibody with the heavy chain of the IP--antibody (Results, Figure 24, E & Figure 29, E, Supplementary, Figure 31), we cannot conclude that observed changes in cFLIP(L) p43 levels originate from lowered cFLIP(L) processing in the DISC or a reduced recruitment of cFLIP(L) to the DISC.

Therefore, finding a primary anti--cFLIP antibody that does not cross--react with the IP will help to clarify this issue. Tests for suitable anti--cFLIP antibodies are ongoing.

II -Analyses of the NMII/DR interaction nature

We attempted to understand the molecular basis of the NMII/DR interaction. On one hand we observed differential interaction intensities comparing unstimulated normal and transformed cells, which might be related to a differential NMII activity in those cells. On the other hand we found that in transformed cells rhTRAIL induced the loss of NMII from DR. Since rhTRAIL was indicated to induce caspase--dependent (apoptosis) and caspase--independent (survival or necrosis) signalling pathways (see 'Introduction'), we decided to explore the rhTRAIL--induced NMII/DR loss in more detail.

MLC2 phosphorylation potentially regulates the NMII/DR interaction.

Aforementioned results demonstrated a stronger NMII/DR interaction and higher MLC2 phosphorylation levels in unstimulated normal BJ than in transformed BJELR cells (Results, Figure 28,A). Moreover, the NMII/DR interaction in normal cells was abrogated if MLC2 phosphorylation was inhibited with the MLCK inhibitor ML7.

Therefore, we hypothesised that the NMII/DR interaction is regulated by MLC2 phosphorylation. Above that, NMHCIIa, NMHCIIb, MLC2 and ßACT were lost from DR upon rhTRAIL addition in transformed BJELR and HA1ER cells but not in normal BJ cells Mg 2+ --ATPase activity through cleavage of PAK2 [START_REF] Lee | Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis[END_REF][START_REF] Fischer | Unique and overlapping substrate specificities of caspase--8 and caspase--10[END_REF], but not PAK1 [START_REF] Lee | Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis[END_REF]. Whilst active full--length PAK2 is signalling towards survival phosphorylating and inhibiting BAD, PAK2 cleavage resulted in a pro--apoptotic, constitutively active kinase [START_REF] Fischer | Unique and overlapping substrate specificities of caspase--8 and caspase--10[END_REF], which induced cell detachment and apoptosis in epithelial HeLa and CHO cells [START_REF] Lee | Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis[END_REF]. How the pro--apoptotic version of PAK2 mediates apoptosis is unknown, but has been suggested to involve the production of reactive oxygen species [START_REF] Lee | Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis[END_REF] or mechanical stress due to cytoskeleton rearrangements [START_REF] Vilas | Posttranslational myristoylation of caspase--activated p21--activated protein kinase 2 (PAK2) potentiates late apoptotic events[END_REF]. Like PAK1, PAK2 phosphorylates and inactivates MLCK [START_REF] Goeckeler | Phosphorylation of myosin light chain kinase by p21--activated kinase PAK2[END_REF] Recently, the loss of TNFR1--internalisation upon NMHCIIb but not NMHCIIa knockdown was reported [START_REF] Flynn | Non--muscle myosin IIB helps mediate TNF cell death signaling independent of actomyosin contractility (AMC)[END_REF], which is a process required for TNFR1 apoptosis to occur. In TRAIL signalling TRAIL DISC internalisation in breast cancer cells accounted for TRAIL--resistance [Zhang and Zhang 2008] whilst this process was required for TRAIL induced apoptosis in malignant hepatocytes [START_REF] Akazawa | Death receptor 5 internalization is required for lysosomal permeabilization by TRAIL in malignant liver cell lines[END_REF].

Based on their difference in NMII/DR interaction, we questioned whether normal BJ and transformed BJELR cells display a differential TRAIL--induced DR5 internalisation pattern. For that we stimulated BJ and BJELR cells with 1µg mL --1 rhTRAIL for different However, how DR4 or DR5 find their way to the plasma membrane is unknown.

Assessment of DR4 cell surface levels in NMHCIIa KD cells will clarify, whether NMIIa at all is involved in DR4--selective transport processes or not.

Our analysis of WCL and the DISC in NMHCIIa KD normal BJ cells showed an increased CASP8 activation (Results, Figure 29, D&E). Whether this increase in CASP8 activation is due to higher numbers of CASP8 molecules being activated per cell or to same quantities of CASP8 molecules are activated in a higher number of cells is currently unclear. In line with our findings, an enhancement of CASP8 recruitment to the FLAG--TRAIL immunoprecipitated DISC was reported if the NMII regulatory kinase CK2, whose activity prevents S100A4/NMHCII binding and NMII filament disassembly, was inhibited [START_REF] Izeradjene | Influence of casein kinase II in tumor necrosis factor--related apoptosis--inducing ligand--induced apoptosis in human rhabdomyosarcoma cells[END_REF], an effect that was not caused by altered DR5 surface levels. Generally, CK2 activity is high in TRAIL resistant and low in TRAIL sensitive cancer cells [START_REF] Shin | Caspase--2 primes cancer cells for TRAIL--mediated apoptosis by processing procaspase--8[END_REF] and CK2 inhibition augments and elicits, respectively, sensitivity to TRAIL--apoptosis in rhabdomyosarcoma [START_REF] Izeradjene | Influence of casein kinase II in tumor necrosis factor--related apoptosis--inducing ligand--induced apoptosis in human rhabdomyosarcoma cells[END_REF], colon carcinoma [START_REF] Izeradjene | Casein kinase II (CK2) enhances death--inducing signaling complex (DISC) activity in TRAIL--induced apoptosis in human colon carcinoma cell lines[END_REF] and prostatic cancer cell lines [START_REF] Wang | Role of protein kinase CK2 in the regulation of tumor necrosis factor--related apoptosis inducing ligand--induced apoptosis in prostate cancer cells[END_REF] and in primary endometrial cacinoma cells [START_REF] Llobet | CK2 controls TRAIL and Fas sensitivity by regulating FLIP levels in endometrial carcinoma cells[END_REF]. These data suggest that active From the receptor side the minimally required DR domain to interact with NMII is unknown. Since the interaction may not be direct or the interaction could require proper protein folding, we will generate N--terminally tagged DR5 mutants, deleting section--wise from its C--terminal end, and express these constructs in normal BJ cells to control the NMII interaction upon immunoprecipitation. Thereby, special focus will be put on the depletion of the beforehand mentioned IQ2--binding motif. under TNFα [START_REF] Zhou | Tumor necrosis factor--alpha induced expression of matrix metalloproteinase--9 through p21--activated kinase--1[END_REF]. The mechanism for how PAK1 protein is stabilised is unknown, but seems to be independent of the kinase catalytic activity and RAS--GTPase binding capacities [START_REF] Zhou | Tumor necrosis factor--alpha induced expression of matrix metalloproteinase--9 through p21--activated kinase--1[END_REF].

NMII activity in

No comparative study between normal and transformed cells exists, which directly demonstrates an altered NMII Mg 2+ --ATPase activity. So far, relative studies only compared phospho--MLC2 levels. Interestingly, contrary to our findings of reduced MLC2 phosphorylation levels, transformation with HRAS or HRASG12V [START_REF] Zhong | Rho--stimulated contractility contributes to the fibroblastic phenotype of Ras--transformed epithelial cells[END_REF] or with the RHO--guanine nucleotide exchange factor (RHO--GEF) DBS [START_REF] Liu | Transformation by the Rho--specific guanine nucleotide exchange factor Dbs requires ROCK I--mediated phosphorylation of myosin light chain[END_REF] was linked to an increase of phospho--MLC2 levels associated with RHOA hyperactivation [START_REF] Zhong | Rho--stimulated contractility contributes to the fibroblastic phenotype of Ras--transformed epithelial cells[END_REF] and enhanced actin stress--fiber formation [START_REF] Liu | Transformation by the Rho--specific guanine nucleotide exchange factor Dbs requires ROCK I--mediated phosphorylation of myosin light chain[END_REF],

respectively. The reason for this discrepancy is most probably due to the fact that normal mammary epithelial cells [START_REF] Zhong | Rho--stimulated contractility contributes to the fibroblastic phenotype of Ras--transformed epithelial cells[END_REF] or normal murine fibroblasts [START_REF] Liu | Transformation by the Rho--specific guanine nucleotide exchange factor Dbs requires ROCK I--mediated phosphorylation of myosin light chain[END_REF] have been transformed, whereas in our case HRAS transformation was carried out in an SV40ER--pretransformed background in which RHOA expression might be already lost. Loss of RHOA activity in HRASG12V--expressing cells has been reported

earlier [START_REF] Izawa | Possible involvement of the inactivation of the Rho--Rho--kinase pathway in oncogenic Ras--induced transformation[END_REF]. In support of this assumption, BJELR cells poorly attach (see 'Results', p99) and do not exhibit actin stress fibers (Supplementary, Figure 35). Future

Western blot analyses will assess total RHOA levels in cells of the stepwise system, as well as CDC42 and RAC1 levels, which counteract RHOA routes.

In transformed BJELR cells we observed increased total MLC2 expression levels, evidenced by an antibody, which recognises all 3 MLC2 isoforms, smMLC2, nmMLC2 and nmlMLC2. This result somewhat contradicts findings that transformation is often associated with the downregulation of cytoskeletal proteins [START_REF] Iida | Overexpression of Plk3 causes morphological change and cell growth suppression in Ras pathway--activated cells[END_REF] and that HRAS--transformation induced the decrease of smMLC2, but not nmMLC2 and nmlMLC2, mRNA and protein levels [START_REF] Kumar | Cytoskeletal changes in human transformed cells: studies on HOS cells[END_REF][START_REF] Kumar | Regulation of smooth muscle alpha--actin promoter in ras--transformed cells: usefulness for setting up reporter gene--based assay system for drug screening[END_REF][START_REF] Gerrits | Gene duplication and conversion events shaped three homologous, differentially expressed myosin regulatory light chain (MLC2) genes[END_REF]. and/or disorganised focal adhesions, so that they adhere poorly to growth surfaces and so that ERK and calpains do not co--localise. This way, ERK cannot activate calpains, MYOD is not downregulated, which would finally explain why total MLC2 expression levels in BJELR cells are higher than in normal BJ cells. Intriguingly, also the expression of cFLIP was shown to be dependent on calpains [START_REF] Benayoun | NF--kappaB--dependent expression of the antiapoptotic factor c--FLIP is regulated by calpain 3, the protein involved in limb--girdle muscular dystrophy type 2A[END_REF] and it was indicated that cFLIP is expressed in myofibers [START_REF] Nagaraju | The inhibition of apoptosis in myositis and in normal muscle cells[END_REF][START_REF] Benayoun | NF--kappaB--dependent expression of the antiapoptotic factor c--FLIP is regulated by calpain 3, the protein involved in limb--girdle muscular dystrophy type 2A[END_REF]. Thus, active NMII fibers and mature focal adhesions seem to be of central importance for TRAIL resistance.

TRAIL-DISC composition in normal and transformed cells. Even though an

ERK--driven stabilisation of MYC and a subsequent MYC--driven upregulation of DR5 and DR4 were reported to account for TRAIL--sensitivity of transformed stepwise system cells [Nesterov et al 2004, Wang et Potential functions of GRB2 and RAF1 at the TRAIL DISC of normal cells.

Likewise it has been reported for TNFR1 [START_REF] Hildt | Identification of Grb2 as a novel binding partner of tumor necrosis factor (TNF) receptor I[END_REF], we found GRB2 to constitutively co--immunoprecipitate with DR5 (Results, Figure 30, B&C) and DR4 (not shown) in normal BJ cells. GRB2 is a communicator that links membrane receptors and membrane--bound proteins to intracellular cytoskeletal regulators, increasing their local concentrations at the membrane and facilitating enzymatic reactions and activation and couples to mitogenesis and cytoskeletal reorganisation [reviewed by [START_REF] Giubellino | Grb2 signaling in cell motility and cancer[END_REF]. Depletion of GRB2 from normal BJ cells decreased overall levels of active RAF1 and MEK1/2, which is in line with findings that the TNFR1/GRB2 interaction was essential for TNFα--dependent activation of RAF1 kinase [START_REF] Hildt | Identification of Grb2 as a novel binding partner of tumor necrosis factor (TNF) receptor I[END_REF]. Contrary to the FAS--DISC, at which RAF1 recruitment was induced upon ligand stimulation and RAF1 was suggested to directly interact with and to be activated by cFLIP(L)p43 [START_REF] Kataoka | The caspase--8 inhibitor FLIP promotes activation of NF--kappaB and Erk signaling pathways[END_REF][START_REF] Park | Opposite role of Ras in tumor necrosis factor-alpha--induced cell cycle regulation: competition for Raf kinase[END_REF], we found RAF1 to constitutively interact with DR5 (Results, Figure 30,C). This suggests that at the DR of normal cells RAF1 might be activated via GRB2/SOS1/RAS in a ligand--independent fashion. Upon TRAIL--stimulation, RAF1 could additionally be induced by cFLIP(L)p43. Whilst RAF1 plays an essential role in maintaining the organization of the cytoskeleton and thus in cell polarity and migration [START_REF] Ehrenreiter | Raf--1 regulates Rho signaling and cell migration[END_REF], GRB2 signalling leads to the activation of MAPK pathways (JNK, p38, ERK1/2). The impact of a putative TRAIL--induced complex II in the activation of these signalling routes therefore might be lower than initially assumed. As previously mentioned, DISC analyses do not allow us to conclude whether FADD, CASP8

and/or cFLIP binding occurs at the same DR than GRB2 and RAF1 binding.

EXTENDED SUMMARY

In this PhD thesis we conducted a study on the transformation--associated mechanisms that lead to the gain of TNF--α--related apoptosis inducing ligand (TRAIL) sensitivity. In that context the cytoskeletal protein nonmuscle myosin heavy chain IIa (NMHCIIa) was identified as entirely novel TRAIL death receptor interactor that proved critical for TRAIL resistance of normal cells. This study was performed at the Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), Illkirch, France.

TRAIL is a cytokine of the TNF--superfamily that binds 4 specific membrane-bound receptors inducing their oligomerisation [START_REF] Wiley | Identification and characterization of a new member of the TNF family that induces apoptosis[END_REF][START_REF] Pitti | Induction of apoptosis by Apo--2 ligand, a new member of the tumor necrosis factor cytokine family[END_REF], Chaudhary et al 1997, Degli--Esposti et al 1997, Pan et al 1997a, 1997b, Sheridan et al 1997[START_REF] Schneider | TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD--dependent apoptosis and activate NF--kappaB[END_REF][START_REF] Degli--Esposti | Cloning and characterization of TRAIL--R3, a novel member of the emerging TRAIL receptor family[END_REF] Resulting close proximity of CASP molecules and/or cFLIP at the DR results in their mutual cleavage and activation. The complex minimally comprising DR, FADD, CAP8/10 and/or cFLIP is called the death inducing signalling complex (DISC) (yellow). Once activated at the DISC, CASP8/10 can directly cleave and activate effector caspases 3 and 7 (CASP3, CASP7) and hence fulfil the so--called extrinsic apoptosis pathway. In certain cells the apoptotic signal is propagated and enhanced via the intrinsic apoptotic cascade. Therefore DISC--activated CASP8/10 proteolytically convert BH3--interacting domain death agonist (BID) to the active, truncated form (tBID) that induces BCL2--antogonist/killer (BAK) and BCL2--associated X protein (BAX) at the mitochondria and in turn results in permeabilisation of the outer mitochondrial membrane and release of proteins such as cytochrome c, second mitochondria--derived activator of caspases (SMAC)/direct inhibitor of apoptosis protein (IAP)--binding protein with low pI (DIABLO). Cytochrome c binds to adaptor molecule apoptotic protease--activating factor 1 (APAF1) that binds to initiator procaspase 9 (CASP9) to form the apoptosome. At the apoptosome, CASP9 is activated leading to processing of CASP3/7 and eventually cell death. The figure is based on the idea of [START_REF] White--Gilbertson | Transformation, translation and TRAIL: an unexpected intersection[END_REF][START_REF] Newsom--Davis | Is TRAIL the holy grail of cancer therapy?[END_REF].

TRAIL is the only human cytokine known to induce death of diverse tumour cells while sparing healthy normal ones [START_REF] Pitti | Induction of apoptosis by Apo--2 ligand, a new member of the tumor necrosis factor cytokine family[END_REF][START_REF] Walczak | Tumoricidal activity of tumor necrosis factor--related apoptosis--inducing ligand in vivo[END_REF] Since DISC formation is at the origin of TRAIL signalling (Figure 1), we hypothesised that as yet unknown DISC interactors may be critically involved in DISC function and that tumourigenic transformation would alter their recruitment, modification and/or expression and thus result in differential TRAIL--sensitivity.

Therefore, we set out to identify novel DISC--interacting proteins that are differentially recruited in normal and tumour cells. We used stepwise human cell--based transformation models [START_REF] Hahn | Creation of human tumour cells with defined genetic elements[END_REF] that enabled a direct comparison of cancer cells with the normal isogenic progenitors (Figure 2). Notably, such a comparative analysis is not possible with established cancer and so--called 'normal' cell lines. While transformed cells derived from stepwise models display tumourigenic features [START_REF] Hahn | Creation of human tumour cells with defined genetic elements[END_REF] and TRAIL sensitivity, the normal human cells were resistant to TRAIL [START_REF] Nesterov | Oncogenic Ras sensitizes normal human cells to tumor necrosis factor--alpha--related apoptosis--inducing ligand-induced apoptosis[END_REF], Wang et al 2005]. Whilst hTERT maintains telomere length and confers replicative immortality to BJ cells, HEK cells require additional SV40ER--encoded elements to reach immortality. Obtained transformed cells (here: BJELR or HA1ER) display cancer cell features like unlimited proliferation, anchorage--independent growth and tumour formation in vivo and are sensitive to TRAIL--induced apoptosis.

The 'results' section includes the protocols for the production and purification of recombinant human TRAIL and for native DISC immunoprecipitation. These protocols required establishment in the laboratory to facilitate analysis of the initial steps of TRAIL signalling in stepwise system cells as well as the identification of novel DISC interactors. In the following DISC analyses are presented showing that both normal and tumour cells formed DR5/DR4 DISC upon TRAIL stimulus. However, a reduced amount of cleaved CASP8 was seen in normal cells indicating poor pro--apoptotic signal intensity. Moreover, FADD recruitment was diminished and cFLIP(L) cleavage was higher in normal cells, suggesting that the resistance to TRAIL arises at the DISC (Figure 3). The scheme indicates the experimental set--up. After immunoprecipitation (IP), proteins were eluted and eluates were split into 2 fractions. One aliquot was separated on SDS--PAGE gels and transferred onto nitrocellulose membranes that were stained with Ponceau S. Observed differential bands were excised from the membranes, proteins were digested and analysed by mass spectrometry (MS). The other aliquot was separated on SDS--PAGE gels, blotted onto nitrocellulose membranes and analysed for protein interactions using specific antibodies (AB). In this way (A) death receptor 5 (DR5) was purified from lysates of BJ and BJELR cells that were left unstimulated (--) or that were stimulated for 1h with 1µg mL --1 recombinant human TRAIL (rhTRAIL) (+). Differential bands visualised with Ponceau S were cut out, digested and given for mass--spectrometrical peptide identification. (B) Western blot analysis of DR5--IP eluates to validate the mass--spectrometric results obtained in (A) (upper panel). Death receptor 4 (DR4) was purified under similar experimental conditions and analysed by Western blot (lower panel). Depicted are representative blots from 1 out of 2 independent experiments. Abrogation of the NMHCII/DR interaction using the nonmuscle myosin II (NMII) inhibitor blebbistatin or the myosin light chain kinase (MLCK) inhibitor ML7 increased the response of normal cells to TRAIL--induced apoptosis (Figure 5). Intriguingly, siRNA--mediated knockdown of NMHCIIa but not NMHCIIb robustly released the TRAIL resistance block in normal cells and was characterised by reduced DR4 recruitment, decreased processing of cFLIP(L) and elevated CASP8 cleavage at the DR5--DISC (Figure 6). Exploration of the signalling cascade dictating differential regulation of the NMHC/DR interaction in normal and transformed cells showed higher NMHC activity in normal cells as indicated by increased MLC2 phosphorylation levels. In transformed cells MLC2-phosphorylating kinase (MLCK) activity is impaired, as supported by the observation that MLCK protein levels were downregulated, MLCK inhibitory kinase (PAK1) protein levels were increased and the circumstance that normal cells survived higher doses of MLCK inhibitor (ML7) levels than transformed cells (Figure 7). Cells were pre--incubated with indicated concentrations of ML7 or solvent (ethanol/water mix at a 1:1 ratio) for 30 min and then co--treated with 1µg mL --1 purified TRAIL. Apoptosis was quantified as 7A6 positivity in FACS analysis after 16h of treatment. Values derive from 2 independent experiments.

Together these data support a model in which the newly identified NMHCIIa interaction with DR maintains TRAIL resistance in normal cells. Moreover, they suggest a functional role of NMHCIIa in the acquisition of TRAIL responsiveness through the process of transformation. Finally, since NMHCIIa has a central role in cell adhesion, our model provides a direct link between the correlation of cell detachment and TRAIL sensitivity described in literature.

Future work will focus on the identification of the domain minimally required for NMHCIIa to interact with DR aiming at understanding the (structural) basis of interaction. The biological significance of this interaction will be confirmed in other existing cell models of neoplastic transformation. Cytoskeletal changes will be investigated in immortalised and pre--transformed cells of the stepwise transformation system to define whether observed differences in transformed cells are mediated by Ras. Unravelling differences in the DISC composition of normal and transformed cells will decipher the molecular basis of TRAIL sensitivity/resistance and provide a solid platform for the exploitation of TRAIL--based anti--cancer therapies.

RÉSUMÉ

Dans ce projet de thèse, nous avons mené une étude sur les mécanismes associés à la transformation cellulaire qui aboutissent à une sensibilité accrue au TNF--α--related apoptosis inducing ligand (TRAIL). Dans ce contexte, la protéine du cytosquelette non-muscle myosin heavy chain IIa (NMHCIIa) a été identifiée comme un tout nouveau partenaire du récepteur de mort de TRAIL. Il est essentiel pour la résistance à TRAIL des cellules normales. Cette étude a été menée à l'Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France dans l'équipe du Dr. Hinrich GRONEMEYER.

TRAIL est une cytokine de la superfamille du TNF qui se lie à 4 récepteurs membranaires spécifiques, induisant leur oligomérisation [START_REF] Wiley | Identification and characterization of a new member of the TNF family that induces apoptosis[END_REF][START_REF] Pitti | Induction of apoptosis by Apo--2 ligand, a new member of the tumor necrosis factor cytokine family[END_REF], Chaudhary et al 1997, Degli--Esposti et al 1997, Pan et al 1997a, 1997b, Sheridan et al 1997[START_REF] Schneider | TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD--dependent apoptosis and activate NF--kappaB[END_REF][START_REF] Degli--Esposti | Cloning and characterization of TRAIL--R3, a novel member of the emerging TRAIL receptor family[END_REF]. Les récepteurs de mort 5 et 4 (DR5, DR4) contiennent un domaine de mort (DD) qui sert à l'assemblage de complexes de signalisation [MacFarlane et al 1997, Pan et al 1997a, Pan et al 1997b[START_REF] Schneider | TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD--dependent apoptosis and activate NF--kappaB[END_REF][START_REF] Degli--Esposti | Cloning and characterization of TRAIL--R3, a novel member of the emerging TRAIL receptor family[END_REF]. Les 2 autres récepteurs de TRAIL sont des récepteurs leurres (DcR1, DcR2) qui possèdent un DD tronqué et ne transmettent pas de signaux [MacFarlane et al 1997, Pan et al 1997b[START_REF] Schneider | TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD--dependent apoptosis and activate NF--kappaB[END_REF], Degli--Esposti et al 1997].

L'oligomérisation des DR après liaison de TRAIL induit une interaction avec des molécules adaptatrices comme Fas--associated death domain (FADD) qui à leur tour recrutent les caspases initiatrices 8 (CASP8) et / ou 10 (CASP10) [Chaudhary et al 1997, MacFarlane et al 1997, Bodmer et al 2000[START_REF] Kischkel | Apo2L/TRAIL--dependent recruitment of endogenous FADD and caspase--8 to death receptors 4 and 5[END_REF], Sprick et al 2000;[START_REF] Kischkel | Death receptor recruitment of endogenous caspase--10 and apoptosis initiation in the absence of caspase--8[END_REF][START_REF] Wang | Caspase--10 is an initiator caspase in death receptor signaling[END_REF][START_REF] Sprick | Caspase--10 is recruited to and activated at the native TRAIL and CD95 death--inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase--8[END_REF]. De la même manière, la protéine anti--apoptotique cellular FLICE--like inhibitory protein (cFLIP) est recrutée au niveau des DR. Elle rentre en compétition avec la caspase 8 pour la liaison de FADD et déclenche alors des voies de signalisation pour la survie [Thome et al 1997, Burns and El--Deiry 2001, Krueger et al 2001[START_REF] Chang | c--FLIP(L) is a dual function regulator for caspase--8 activation and CD95--mediated apoptosis[END_REF], Jin et al 2004]. Les récepteurs de TRAIL, FADD et les caspases initiatrices constituent le complexe de signalisation de mort (DISC) (Figure 1) [Bodmer et al 2000]. Alors que la formation du DISC dans les cellules transformées est bien étudiée, on ne dispose d'aucune information sur la composition du DISC natif dans les cellules normales. TRAIL est la seule cytokine humaine connue pour induire la mort des cellules tumorales tout en épargnant les cellules normales [START_REF] Pitti | Induction of apoptosis by Apo--2 ligand, a new member of the tumor necrosis factor cytokine family[END_REF][START_REF] Walczak | Tumoricidal activity of tumor necrosis factor--related apoptosis--inducing ligand in vivo[END_REF]. L'abrogation de l'interaction NMHCII / DR en utilisant la blebbistatine, un inhibiteur de la myosine non musculaire (NMII) ou ML7, un inhibiteur de la kinase de la chaîne légère de myosine (MLCK) augmente l'apoptose induite par TRAIL dans les cellules normales (Figure 5). mots-clés: TRAIL, DISC, cellules normales, apoptose, spécifique de tumeur, cytosquelette, nonmuscle myosin heavy chain IIa, NMHCIIa

Abstract

The cytokine TRAIL is a promising cancer therapeutic candidate as it induces apoptosis selectively in transformed cells. TRAIL-induced clustering of its receptors (DR) is essential for the formation of the DISC complex, which induces cell death. The mechanism for TRAIL's tumour selective effect is largely unknown. We identified the cytoskeleton proteins non-muscle myosin heavy chain IIa, IIb (NMHCIIa, NMHCIIb), myosin regulatory light chain (MLC2) and ß-actin as novel DR-interactors. An initially weak and TRAIL-induced abrogation of NMHCII/DR interaction correlated with efficient DISC formation in tumour cells. In contrast, a robust NMHCII/DR interaction that was sustained upon TRAIL stimulus was accompanied by incomplete DISC arrangement. Weakening the NMHCII/DR interaction in normal cells using chemical inhibitors enhanced TRAIL-induced apoptosis. Intriguingly, siRNA-mediated NMHCIIa-but not NMHCIIb depletion potently released the TRAIL resistance block in normal cells and influenced DISC composition. Reduced NMHCII/DR interaction in transformed cells was characterised by diminished MLC2 phosphorylation levels and altered protein expression levels of upstream regulatory kinases. Our results suggest that normal cell resistance to TRAILapoptosis is based on the interaction of cytoskeleton components with the DR that is impaired upon transformation. Since NMHCII function in cell adhesion and migration, it will be interesting to study possible roles of the NMHCII/DISC interaction in cell detachment in addition to modulating TRAIL sensitivity; moreover this link may provide clues as to the cause of TRAIL resistance in some cancers.

keywords: TRAIL, DISC, normal cells, apoptosis, tumour-selective, cytoskeleton, non-muscle myosin heavy chain IIa, NMHCIIa

Figure 1 :

 1 Figure 30: GRB2 and RAF1 binding to DR5 and effects of GRB2 siRNA knockdown on cell proliferation, TRAIL apoptosis and TRAIL--induced caspase cleavage in normal BJ cells .................................................................................................................................................125 SUPPLEMENTARY Figure 31: DR5--, DR4--and CASP8--DISC formation of normal BJ and transformed BJELR cells and the interaction with NMHCIIa and NMHCIIb...................................................................127 Figure 32: Interaction of NMHCIIa and NMHCIIb with DR5 of transformed HA1ER cells.............128 Figure 33: Identification of novel TRAIL DR5--DISC interactors by in--solution digestion of whole DR5--DISC--IP eluates of normal BJ and transformed BJELR cells followed by mass spectrometry ...............................................................................................................129 Figure 34: Apoptosis induction at high levels of myosin light chain kinase (MLCK) inhibitor ML7 in normal BJ and transformed BJELR cells. ...........................................................130 Figure 35: Actin stress fiber formation in normal BJ and transformed BJELR cells .........................130 WORK IN PROGRESS Figure 36: Fragmentation of NMHCIIa, NMHCIIb and ßACT during rhTRAIL apoptosis and NMII/DR5 interaction in transformed BJELR cells with pan--caspase inhibition ...............135 Figure 37: Caldesmon 1 and calmodulin co--purify with DR5 in normal BJ and transformed BJELR fibroblasts.................................................................................................................137 Figure 38: DR5 internalisation and DR5 surface levels upon blebbistatin treatment and NMHCIIa siRNA knockdown in normal BJ cells.................................................................................139 Figure 39: NMII/DR5 interaction in pre--transformed BJEL and transformed BJELR cells ...........140 DISCUSSION & PERSPECTIVES Figure 40: Proposed model of the NMIIa/DR interaction regulating the cFLIP(L)--mediated switch between morphogenic and apoptotic TRAIL signalling in normal (N) and transformed (T) cells ....................................................................................................................................149

  [http://fda.gov, http://clinicaltrials.gov]. One example targeting for instance cancer cells self--sufficiency in growth signals is the development of monoclonal antibodies promoting internalisation and degradation of epidermal growth factor receptors (EGFR), which are transmembrane tyrosine kinase receptors regulating cell proliferation and survival. In cancer cells EGFR are commonly overexpressed and either signal autonomously via ligand secretion through the cancer cell itself, or they even signal in absence of the ligand. Inactivating EGFR signalling thus inhibits proliferative signals required by the cancer cell and induces its apoptosis [reviewed by Shawver et al 2002, Hynes and Lane 2005].

Figure 2 :

 2 Figure 2: Hallmarks of cancer as defined by Hanahan and Weinberg 2000. Targeted strategies for cancer therapy are under development that specifically aim at one or several of these features. The tumour mass in the middle was taken from [Bode and Dong 2009].

Figure 3 :

 3 Figure 3: Extrinsic and intrinsic apoptosis pathways. The extrinsic pathway is activated by binding of TNF cytokine family members (here: TRAIL) to their death receptors (here: DR5, DR4), leading to the formation of the death inducing signalling complex (DISC) (yellow). The intrinsic apoptosis pathway is stimulated by severe cell stress, such as DNA or cytoskeletal damage. Unlike the intrinsic pathway, the extrinsic pathway operates independently of tumour suppressor p53 and can activate the mitochondrial pathway to amplify the apoptotic signal. The figure is based on the idea of [White--Gilbertson et al 2008, Newsom--Davis et al 2009].

  al 2006] that connects the extrinsic apoptosis pathway to the mitochondria. Truncated BID (tBID) translocates to the mitochondrial membrane where it promotes aggregation of the BCL2 protein family members BAX and BAK leading to mitochondrial outer membrane permeabilisation (MOMP), as described in the following section.The intrinsic apoptosis pathway. The signal triggering the intrinsic apoptosispathway is elicited inside the cell and comprises stimuli such as DNA or cytoskeletal damage caused by agents such as ionising radiation, heat shock, toxins, cell detachment, bacterial or viral infection and oncogenic transformation. Other stimuli triggering this cascade during development, such as growth factor deprivation or decline in tissue morphogen concentrations as well as during prolonged stress of the endoplasmatic reticulum due to accumulation and aggregation of unfolded proteins [reviewed by[START_REF] Ashkenazi | Directing cancer cells to self--destruct with pro--apoptotic receptor agonists[END_REF]]. The efflux of effector proteins during MOMP represents a crucial step in the intrinsic apoptosis pathway that is mainly regulated by members of the BCL2 protein family, which are divided into anti--apoptotic (e.g. A1, BCL2, BCLW, BCLXL, MCL1, promote survival) pro--apoptotic (e.g. BAX, BAK, BCLXS, BOK, activate apoptotic effector pathway) and BH3--only death proteins (e.g. BAD, BID, BIK, BIM, BMF, HRK, NOXA, PUMA, initiate apoptosis) [reviewed by[START_REF] Kelly | The role of Bcl--2 and its pro--survival relatives in tumourigenesis and cancer therapy[END_REF]. Whilst anti-apoptotic and BH3--only BCL2 proteins regulate the interaction of BCL2 proteins amongst each other, the pro--apoptotic BCL2 family members BAX and BAK insert into the mitochondrial membrane to form pores, leading to MOMP. This enables the release of apoptogenic factors such as cytochrome c, inhibitor of apoptosis (IAP), second mitochondria-derived activator of caspase/direct IAP binding protein with low pI (SMAC/DIABLO) and omi stress--regulated endoprotease/high temperature requirement protein A2 (OMI/HTRA2). After its release, cytochrome c binds to apoptotic protease-activating factor 1 (APAF1) to form the apoptosome, which recruits initiator procaspase--9 (CASP9) promoting its self--activation. Activated CASP9 cleaves the downstream effector proteases CASP3 and CASP7, which cleave various intracellular substrates leading to cell death. Notably, CASP9, CASP3 and CASP7 can be inhibited by direct binding of IAP family members (XIAP, cIAP1, cIAP2, IAP2, MLIAP, NAIP, survivin and apollon) to their active site or by enhancement of CASP proteasome degradation. In turn, IAP proteins can be inactivated intrinsically by SMAC/DIABLO and OMI/HTRA2

  and Huntington's disease[START_REF] Sanchez Mejia | Caspases in Huntington's disease[END_REF] or the augmentation of apoptosis in self--reactive T cells in autoimmune diseases [reviewed by[START_REF] Prasad | Apoptosis and autoimmune disorders[END_REF] and in malignant cells in cancer [reviewed by[START_REF] Gerl | Apoptosis in the development and treatment of cancer[END_REF] Vaux 2005, Ashkenazi 2008]. Since the human body on average generates 60 billion cells per day, an equal number of cells must die to maintain cell homeostasis [reviewed by[START_REF] Cotter | Apoptosis and cancer: the genesis of a research field[END_REF]. Defective or inefficient apoptosis is therefore disastrous and represents one out of six acquired hallmarks of cancer cells (Preamble Figure2)[START_REF] Hanahan | The hallmarks of cancer[END_REF]. Many cancers that evade from apoptosis carry inactivating mutations in genes such as the transcription factor tumour suppressor protein 53 (p53) [reviewed by[START_REF] Kinzler | Lessons from hereditary colorectal cancer[END_REF], which is instrumental in the activation of the intrinsic apoptosis pathway. For instance, upon DNA damage, p53 accumulates, leading to the expression of cell cycle arresting proteins.

Figure 4 :

 4 Figure4: Classification of TNF superfamily members and protein sequence homologies of TRAIL versus FASL and TNFα, respectively. (A) Overview of TNF superfamily members classified according o their roles in inflammation, cellular proliferation, apoptosis and morphogenesis. All TNF superfamily members exhibit pro-inflammatory activity initiating the transcription factor nuclear factor kappa--B (NFKB) (red circle). Certain members display proliferative activity on hematopoietic cells triggering various mitogen-activated kinases (MAPK) (light blue). Other representatives, such as tumour necrosis factor alpha (TNFα), FAS ligand (FASL), and TNFα-related apoptosis inducing ligand (TRAIL, encircled in red) control apoptosis (dark blue) and regulate morphogenesis and differentiation (green). The figure is taken and modified from[START_REF] Aggarwal | Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey[END_REF]. (B) Graphic summary for the protein sequence alignments of human TRAIL and FASL (upper black and green lane) and human TRAIL and TNFα (lower black lane) using NCBI--BLAST Align[START_REF] Altschul | Basic local alignment search tool[END_REF].

  aiming to enhance TRAIL's biological activity [reviewed by[START_REF] Schneider | Apoptosis induced by death receptors[END_REF]. Thus, the structure of TRAIL is essential for TRAIL's selectivity to tumour versus normal cells. The indispensability of a native and untagged TRAIL structure for TRAILs tumour--selectivity and safety has also been proven in vivo during preclinical studies. Initially observed TRAIL--toxicity towards mice xenografted with human hepatocytes was clearly related to exogenously added TRAIL amine tags[START_REF] Hao | TRAIL inhibits tumor growth but is nontoxic to human hepatocytes in chimeric mice[END_REF], reviewed by Ashkenazi et al 2008]. Therefore, next to humanised antibodies specifically targeting DR5 or DR4, only one rhTRAIL variant is accepted and tested in clinical trials. This version is based on the human protein's natural sequence (extracellular amino acid residues 114--281) and does not carry an exogenous tag. The exact same untagged rhTRAIL version is used in this study.

Figure 5 :

 5 Figure 5: Current model for TRAIL signalling. For details see text. Reprinted by permission from Macmillan Publishers Ltd: [Oncogene] (Gonzalvez and Ashkenazi 2010), copyright (2010).

  FAS--apoptosis[START_REF] Schneider--Brachert | Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles[END_REF][START_REF] Lee | The role of receptor internalization in CD95 signaling[END_REF] and TRAIL binding induces rapid internalisation of both DR5 and DR4 via clathrin--dependent [Austin et al 2006] and independent [Kohlhaas et al 2007] pathways, inhibition of endocytosis does not prevent FADD or CASP8 recruitment in various type I signalling cells [Austin et al 2006, Kohlhaas et al 2007]. Thus, unlike TNFR1 and FAS, DR5 and DR4 do not require TRAIL--induced internalisation for DISC formation and apoptosis at least in type I cells [Austin et al 2006, Kohlhaas et al 2007]. If receptor internalisation is required in type II signalling cells is unknown. Apart from TRAIL--induced internalisation, DR5 and DR4 can undergo constitutive clathrin--dependent endocytosis as part of a desensitising mechanism in leukemia, colon and breast cancer cells [Jin et al 2004, Cheng et al 2006, Zhang and Zhang 2008].

  that one FADD molecule binds to one receptor recruiting one molecule of CASP8, CASP10 or cFLIP and that single DISCs are clustered by receptor [(FAS)[START_REF] Scott | The Fas--FADD death domain complex structure unravels signalling by receptor clustering[END_REF] and/or FADD interactions (Figure5) [(FAS)[START_REF] Carrington | The structure of FADD and its mode of interaction with procaspase--8[END_REF]. Now, this model appears to require a complete reassessment, since recent quantitative mass spectrometrical analyses have shown that FADD is consistently substoichiometric relative to FAS, DR5 and DR4 or DED--only proteins[START_REF] Schleich | Stoichiometry of the CD95 Death--Inducing Signaling Complex: Experimental and Modeling Evidence for a Death Effector Domain Chain Model[END_REF][START_REF] Dickens | A Death Effector Domain Chain DISC Model Reveals a Crucial Role for Caspase--8 Chain Assembly in Mediating Apoptotic Cell Death[END_REF]. Strikingly, there was up to 9--fold more CASP8 than FADD in the TRAIL--DISC and on average only 1 FADD molecule per 3 receptors detected. Therefore, a DISC structure has been proposed, which relies on the formation of CASP8 chains (Figure6). Indeed, CASP8, CASP10 and cFLIP can interact with each other via their second DED domain (DED2)[START_REF] Irmler | Inhibition of death receptor signals by cellular FLIP[END_REF][START_REF] Wang | Caspase--10 is an initiator caspase in death receptor signaling[END_REF] and mutating a specific motif within the CASP8 DED2 prevented chain formation, CASP8 activation and TRAIL--apoptosis [Dickens et al 2012]. Interestingly, Dickens et al could not detect any CUL3 in the native DISC of type I (BJAB) or type II signalling (Z138, Jurkat) cells and detected only very low CUL3 ratios in the DISC of type II HeLa cells. Therefore, the relevance of CUL3--mediated CASP8 ubiquitinylation and subsequent DISC localisation into ubiquitin--rich foci remains to be established (Figure 5). Moreover, mass--spectrometry analysis of the TRAIL DISC identified CASP10 only in type I signalling BJAB at much lower abundance than CASP8, from Dickens et al concluded that CASP10 does not have an obligatory role in TRAIL--DISC signalling. Nonetheless, one has to consider that the timepoint or target of the immunoprecipitaton was inappropriate to detect CUL3 and CASP10 or that DISC composition simply depends on the kind of stimulus applied to the receptors, as it was indicated for FAS signalling [Chaigne--Delalande et al 2009]. Finally, Dickens et al noted that a structural CASP8 rearrangement would be required to unmask CASP8 DED2 to facilitate CASP8 chain formation at DR5 and DR4. Amongst the options for how this structural rearrangement could be accomplished, it was hypothesised that also the cytoskeleton might play a role, which was not further specified [Dickens et al 2012].

  -apoptotic TRAIL-DISC components. Several proteins oppose TRAIL-apoptotis signalling at the level of the DISC, interfering with efficient initiator CASP activation in the DISC and/or activating pro--survival pathways (see also 'TRAIL non-apoptotic signalling and complex II formation'). Amongst those proteins are 3 further TRAIL receptors, decoy receptor 1 and 2 (DcR1, DcR2) [Degli--Esposti et al 1997a, Degli--Esposti et al 1997b] and soluble osteoprotegerin (OPG), to which TRAIL binds besides DR5 and DR4. Structurally DcR1 and DcR2 resemble DR5 and DR4, but whilst DcR1 is a glycosylphosphatidylinositol (GPI)--linked receptor that lacks the entire intracellular domain inclusively the DD, DcR2 possesses a truncated, dysfunctional DD (Figure 3). Therefore, TRAIL binding to DcR1 or DcR2 fails to elicit apoptosis. The mode of how DcR1 and DcR2 interfere with DR5-- and DR4--stimulated apoptosis in the DISC appears to differ. Whilst DcR1 localises to lipid rafts where it captures TRAIL away from the DISC, DcR2 competes with DR4 for DR5 binding upon TRAIL stimulus inhibiting efficient recruitment, cleavage and activation of initiator CASP at the DISC [Merino et al 2006]. Notably, the decoy function of DcR1 and DcR2 has so far been shown in overexpression studies only [Degli--Esposti et al 1997a, Degli--Esposti et al 1997b, Merino et al 2006].

Figure 7 :

 7 Figure 7: Scheme of Hahn and Weinberg's stepwise transformation model. Normal cells (here: human foreskin fibroblasts (BJ) or human embryonic kidney cells (HEK)) were gradually transformed by introducing the human telomerase catalytic subunit (HTERT), simian virus 40 early region (SV40ER) and a constitutively active HRAS mutant (HRASG12V). Whilst HTERT maintains telomere length and confers replicative immortality to BJ cells, HEK cells require additional SV40ER--encoded elements to reach immortality. Obtained transformed cells (here: BJELR or HA1ER) display cancer cell features like unlimited proliferation, anchorage--independent growth and tumour formation in vivo and are sensitive to TRAIL--induced apoptosis.

Figure 9 :

 9 Figure 9: Regulation of the actin cytoskeleton motility by RHO--family GTPases. For details see text. Reprinted by permission from Macmillan Publishers Ltd: [EMBO Reports] (Ahmadian et al 2002), copyright (2002).

Figure 10 :

 10 Figure10: Localisation of actin filaments within the cell. The cortical actin is a loosely organised network of actin filaments underneath the plasma membrane. Actin stress fibers can span the whole cell. Actin filaments can also be organised to form cell extensions such as podosomes, lamellipodia, filopodia, microvilli and membrane ruffles. Myosins (green) confer contractability to the actin fibers and regulate their assembly. Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Microbiology][START_REF] Taylor | Subversion of the actin cytoskeleton during viral infection[END_REF](Taylor et al ), copyright (2011)).

  by[START_REF] Ostap | Tropomyosins as discriminators of myosin function[END_REF]. Another study revealed an SV40--mediated decline in α--actinin expression levels[Glück et al 1993], a protein that cross--links and anchors actin fibers to a variety of intracellular structures.Interestingly, recent studies compared the proteomes of BJ stepwise system cells at each step of the genetic modification towards full transformation[Pütz et al 2010, Pütz et al 2012]. Most drastic alterations in protein expression levels were revealed upon SV40ER introduction. Surprisingly, in the first study only 6 cytoskeletal proteins were found deregulated, of which in turn only one (ßACT) is an actin cytoskeleton protein[Pütz et al 2010]. In a consecutive study the same authors applied a more powerful SILAC--based 2D--DIGE approach and found the actin filament destabilising gelsolin and CALD (next to 3 further actin cytoskeleton proteins) downregulated upon SV40 introduction[Pütz et al 2012]. The contribution of the single SV40ER proteins to the drop in tropomyosin, gelsolin or CALD expression is not clarified. Since SV40 LT localises to the cells nucleus to inhibit pRB, p107, p130 and p53, one could speculate that SV40--associated downregulation of tropomyosin, gelsolin or CALD is mediated by LT. However, only little is known about LT action on the actin cytoskeleton or actin--related proteins and the introduction of LT alone did not induce alterations in actin fibers of rat embryo fibroblasts[START_REF] Graessmann | Simian virus 40 small--t protein is required for loss of actin cable networks in rat cells[END_REF]. Contrary, SV40 ST immediately engaged the actin cytoskeleton during cell transformation and its single expression sufficed to induce loss of actin filaments[START_REF] Graessmann | Simian virus 40 small--t protein is required for loss of actin cable networks in rat cells[END_REF]. Via the inhibition of PP2A, ST protein expression resulted in the rearrangement of F--actin networks in epithelial MDCK cells [Nunbhakti--Craig et al 2003]. Actin cytoskeleton rearrangements in ST expressing cells included RAC1--induced membrane ruffling and formation of lamellipodia, CDC42-initiated formation of filopodia, and loss of RHOA--dependent stress fibres, which coincided with increased RAC1 and CDC42 expression levels and decreased RHOA levels in those cells (Figure 11) [Nunbhakdi--Craig et al 2003]. Importantly, expression of an ST mutant unable to interact with PP2A failed to induce the marked disorganization of F-actin observed in MDCK cells expressing wildtype ST [Nunbhakdi--Craig et al 2003].

[

  [START_REF] Arroyo | Involvement of PP2A in viral and cellular transformation[END_REF][START_REF] Sontag | Regulation of cell adhesion by PP2A and SV40 small tumor antigen: an important link to cell transformation[END_REF][START_REF] Virshup | From promiscuity to precision: protein phosphatases get a makeover[END_REF][START_REF] Basu | PP2A in the regulation of cell motility and invasion[END_REF]. It should nevertheless be noted that MYC is directly dephosphorylated by PP2A, and inhibition of PP2A activity by ST stabilised MYC[START_REF] Yeh | A signalling pathway controlling c--Myc degradation that impacts oncogenic transformation of human cells[END_REF]. Although a recent publication using less differentiated, human mammosphere--derived epithelial cells indicated that the introduction of only hTERT and SV40ER was sufficient for tumourigenic conversion[START_REF] Paranjape | Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties[END_REF], cancerous transformation of differentiated human mammary epithelial cells usually requires additional activation of strong oncogenic signalling.

Figure 11 :

 11 Figure 11: Influence of SV40 ST on RHO--family GTPase--mediated modelling of the actin cytoskeleton. SV40 ST upregulates the RHO--GTPases RAC1 and CDC42 and blocks RHOA--mediated signalling. Reprinted (with modifications) by permission from Macmillan Publishers Ltd: [Nature Reviews Microbiology] (Taylor et al 2011), copyright (2011).

  the intrinsic apoptosis pathway. Evidence for an active role of the actin cytoskeleton to induce or to inhibit apoptosis are manifold and refer to actin itself as well as to actin--binding proteins. Initially it was observed that stabilisation of actin filaments (F--actin) by drug--mediated inhibition of actin depolymerisation (jasplakinolide) induced increased CASP3 activation and mitochondria--dependent apoptosis in different transformed and normal human cells [Suria et al 1999, reviewed by Franklin--Tong and Gourlay 2008]. Accordingly, disruption of the actin cytoskeleton (cytochalasin D) inhibited nitric oxide--induced apoptosis in articular chondrocytes [Kim et al 2003], indicating that stabilisation of F--actin filaments initiated apoptosis. However, partially in the same cell types apoptosis could also be triggered by the destabilisation of F--actin structures using actin--depolymerising agents (cytochalasin D, latrunculin B, sphinxolide) [reviewed by Franklin--Tong and Gourlay 2008], which in some cases was shown to involve mitochondrial cytochrome c release [Suria et al 1999, Paul et al 2006]. It is not known under which conditions F--actin stabilisation or depolymerisation influences entry into apoptosis and there is only one mechanistic explanation for actin alteration--stimulated activation of BCL2 proteins available. A pro--apoptotic BCL2 member (BMF) was found to be sequestered with actin-associated myosin V motors and was released from the actin cytoskeleton upon cell detachment or cytochalasin D treatment resulting in mitochondrial--dependent apoptosis [Puthalakhat et al 2001].

  addition to actin itself, several actin--binding proteins have been studied in their relation to mammalian cell apoptosis. These include cofilin (F--actin depolymerising), thymosin β (globular actin sequestering), coronin 1 (F--actin branching), filamin (F--actin branching), gelsolin (actin severing and F--actin capping), tropomyosin (actin stabilising) and myosin II (actin filament contraction or bundling)[reviewed by[START_REF] Tong | A role for actin in regulating apoptosis/programmed cell death: evidence spanning yeast, plants and animals[END_REF][START_REF] Leadsham | Apoptosis and the yeast actin cytoskeleton[END_REF][START_REF] Desouza | The actin cytoskeleton as a sensor and mediator of apoptosis[END_REF]. For reasons of complexity, the role of actin--branching proteins (coronin, filamin) in apoptosis is not introduced here and can be read elsewhere[START_REF] Desouza | The actin cytoskeleton as a sensor and mediator of apoptosis[END_REF].Likewise it is unknown at which cell state F--actin stabilisation or depolymerisation induces apoptosis, data for certain actin filament severing, capping and depolymerising proteins (cofilin) exist that support both, a protective as well as an apoptosis--promoting function [reviewed by[START_REF] Desouza | The actin cytoskeleton as a sensor and mediator of apoptosis[END_REF]. Interestingly however, is that actin depolymerising cofilin was shown to translocate to the mitochondrial membrane in response to the kinase inhibitor staurosporin, which resulted in cytochrome c release. Expression of a phosphorylated and inactive cofilin mutant abolished this mitochondrial targeting of cofilin and apoptosis, emphasising the requirement for active de--phosphorylated cofilin in apoptosis[START_REF] Chua | Mitochondrial translocation of cofilin is an early step in apoptosis induction[END_REF]. Cofilin is dephosphorylated by PP2A, which lead to decreased F--actin depolymerisation rates inhibited of actin dynamics and reduced migration of human adenocarcinoma A549 cells, whilst chemical PP2A inhibition (calyculin) restored motility[START_REF] Oleinik | ALDH1L1 inhibits cell motility via dephosphorylation of cofilin by PP1 and PP2A[END_REF].

  were capable to induce apoptosis via the nuclear localization and activation of DNAse I [Chhabara et al 2005].Finally, also several tropomyosin isoforms were evidenced to regulate apoptosis, mainly by coordinating the interaction of other actin--binding proteins with actin [reviewed by[START_REF] Desouza | The actin cytoskeleton as a sensor and mediator of apoptosis[END_REF]. Notably, tropomyosin 5 (TM5) expression in neuroepithelial cells was found to induce the recruitment of non--muscle myosin IIa (NMIIa) to stress fibers, resulting in reduced cell migration[START_REF] Bryce | Specification of actin filament function and molecular composition by tropomyosin isoforms[END_REF]. Downregulation of another isoform, tropomyosin 1 (TM1), in mammary epithelial carcinomas was correlated with an increased resistance to detachment--induced apoptosis (anoikis)[Raval et al 2003]. Restoring Tm1 expression in cultured mammary carcinoma cell lines (MCF7 and MBAMB231) led to the generation of distinct actin stress fibers and re--sensitised cells to anoikis[START_REF] Bharadwaj | Resensitization of breast cancer cells to anoikis by tropomyosin--1: role of Rho kinase--dependent cytoskeleton and adhesion[END_REF]. Therefore, TM1

  it was indicated that actin filament disruption with jasplakinolide treatment of normal epithelial cells induced apoptosis and DISC formation at the FAS receptor in the absence of FASL[START_REF] White | Initiation of apoptosis by actin cytoskeletal derangement in human airway epithelial cells[END_REF]. Contrary, in type I SKW6.4 and H9 lymphoblastoma cells FADD recruitment to FAS, as well as FAS internalization was concluded to require intact actin fibers [Algeciras--Schimnich et al 2002], although the applied disruption of actin fibers with latrunculin A at least in H9 cells lead to reduced FADD expression and total FAS expression was not controlled. Evidence for an actin cytoskeleton--dependent regulation of TNF (and -related?) protein expression exists, since F--actin disruption using cytochalasin D blocked LPS--induced expression of TNFα in monocytes[START_REF] Rosengart | The actin cytoskeleton: an essential component for enhanced TNFalpha production by adherent monocytes[END_REF] and RAC1 siRNA depletion reduced FASL mRNA levels in Jurkat cells[START_REF] Ramaswamy | Cutting edge: Rac GTPases sensitize activated T cells to die via Fas[END_REF]. In line with Algeciras--Schimnich study, in FASL--sensitive, type--I activated CD4+ T lymphocytes[START_REF] Parlato | CD95 (APO--1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway[END_REF] and HIV-specific CD8+ T--cells[Petrovas et al 2007a] actin filament disruption with cytochalasin D and/or latrunculin A reduced percentages of FAS--induced annexin V--positive cells[START_REF] Parlato | CD95 (APO--1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway[END_REF], Petrovas et al 2007a] and disordered the polarised FAS distribution

[

  [START_REF] Hebert | Rho--ROCK--dependent ezrin--radixin-moesin phosphorylation regulates Fas--mediated apoptosis in Jurkat cells[END_REF]. Finally, the question of whether in type II signalling cells ezrin loss influences FAS--apoptosis or not was not convincingly solved by the Kuo study, as ezrin knockdown efficiencies in type II cells appear lower than in type I cell and loading controls seem to fluctuate. Interestingly, in type I signalling H9 lymphoblastoma cells time--course analysis of the FAS--DISC demonstrated that with increasing time of FASL-stimulation and DISC formation, ezrin was lost from FAS. In addition, ezrin KD enhanced FAS--apoptosis[START_REF] Kuo | Ezrin is a negative regulator of death receptor--induced apoptosis[END_REF]. Contrary, in type II Jurkat cells the interaction of ezrin (and moesin) with FAS and ezrin and moesin phosphorylation levels increased over time of FASL treatment. Knockdown of either ezrin or moesin did not inhibit FADD or CASP8 association with FADD, but decreased CASP8 cleavage and FAS--mediated apoptosis[START_REF] Hebert | Rho--ROCK--dependent ezrin--radixin-moesin phosphorylation regulates Fas--mediated apoptosis in Jurkat cells[END_REF]. These results indicate that the FASL--induced ezrin regulation and maybe even basal ezrin activity seem to fundamentally differ in type I and type II signalling cells. ERM proteins and their interaction with other proteins are regulated by phosphorylation/dephosphorylation events. ERM proteins are activated by RHOA/ROCK--mediated phosphorylation, whilst they are inactivated via constitutively active RAC1--stimulated dephosphorylation. Treatment of type II signalling Jurkat cells with aplidin, an actin cytoskeleton reorganising agent that acts via the activation of RAC1 [Gonzalez--Santiago et al 2006], lead to the clustering of FAS, DR5, FADD, CASP8, CASP10, ezrin, moesin, RHOA and RHO--GDP dissociation inhibitors (RHO--GDI) into lipid rafts and FAS--dependent apoptosis. Pretreatment of cells with actin cytoskeleton disrupting cytochalasin B or jasplakinolide in turn prevented aplidin--induced apoptosis and FAS--clustering into 'caps' [Gajate and Mollinedo 2005]. Similarly, in type I signalling T cell receptor (TCR)--stimulated CD4+ T cells were sensitive to FASL--apoptosis, which correlated with TCR--induced FAS translocation to lipid rafts and ERM protein dephosphorylation. RAC1 knockdown reduced FAS antibody--induced apoptosis in TCR-activated T cells [Ramaswamy et al 2007]. Thus, actin and actin--linking proteins regulate aplidin-- or TCR-- induced translocation of DISC components into lipid rafts via the activation of RAC1 and in this way it might prone cells for FAS--apoptosis. Of note, also RHOA signalling and the actin--remodelling proteins filamin and cofilin are used in actin--dependent receptor clustering processes [Jimenez--Baranda et al 2007, Yoder et al 2008]. One model for FAS resistance was proposed based on the lack of FAS internalisation as a result of a defect in in the regulation of ezrin. In fibroblasts and fetal liver cells it was shown that FAS stimulation induced RAF1/ROCK2 clusters. Thereby, RAF1 which interacts directly with cFLIP [Kataoka et al 2000], restrained ROCK2 activity and therefore ezrin phosphorylation required for FAS/actin interaction, FAS internalisation and hence FAS--DISC formation [Piazzolla et al 2005]. Since RAF1, which plays an essential role in maintaining the cytoskeleton organisation and thus in cell polarity and migration [Ehrenreiter et al 2005], counteracts apoptosis additionally through inhibition of the proapoptotic kinases ASK1 and MST2 [reviewed by Matallanas et al 2011], it will be interesting to decipher the contribution of each these kinases in FASL--apoptosis. Concludingly, it remains to point out that even though no co-immunoprecipitation data of actin or ezrin for the other TNF death receptors TNFR1, DR5 and DR4 exist, an association of DR5 and DR4 with the cytoskeleton in general or with actin and ezrin is often described in literature. Given the current unclear picture for the actual role of actin and ezrin in FAS--signalling, the role of these proteins in TRAIL-signalling remains to be elucidated. Less contradictory, probably because not as much studied examples for the regulation of TNF cytokine--induced apoptosis by cytoskeletal proteins are for instance thymosin ß, which enhanced TNFα--induced apoptosis when overexpressed in normal fibroblasts [Hall et al 1995], or gelsolin which inhibited FAS--mediated apoptosis of overexpressed in type II Jurkat cells [Ohtsu et al 1997]. Importantly, the actin--binding and crosslinking non--muscle myosin II (NMII), which conveys contractile properties to F--actin, has been marginally implicated in the extrinsic apoptosis regulation. In case of TNFα--signalling, NMII activity, measured at the level of MLC2 phosphorylation, was linked to the transport of TNFR1 from the trans--Golgi--network to the plasma membrane. If NMII were activated overexpressing the NMII regulatory kinase MLCK in normal epithelial Madin--Darby canine kidney (MDCK) cells, or treating cells with ionomycin or endotoxin, cell surface levels of TNFR1 transport was increased. Moreover, MLCK overexpression accelerated TNFα--induced apoptosis, whilst a dominant--negative (DN) MLCK version delayed apoptosis [Jin et al 2001]. Notably, apoptosis was quantified as the number of viable cells still attached after TNFα--treatment and total apoptosis outcomes were not reduced by DN--MLCK expression. Another study conducted in normal pulmonary endothelial cells reported that NMII activity promoted TNFα-apoptosis and observed a TNFα--induced increase in MLC2 phosphorylation levels. Inhibition of MLCK (ML7) or ROCK (Y--27632) attenuated TNFα--induced stress fiber formation as well as relative percentages of apoptotic cells positive for annexin V and percentages of CASP8 activity [Petrache et al 2001]. Distinguishing between the two NMII isoforms NMIIa and NMIIb in HeLa cells, immunoprecipitation of NMIIb revealed co--purification of clathrin. Moreover, siRNA--mediated NMIIb depletetion resulted in consistent TNFR1 surface levels upon TNFα--stimulus as well as impaired CASP8 cleavage. Therefore, NMIIb was concluded to function in TNFR1 internalisation. Protein depletion of NMIIa neither reduced levels of TNFR1 internalisation nor CASP8 activation. The apoptosis--promoting function of NMIIb was deduced to occur independent of the actin myosin contractility, as neither NMII (blebbistatin) nor actin depolymerisation (cytochalasin D) inhibited CASP8 or CASP3 cleavage. Even though it was attempted, TNFR1 could not be co--immunoprecipitated with NMIIb [Flynn and Helfman 2010]. Similar to TNFα signalling, FAS--receptor clustering appeared to be actin-but not NMII--dependent, as the myosin inhibitor 2--3--butanedione monoxime showed no effect in suspension cells (SKW6.4 B cell lymphoma) [Algeciras--Schimnich et al 2002]. Non-muscle myosins II (NMII) Myosins are actin--based ATP--driven motor proteins of which there are as yet 12 classes identified in humans (Figure 13). Most of what is known about myosin function and regulation was studied on class II myosins, which are unique compared to other myosins for their capacity to form filaments [reviewed by Sellers 2000]. Members of the myosin II family are hexameric proteins consisting of 2 heavy chains, 2 essential (ELC) and 2 regulatory light chains (MLC2). Myosins II are distinguished in dependence of their heavy chain, encoded by 13 different genes. The sequence of the motor domain, located in the head domain of the heavy chain, subdivides myosins II into skeletal/cardiac and muscle/non--muscle isoforms. With few exceptions, the single muscle and the 3 different non--muscle heavy chains are ubiquitously expressed in muscle and non--muscle cells, although to varying concentration levels [reviewed by Eddinger and Meer 2007]. Contrary to the heavy chains, nothing is known about a functional difference of the 3 distinct MLC2 isoforms expressed in non--muscle cells. The 3 human MLC2 genes MYL9, MYL12A and MYL12B encode smooth muscle MLC2 (smMLC2), non--muscle--like MLC2 (nmlMLC2) and non--muscle MLC2 (nmMLC2), whose protein sequences are highly homologue, so that no antibody exists that can distinguish between these isoforms [Gerrits et al 2012]. The non--muscle myosins II (NMII), which are implicated in such diverse processes as adhesion, migration, vesicle transport from the Golgi, receptor capping [Jimenez--Baranda et al 2007, Yoder et al 2008, reviewed by Maravillas--Montero and Santos--Argumedo 2012], endocytosis [Rey et al 2007], apoptosis (as described in a previous section) and virus entry [Arii et al 2010, reviewed by Taylor et al 2011] and egress [van Leeuwen et al 2002], will be described in more detail in this chapter.

Figure 13 :

 13 Figure 13: Phylogenetic tree of the human myosin family. Structural features of the single myosin domains are indicated as illustrated in the legend below the tree. Class II myosins are unique for their filament formation capacity and are subdivided into muscle and non--muscle forms according to their motor domain sequence. The figure is taken from [Peckham and Knight 2009].

Figure 14 :

 14 Figure 14: Non--muscle myosin II (NMII) molecule structure and heavy chain (NMHCII) protein homology. (A) Schematic structure of an NMII molecule indicating the main NMHCII (yellow) domains. (B) Protein sequence alignment of the human heavy chain isoforms NMHCIIa, IIb and IIc. (C) Graphical summary of the protein sequence alignments of human NMHCIIa versus IIb (upper red lane) and human NMHCIIa versus IIc (lower red lane). For alignments in (B) and (C) NCBI--BLAST Align [Altschul et al 1990] was used.

Figure 18 :

 18 Figure18: NMII filament disassembly is promoted by non--covalent binding of regulatory proteins or NMHCII phosphorylation. Proteins like S100A4 (blue) bind to the non--helical NMHCII tail, which is essential for NMII bipolar filament formation (left). Phosphorylation (yellow) of NMHCII by various kinases (for instance PAK1) (red) destabilises the electrostatic interactions between the coiled--coil NMHCII rods. The figure is based on the idea of[START_REF] Clark | Myosin II and mechanotransduction: a balancing act[END_REF].

o

  By applying commonly used immunoprecipitation, Western blot and mass spectrometry methods, the DISC composition of TRAIL--resistant normal cells and TRAIL--sensitive transformed cells was defined. The individual subgoals included in this thesis were: o To overexpress, affinity--purify and biologically validate the extracellular, soluble portion of untagged recombinant human TRAIL (amino acids 114--281). o To immunoprecipitate and analyse the native DISC composition of normal and transformed cells using antibodies directed against single DISC components. To identify differentially represented factors in the DISC of TRAIL-insensitive and TRAIL--sensitive cells by mass spectrometry. o To scrutinise identified factors regarding their impact on tumour--selective apoptosis using pharmacological inhibition and knockdown strategies.MATERIALS & METHODSAntibodies. For immunoprecipitation (IP) anti--CASP8 p20 (C--20) (Santa Cruz), anti--DR4 (R&D), anti--DR5 (Diaclone or R&D), anti--immuneglobulin G (IgG) (human IgG, in--house purification) and anti--TRAIL (R&D) were used. For Western blot analysis anti--ßACT (ACTBD11B7) (Santa Cruz), anti--phospho BAD (Ser112) (Cell Signaling), anti--Bid (Cell Signaling), anti--CALD (Sigma), anti--CALM (Upstate), anti--cleaved CASP3 (ASP175) (Cell Signaling), anti--cleaved CASP7 (Asp198) (Cell Signaling), anti--CASP8 (1C12) (Cell Signaling), anti--CASP9 (Cell Signaling), anti--CASP10 (MBL), anti--cFLIP, anti--DcR1 (Chemicon), anti--DcR2 (Chemicon), anti--DR4 (Chemicon), anti--DR5 (Sigma), anti--FADD (BD Laboratories), anti--MEK1/2 (Cell Signaling), anti--phospho MEK1/2 (Ser217/221) (Cell Signaling), anti--MLC2 (Cell Signaling), anti--phospho MLC2 (Thr18/Ser19) (Cell Signaling), anti--MLCK (Sigma), anti--NMHCIIa (Sigma), anti--NMHCIIb (Sigma), anti--PAK1 (Cell Signaling), anti--PARP (Cell Signaling), anti--RAF1 (BD Biosciences), anti--phospho RAF1 (Ser338) (Cell Signaling), anti--ROCK1 (BD Biosciences), anti--TIF1a (in--house generated mouse monoclonal AB), anti--TRADD (Santa Cruz), anti--TRAIL (R&D). Secondary antibodies produced in chicken (Santa Cruz) were used when revealing for proteins in inputs or IPs. Secondary antibodies used for Western blot analysis of whole cell lysates originated from Santa Cruz or Cell Signaling. For fluorescence--activated cell sorting (FACS) analyses phycoerythrin (PE)--labeled anti--Apo2.7 (Beckman Coulter), anti--DR5--PE (Diaclone) and anti--IgG1--PE (Beckman Coulter) were applied. Apoptosis assays. The mitochondrial membrane protein 7A6 was used as apoptosis marker. The protein of unknown function is also called APO2.7 after the monoclonal antibody recognising the protein. If human cells undergo cell death by

Figure 19 :

 19 Figure 19: Experimental set--up and principle of 7A6 apoptosis assays. (A) Cells used in apoptosis assayswere plated or the cell culture medium was renewed (Δ) 24h prior the start of rhTRAIL treatment. For 16h cells were incubated with rhTRAIL, collected, immunostained for the antigen 7A6 and analysed by FACS. (B) The outer mitochondrial membrane of viable cells is negative or weakly positive for the protein 7A6, which translocates to the outer mitochondrial membrane upon apoptotic insult. Permeabilisation of apoptotic cells allows cell entry of the 7A6--specific monoclonal antibody Apo2.7 that is directly labeled with a fluorescence dye. (C) Representative FACS plots of normal (BJ) cells that were transfected with lipofectamine (mock), scrambled control siRNA (SCR) or siRNA targeting non--muscle myosin heavy chain IIa (NMHCIIa). Cells were left untreated (no rhTRAIL) or were treated with 1µg mL --1 rhTRAIL for 16h (16h rhTRAIL). Plots depict total cell populations that were gated for analysis in black and cell populations plotted versus fluorescence in blue. Gated blue cell populations are positive for 7A6 (Apo2.7 fluorescence) and cell percentages are given in red. Plots to the very right show fluorescence intensities of 7A6 positive cells in an overlay, whereby mock, respectively SCR control cells are depicted in black and SCR, respectively NMHCIIa knockdown cells are shown in red.

  Escherichiacoli strains were used: BL21(DE3) was transformed when aiming for protein production and DH5α served for the amplification of plasmid DNA. 1μg plasmid DNA was mixed with 100μL KCM buffer [100mM potassium chloride (KCl), 30mM calcium chloride (CaCl2), 50mM magnesium chloride (MgCl2)] and chilled on ice for 10 min to enable proper dissolution of the DNA molecules prior to transformation. The DNA mix was then added to 100μL competent bacteria that were thawed on ice. Keeping the DNA/bacteria mix on ice for 20min, DNA will stick to the bacterial membrane, which is then incorporated into the bacterium upon thermic shock performed for 10min at RT.Transformed bacteria were resuspended in 1mL LB medium, incubated for 1h at 37°C to allow recovery from the stress of transformation and finally 75μL of bacteria were spread on Luria Brothe (LB)--agar plates supplemented with the respective selection antibiotics encoded in the plasmid to enable clonal selection.Bacterial expression of rhTRAIL. The basic protocol for the bacterial expression and purification of rhTRAIL originates from[START_REF] Kim | Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells[END_REF] and shall briefly be described in the following. Prior to large--scale production of rhTRAIL, an induction test was performed to choose the bacterial clone with highest rhTRAIL productivity.Therefore, 3mL LB cultures supplemented with selection antibiotic were inoculated with different bacterial clones and grown ON at 37°C and 190rpm. The following day 10mL LB medium cultures containing selection antibiotics and 100µM zinc sulfate (ZnSO4) to stabilise rhTRAIL's homotrimeric structure were inoculated with 10µL of the ON starter culture. Bacterial cultures were grown at 18 to 21°C and 190rpm to prevent aggregation and formation of insoluble inclusion bodies upon overexpression of the recombinant protein, which is counterproductive to purification of native proteins. Once the culture has reached an optical density (OD) of 0.3 to 0.6, the bacterial expression of the rhTRAIL gene was induced adding the lactose analog isopropyl β--D--1-thiogalactopyranoside (IPTG) to a final concentration of 0.5mM. The cultures were induced at RT and 190rpm until the OD has doubled (≈ 14h). Aliquots of un--induced and induced cultures were taken, whereby the aliquot volume was calculated according to the measured OD. Bacteria were pelleted, washed once with ice--cold PBS, lysed in identical volumes of lysis buffer and analysed on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels that were stained with Coomassie brilliant blue. The bacterial clone with the strongest rhTRAIL protein band was chosen for large--scale rhTRAIL production and purification. Therefore, 3x 1L LB medium cultures containing selection antibiotics and 100µM ZnSO4 were inoculated with 1mL of culture used for the induction test and grown at 18--20°C and 190rpm. When the culture has reached an OD of 0.3--0.6 (which can take up to 23h since bacteria transformed with the untagged rhTRAIL grow slower than bacteria transformed with 6x histidine (His)-tagged rhTRAIL), rhTRAIL gene expression was induced with 1mM IPTG at 18--20°C and 190rpm until the OD has doubled (≈ 26h). Like in the induction test, also at large scale rhTRAIL expression aliquots of the un--induced and induced cultures were analysed for expression efficiency. Bacterial cultures were centrifuged at 6000rpm and 4°C for 30min and cell pellets were stored at --80°C until protein purification.Bacterial lysis and batch-purification of rhTRAIL. Due to it's 6 histidine (His)residues, untagged soluble rhTRAIL comprising amino acids 114 to 281 can be purified via nickel(Ni 2+ ) affinity purification procedures[START_REF] Ashkenazi | Safety and antitumor activity of recombinant soluble Apo2 ligand[END_REF][START_REF] Kim | Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells[END_REF], The Qiaexpressionist 06/2003]. Bacterial cell pellets were defrozen on ice and resuspended in one--fortieth volume of ice--cold lysis buffer [50mM monobasic sodium phosphate monohydrate (NaH2PO4*H2O), 300mM sodium chloride (NaCl), 5mM imidazole, 5mM dithiothreitol (DTT), pH 8.0] related to the initial bacterial culture. Once the complete pellet was resuspended upon repeated pipetting up and down, the bacterial cell wall was weakened by adding lysozyme to a final concentration of 200µg to 1mg ml --1 and incubating the mixture for 30min on ice and subsequently for 5min at 37°C. Then, bacterial cell walls were ruptured sonicating the mixture at 30% intensity for 2 min on ice, whereby every 10s of sonication were followed by 10s breaks to prevent the solution from heating and hence rhTRAIL from denaturation. Upon addition of 1--fold the volume of lysis buffer --related to the initial lysis buffer volume-- the 2min sonication procedure was repeated, keeping the 10s intervals. Fully homogenized lysates then were cleared from bacterial debris ultracentrifuging at 35,000rpm and 4°C for 40min. Resulting supernatants were recovered as crude extracts. At each step of bacterial lysis and rhTRAIL purification a constant aliquot volume was sampled for an SDS--PAGE analysis to control protein solubility and purity. As Ni 2+ ions of commercially available HiTrap columns chelated when using lysis buffers containing 5mM DTT, native rhTRAIL was purified in batch purification procedure using Ni 2+ --nitriloacetic acid (NTA) agarose beads (Qiagen, Hilden, Germany) in accordance to manufacturer's instructions.In brief, cooled Ni 2+ --NTA slurry was equilibrated washing the beads 4x with ice--cold double--distilled water (ddH2O) water and 5x with ice--cold lysis buffer. For each washing step at least 4x the bead volume was used and between the washing steps Ni 2+ --NTA beads were pelleted centrifuging at 4°C and 1000xg for 10s. Equilibrated Ni 2+ --NTA beads were mixed with crude extract in a 1:4 ratio and gently tilt over on a head--to--head shaker at 4°C ON. The following day Ni 2+ --NTA beads, to which rhTRAIL has bound, were sedimented and resulting supernatant containing unbound rhTRAIL, herein referred to as flow through, was mixed a second time with equilibrated Ni 2+ --NTA beads for 1h at 4°C on a head--to--head shaker. The pelleted Ni 2+ --NTA beads of both rhTRAIL binding rounds were pooled and washed 7x with ice--cold lysis buffer using at least 4x the beads volume. To liberate rhTRAIL from it's binding to the Ni 2+ --ions, the beads are washed with excess concentrations of imidazole, an organic compound that builds a side chain of His and therefore is especially abundant in this amino acid. Excess imidazole washings displaces His residues from nickel co--ordination, freeing the His--rich rhTRAIL. For rhTRAIL elution Ni 2+ --NTA beads were incubated 5x with ice--cold elution buffer [50mM Na2PO4*H2O, 300mM NaCl, 40mM imidazole, 5mM DTT, pH 8.0] whereby each time 4x the beads volume was used. Resulting eluates were not pooled but separately dialysed in a 3 KDa cut--off dialysis tube (Spectra/Por Spectrum Laboratories) versus ice--cold PBS containing 5mM DTT carefully stirring at 4°C ON. The dialysis buffer was renewed 12h and 18h after dialysis start and it was observed that the degree of protein precipitation during dialysis decreased if the initial eluate protein concentration was low. Upon dialysis, concentrations of single rhTRAIL eluates were determined by using the Bradford assay and aliquots taken during all steps of the lysis and purification procedure were analysed for protein solubility and purity on SDS--PAGE gels stained with Coomassie brilliant blue. Once the purity of the rhTRAIL preparations was confirmed, the single eluates were tested for their biological activity in in vitro apoptosis assays and surface plasmon resonance assays.Cell culture conditions. BJAB suspension cells were grown at densities between 4x10 5 -1.6x10 6 cells ml --1 medium [Roswell Park Memorial Institute medium (RPMI) 1640 without Hepes, 10% heat--inactivated FCS #K3398, freshly supplemented 2mM glutamine]. Cells were diluted in fresh medium latest every second day removing half of the consumed medium and cells and replacing the volume with fresh medium. If cells were grown to densities above 1.2 x10 6 cells, the dilution was performed in consecutive steps allowing the cells to adapt to the lower cell density for at least 2h. Cell viability was routinely checked with trypan blue and only cells with a viability of >90% were used in experiments. Normal BJ fibroblasts and transformed BJELR cells were cultured in the same medium [Dulbecco's Modified Eagle medium (DMEM)/M199 (4:1), 10% heat-inactivated FCS #97] and split every 2d. BJ cells were plated at a density of 8x10 3 cells and BJELR cells were plated at densities ranging from 1.15--1.6x10 4 cells per cm 2 growth surface, depending on their proliferation rate they exhibited in the applied FCS.

  stepwise cells. Therefore, for DISC--IP experiments 10x10 6 stepwise system cells were utilised for equal volumes of beads and identical amounts of IP--ABs. Cells were scraped in warm medium and immediately transferred to pre--cooled tubes containing 5x the volume ice--cold phosphate--buffered--saline (PBS), to stop DISC formation and to preserve the DISC. Cells were spun down at 200xg and 4°C for 5 min and washed once with ice--cold PBS. Cell pellets were resuspended by careful pipetting up and down in 1.1 mL ice--cold lysis buffer [30 mM Tris--hydrochloride (HCl) pH7.4, 150mM NaCl, 5mM KCl, 10% glycerol, 2 mM EDTA] freshly supplemented with complete, EDTA--free protease inhibitor cocktail (Roche, Mannheim Germany, # 11873580001), phosphatase inhibitors (PhosStop, Roche) and 1% Triton--X100 (BioRad) for the lysis of membrane lipids.Samples were incubated on a head--to--head shaker at 4°C or on ice for 30 min. Obtained lysates were spun at 20800xg and 4°C for 15 min to clear away debris. Meanwhile protein G sepharose beads (GE Healthcare) were equilibrated by washing the beads 3x with lysis buffer supplemented with Triton--X100 and spinning the beads at 106xg and 4°C for 2 min. The lysate supernatants were pre--cleared with 50μL of equilibrated G-protein sepharose beads for at least 1h at 4° on a head--to--head shaker. A volume of 50µL G--protein Sepharose beads was coupled with 4µg DR5 IP--antibody (AB), 1.4µg DR4 IP--AB or 0.6µg CASP8 IP--AB for a minimum of 2h at 4°C on a head--to--head shaker. Pre-cleared lysates were centrifuged at 106xg for 2 min to pellet the sepharose beads.

'

  inputs' were supplemented with 10--fold concentrated Laemmli buffer [100mM Tris pH 8.0, 25% SDS, 50% 2--mercaptoethanol]. Both 'inputs' and 'IP' were either immediately analysed by Western blot or stored at --80°C. For analysis samples were defrozen on ice and then heated at 75°C for 10 min prior to loading equal volumes onto SDS--PAGE gels.DR5 surface level determination. Per condition and treatment 1x10 5 normal BJ or transformed BJELR cells were used and per condition and treatment cell autofluorescence (no staining), DR5 surface levels (DR5--PE stain) and IgG surface levels (IgG--PE stain) was assessed. After trypsinisation cells were collected in FACS--tubes containing 1.5mL ice--cold PBS supplemented with 2.5% FCS. Cell culture wells were washed with ice--cold PBS and 2.5% FCS. Then, cells were spun down at 240xg and 4°C for 5 min and incubated in ice--cold PBS containing 2.5% FCS 30min on ice. After an additional centrifugation step at 240xg and 4°C for 5 min, cells were stained in a total volume of 30µL PBS and 2.5% FCS complemented with 5µL DR5--PE antibody solution, respectively 5µL IgG--PE or were left unstained. Cells were incubated in the dark on ice for 1h. To each tube 250µL ice--cold PBS containing 2.5% FCS was added and cells were immediately analysed by FACS. Importantly, cells were kept to all times on ice and cautiously pipettet up and down prior to FACS analysis, to revert cell clumping. After the first analysis 0.25µg mL --1 propidium iodide was added to each sample and cells were again passed through the FACS, which enables discrimination of viable cells and thus ensures analysis of surface stain. Resulting mean (or median) fluorescence intensities (MFI) of the second viable cell analysis were taken for analysis. Values were normalised to the rescpective autofluorescence. DR5 internalisation analyses. Principally, DR5 internalisation experiments were performed as described under 'DR5 surface level determination', except that cells were treated with 1µg mL --1 rhTRAIL for 5 to 60min. Addition of rhTRAIL was started at the latest time--point and was continued to earliest time--point to enable simultaneous sample collection. Recorded MFI was normalised to autofluorescence and resulting MFI in absence of rhTRAIL was set to 100%. Each further MFI value obtained at the single rhTRAIL incubation time--points were subtracted from the initial 100% of DR5 surface level.Generation of competent bacteria. Initial 3mL starter cultures were grown inLB--medium ON at 37°C. Bacteria were diluted 1:100 in fresh LB medium and grown at 37°C to an OD of 0.6. Bacterial cells then were pelleted for 5min at 3000xg and 4°C, resuspended in 1/20 th volume of competent medium [LB medium supplemented with 10% polyethylene glycol (PEG) 3350, 5% DMSO, 10mM MgCl2, 10mM magnesium sulfite (MgSO4)] and incubated for 10min on ice. Competent cells were then supplemented with 10% glycerol, distributed in 100μL aliquots, frozen in liquid nitrogen and stored at --80°C.Immunocytochemistry. Cell medium was removed and cells were fixed with ice--cold 4% paraformaldehyde (PFA) for 10min at RT. After fixation, cells were rinsed 3 times with ice--cold PBS for 5min each. Thereafter, cells were blocked and permeabilised in 1% (w/v) bovine serum albumin (BSA) and 0.05% (w/v) saponin in PBS for 1h at RT.Staining was performed with 6.25nM rhodamine--phalloidine (Cytoskeleton) and Hoechst, diluted in PBS containing 1% BSA and 0.05% saponin for 30min at 37°C. After 3 consecutive washes with ice--cold PBS, cells were mounted in ProLong Gold Antifade Reagent (Invitrogen). Mounting medium was hardened for 24h at RT before samples were analysed by fluorescence microscopy.Mass spectrometry. On--membrane digestion of protein bands was performed according to described protocols [Luque--Garcia et al 2006] and in--solution digestion of IP--eluates according to IGBMC standards. Modifications of the original protocols shall be described herein. In case of on--membrane digestion experiments, the Ponceau S stained nitrocellulose membranes were destained in ddH2O. To avoid sticking of trypsin to membrane pieces, non--specific protein binding sites on the membrane were blocked in 0.5mL 0.5% (w/v) polyvinylpyrrolidone (PVP--40) prepared in 100mM acetic acid at 37°C for 30min. Membrane pieces were washed 6--10 times with 1mL ddH2O prior the digestion in 20µL trypsin solution [12.5ng µL --1 sequencing grade modified trypsin (Promega) dissolved in 25mM ammonium bicarbonate (NH4HCO3) pH 8--8.5] at 37°C for 16h. Digested samples were vacuum--dried, digested peptides were resuspended in 40µL H2O, sonicated for 10min and 1µL of each sample was injected into the ion--trap mass spectrometer that allows protein identification according to its sequence. Results were aligned with two different search engines, Mascot and Sequest. Whilst Mascot integrates searches for peptide molecular mass data and amino acid sequence data, Sequest identifies proteins only by comparison of peptide mass generated from a protein digested with a specific enzyme. Importantly, we considered a protein to be identified if at least 2 peptides were identified per search engine and were judged significant if Mascot scores were ≥68 or XCorr scores were ≥2[START_REF] Perkins | Probability--based protein identification by searching sequence databases using mass spectrometry data[END_REF][START_REF] Washburn | Large--scale analysis of the yeast proteome by multidimensional protein identification technology[END_REF].Microscopic live-cell imaging. Normal BJ and transformed BJELR fibroblasts were grown under beforehand described regular cell culture conditions and imaged using a video--microscope (Leica DM IRE2). Recordings were processed with MetaMorph software.Non-reducing (native) SDS-PAGE. For native gel analysis of rhTRAIL all devices were freed of SDS cleaning with a lot of water and some ethanol afterwards. The running buffer [60mM Tris--HCl, 40mM cyclohexylamino propanesulfonic acid (CAPS), pH 7.0] was cooled to 4°C and cooling of buffer, separating gel [12%/0.32% (v/v) acrylamide/Bis, 60mM Tris--HCl, 40mM CAPS, 0.1% (w/v) ammonium persulfate (APS), 0.1% (v/v) Temed] and stacking gel [4%/0.1% acrylamide/Bis, 60mM Tris--HCl, 40mM CAPS, 0.1% (w/v) APS, 0.1% (v/v) Temed] was ensured during the gel run placing the running chamber on ice. Gels were pre--run with reversed cathode and anode at 100V for 40min. Samples were mixes with loading buffer [25% glycerol, 60mM Tris--HCl, 40mM CAPS, 0.005% bromophenol blue solution] and 5µg protein and 5µL of isoelectric focusing (IEF) marker were loaded per slot. Protein separation was performed at 100V for 6.5h.Pan-CASP inhibition. For the inhibition of CASP activation, we used the pan--CASP inhibitors zVAD (R&D). In pre--tests increasing concentrations (50, 100 and 200µM) of zVAD or equivalent concentrations of solvent (DMSO) were mixed into the medium of transformed BJELR cells for 30min prior to challenging the cells with 1µg mL --1 rhTRAIL for 1h. Cells were lysed, and whole cell lysates were analysed by Western blot for CASP8, CASP10 and CASP3 cleavage. For DR5--IP experiments under pan--caspase inhibition 100µM zVAD, respectively DMSO was added to the cells and the DISC was purified 1h after the start of 1µg mL --1 rhTRAIL treatment.

  DNA purification. For the purification of plasmid DNA the NucleoBond Xtra Kit (Macherey--Nagel) was used in accordance to the manufacturers recommendation.SDS-PAGE. To separate protein mixtures according their molecular weight, SDS--PAGE was performed. Therefore, running buffer [25mM Tris--base, 50mM glycine],separating gels [8--12%/0.21--0.32% (v/v) acrylamide/Bis, 25mM Tris--base, 50mM glycine, 0.05% (w/v) APS, 0.1% (v/v) Temed] and stacking gel [4%/0.1% (v/v) acrylamide/Bis, 25mM Tris--base, 50mM glycine, 0.05% (w/v) APS, 0.1% (v/v) Temed] were utilised. Gels were run at 80--120V.SDS-PAGEgel and nitrocellulose membrane stainings. Coomassie brilliant blue stainings of SDS--PAGE gels was performed boiling gels 2min in staining solution [2.5mg mL--1 coomassie blue R250, 50% ethanol, 10% acetic acid]. Excess staining solution was removed shaking gels in destaining solution [20% ethanol, 7% acetic acid].Nitrocellulose membranes were stained for 10s in commercially available Ponceau S solution (Pierce). After removal of surplus stain with water, membranes were de-stained in salty buffer solution, usually TBST. siRNA transfection. The siRNAs against NMHCIIa, NMHCIIb, GRB2 and the scrambled control were purchased from Qiagen. Target proteins were knocked down by reversed transfection. Therefore, per cm 2 cell culture surface 1.17µL lipofectamine (RNAiMax, Invitrogen) was mixed to a total volume of 26µL with Opti--MEM (Gibco).Likewise, per cm 2 growth surface the respective siRNA was diluted to a final concentration of 15nM in a total volume of 26µL Opti--MEM. Onto each cm 2 cell culture surface 47µL of the lipofectamine/siRNA mix was spread equally to cover the whole surface. Then, per cm 2 surface area 1.25x10 4 normal BJ or 2.5x10 4 transformed BJELR cells resuspended in 200µL cell culture medium were immediately plated onto the siRNAs covering the bottom of the cell culture vessel. Upon incubation of cells for 24h, the transfection reagents were removed from the cells and replaced by fresh culture medium. Usually, knockdown was allowed to proceed for 2d before cells were collected or used in experiments. If the knockdown was prolonged to 4d, cell culture medium again was renewed 3d after the start of transfection.Surface plasmon resonance (Biacore) assays. Binding assays of rhTRAIL to its specific and unspecific receptors were performed as published elsewhere[START_REF] Pavet | Multivalent DR5 peptides activate the TRAIL death pathway and exert tumoricidal activity[END_REF].Western blot analysis. The transfer of proteins from SDS--PAGE gels to nitrocellulose membranes (Whatman) was performed in transfer buffer [25mM Tris-base, 50mM glycine, 20% (v/v) ethanol] at 300mA (and 150V) for 1.5h while cooling on ice. Nitrocellulose mambranes were routinely stained with Ponceau S to control equal protein transfer. After removal of Ponceau S coloration using Tris--buffered saline (TBS)[140mM NaCl, 2.7mM KCl, pH 7.4], unspecific protein binding sites the membrane was blocked in 5% milk powder (w/v) dissolved in TBS supplemented with 0.1% Tween 20 (TBST). To ensure proper dissolving, blocking solutions were heated to 80°C while stirring for at least 1h and then cooled down prior to use. Primary antibodies were applied in 5% milk and TBST or 5% BSA and TBST at 4°C ON moving on a head--to-head shaker. Membranes were then washed 3 times for 5 min each shaking in TBST and incubated with secondary antibody diluted in 5% milk and TBST at RT for 1h on a head-to--head shaker. Unbound secondary antibody was removed washing membranes 3 times for 5 min each shaking in TBST. Protein signals were detected incubating the membranes with enhanced chemiluminescence (ECL) reagent (GE Healthcare or Thermo Fisher Scientific) and exposing them to light--sensitive Hyperfilms (Hyperfilm MP, GE Healthcare).

  associated with the loss of NMII/DR interaction and an increased response of normal cells to TRAIL apoptosis. Moreover, the deregulation of kinase expression levels in transformed cells controlling the NMII Mg 2+ --ATPase activity will be shown. Finally, in the fourth section we will describe how NMHCIIa but not NMHCIIb siRNA--induced protein loss removes the resistance block towards TRAIL--apoptosis in normal cells and how NMHCIIa loss alters the composition of the DISC.According to literature, NMII function in cell adhesion and cell detachment is implicated in sensitivity to TRAIL--apoptosis. Above that, localisation and recruitment of signalling molecules (amongst others) to focal adhesions positively regulating fibroblast proliferation, depends on NMII Mg 2+ --ATPase activity. Hence, we investigated whether anti--apoptotic TRAIL DISC--interacting proteins exist whose recruitment depends on NMII in normal cells. And so, the final results section will introduce the proliferation signalling adapter growth factor receptor--bound protein 2 (GRB2) and RAF1 proto-oncogene serine/threonine--protein kinase (RAF1) as constitutive DR5 interactors and uncover their NMII--independent binding to DR5.

Figure 20 :

 20 Figure 20: Expression, purification and subunit structure of recombinant human TRAIL (rhTRAIL). (A) Amino acid sequence of full--length human TRAIL. Indicated are the cytoplasmic (blue), transmembrane (red) and extracellular (black) domains as well as the extracellular portion that was expressed as untagged soluble recombinant human TRAIL (rhTRAIL, bold). (B) Coomassie stained 15% SDS--PAGE gel to control expression efficiencies of various Escherichia coli strain BL21DE3 clones expressing the 18.59 KDa rhTRAIL upon 24 hours induction with 0.5mM isopropyl--beta--D--thiogalactopyranoside (IPTG). (C) SDS--PAGE gel containing equal volume loads of the single Ni 2+ affinity chromatography--based rhTRAIL purification steps. (D) The rhTRAIL was purified by Ni 2+ affinity chromatography, dialysed and then analysed by non--reducing (native) SDS--PAGE and silver staining. A commercially available, untagged TRAIL (cTRAIL) was included for comparison. The gel displays monomeric, dimeric and trimeric rhTRAIL, latter being considered as the biologically active (here: apoptosis--inducing) structure.

  other TNF receptor family members, we performed surface plasmon resonance (SPR) experiments and determined rhTRAIL binding affinities towards each single receptor. In these SPR experiments sensor plates were used onto which the specific TRAIL receptors (DR5, DR4, DcR2 or DcR1) or unspecific TRAIL receptors of the TNF receptor family (FAS or receptor activator of NFKB (RANK)) were immobilised. The surfaces were objected to a constant flow of aqueous buffer into which varying rhTRAIL concentrations were injected. As rhTRAIL binds to the receptors the accumulation of rhTRAIL on the sensor plate results in an increase in the refractive index measured by an optical method. This change in refractive index is measured in real time, and the result plotted as response or resonance units (RU) versus time. As the continuous flow of running buffer over time washes rhTRAIL away, this again changes the measured refractive index. From these measurements the association (Kon) and dissociation constants (Koff) then can be calculated describing the binding affinity between rhTRAIL and its respective receptor (affinity = Kon/Koff). SPR experiments revealed that purified rhTRAIL in vitro binds to the TRAIL receptors DR5, DR4, DcR1 and DcR2 (Figure21, A). Association constants were determined for all four TRAIL receptors being 0.25nM for DR5, 1.9nM for DR4, 0.6nM for DcR1 and 0.44nM for DcR2, whereby all association constants were calculated from six different binding curves generated at varying rhTRAIL concentrations, except the one of DR4 which was calculated only from three different binding curves[START_REF] Pavet | Multivalent DR5 peptides activate the TRAIL death pathway and exert tumoricidal activity[END_REF].Accordingly with previously reported data for other soluble rhTRAIL preparations[Degli--Esposti et al 1997[START_REF] Emery | Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL[END_REF][START_REF] Truneh | Temperature--sensitive differential affinity of TRAIL for its receptors. DR5 is the highest affinity receptor[END_REF][START_REF] Hymowitz | A unique zinc--binding site revealed by a high--resolution X--ray structure of homotrimeric Apo2L/TRAIL[END_REF], Lee et al 2005], rhTRAIL displays a slightly stronger affinity for DR5, intermediate for the DcRs and lowest for DR4 (DR5>DcR2>DcR1>DR4). Importantly, in surface plasmon experiments purified rhTRAIL did not bind to the receptors of other TNF family members, such as FAS and RANK (Figure21, A). The specificity of ligand--receptor recognition hence could be maintained for the purified rhTRAIL.

  next goal was to evaluate whether rhTRAIL induced apoptosis in accordance to its apoptotic signalling model[START_REF] Newsom--Davis | Is TRAIL the holy grail of cancer therapy?[END_REF] and to define the time--point of maximal CASP8 cleavage that is required for optimal timing of DISC--IPs. For that purpose, BJAB DR5--/+ and BJAB DR5--/--cells were stimulated with 1µg mL --1 rhTRAIL for different time spans, whole cell lysates (WCL) were collected and CASP8 activation was analysed by Western blot. Stimulation of BJAB DR5--/+ with purified rhTRAIL induced a maximal cleavage of CASP8 after 1h of treatment. Contrary, neither the large subunit of CASP8 p43/p41 nor the p18 catalytic subunit were evidenced in untreated BJAB DR5--/+ cells and treated BJAB DR5--/--cells (Figure21, C). Thus in BJAB cells purified rhTRAIL initiates apoptosis via DR5--dependent CASP8 activation.Since CASP8 activation represents only the first level of extrinsic DR--mediated apoptosis, we explored if rhTRAIL initiates the full apoptotic cascade in BJAB cells. For that, BJAB DR5--/+ and BJAB DR5--/--cells were treated with 1µg mL --1 rhTRAIL for 1h and activation of initiator CASP8 and CASP9, full--length BID, and effector CASP3 and CASP7 was analysed by Western blot (Figure21, D). Whereas no changes were detected in BJAB DR5--/--cells, in BJAB DR5--/+ cells 1h of rhTRAIL treatment induced maximal processing of initiator CASP8 and was sufficient to elicit effector CASP3 and CASP7 cleavage. In line with previous reports[START_REF] Menke | Distinct TRAIL resistance mechanisms can be overcome by proteasome inhibition but not generally by synergizing agents[END_REF], we observed that the death signal in BJAB DR5-- /+ cells is enhanced via the intrinsic apoptotic pathway. Particularly, a decline of the full-length BID signal as well as processing of CASP9 was observed. Although BID is cleaved by CASP8, BJAB cells are type I signalling cells, since BID knockdown neither inhibited nor decreased rhTRAIL--induced apoptosis[START_REF] Menke | Distinct TRAIL resistance mechanisms can be overcome by proteasome inhibition but not generally by synergizing agents[END_REF].

Figure 21 :

 21 Figure 21: Biological activity of purified untagged recombinant human TRAIL (rhTRAIL). (A) Re-presentative binding curves for rhTRAIL to its specific receptors (DR5, DR4, DcR1, DcR2) and to other TNF family receptors (FAS, RANK) determined by surface plasmon resonance experiments at 25nM rhTRAIL. Binding curves were recorded with varying rhTRAIL concentrations in 3 (DR4) to 6 (all other

  Despite these basic technical considerations the intact DISC was successfully immunoprecipitated targeting DR5, DR4 or CASP8 (Figure22). In accordance to the prevalent signalling model rhTRAIL stimulation of BJAB cells induced the formation of DR5/DR4 heterocomplexes, recruitment of FADD and activation of CASP8 at the receptors. TRAIL receptors contain a PLAD allowing ligand--independent homotypic and heteroypic receptor interactions[START_REF] Clancy | Preligand assembly domain--mediated ligand-independent association between TRAIL receptor 4 (TR4) and TR2 regulates TRAIL--induced apoptosis[END_REF]. Therefore we investigated whether preformed DR5/DR4 heterocomplexes exist prior to ligand addition. In the absence of rhTRAIL DR5--IP does not co--immunoprecipitate DR4 and DR4--IP does not co-immunoprecipitate DR5 (Figure22, A--B). Thus, in the absence of ligand there are no pre--formed DR5/DR4 heterocomplexes present in BJAB cells. In line with the apoptotic signalling model[START_REF] Gonzalvez | New insights into apoptosis signaling by Apo2L/TRAIL[END_REF][START_REF] Dickens | A Death Effector Domain Chain DISC Model Reveals a Crucial Role for Caspase--8 Chain Assembly in Mediating Apoptotic Cell Death[END_REF][START_REF] Schleich | Stoichiometry of the CD95 Death--Inducing Signaling Complex: Experimental and Modeling Evidence for a Death Effector Domain Chain Model[END_REF] rhTRAIL induces FADD recruitment to the DISC and CASP8 cleavage at the DISC (Figure22, A--C). Whether DR5 and/or DR4 form homotrimeric complexes in absence of TRAIL cannot be investigated using this approach.

Figure 22 :

 22 Figure 22: The TRAIL--DISC of BJAB cells. Immunoprecipitation (IP) of the DISC targeting (A) death receptor 5 (DR5), (B) death receptor 4 (DR4) or (C) caspase 8 (CASP8) from unstimulated (--) and rhTRAIL--stimulated (+)Burkitt's lymphoma cells (BJAB) cells. Wildtype BJAB cells monoallelic for DR5 (BJAB DR5--/+ ), DR5 negative cells (BJAB DR5--/--) or DR5(S) rescue cells (BJAB DR5--/(S) ) were incubated with 1μg mL --1 purified rhTRAIL for 1h. Lysates originating of each cell line and treatment were split into 3 fractions to immunoprecipitate either DR5, DR4 or CASP8. Immunoprecipitates were run on 12% SDS--PAGE gels and analysed by Western blotting. Depicted are representative blots from 1 out of 2 independent experiments.

  By the time tumour explants are taken, of which patient--derived cancer cell lines are generated, tumour cells usually have accumulated genetic alterations that exceed the mutations required for cancer cell initiation. Moreover, if the tumour has metastasised, no 2 tumour samples from the same patient are genetically identical[START_REF] Gerlinger | Intratumor heterogeneity and branched evolution revealed by multiregion sequencing[END_REF]. And, as tumour cells tend to dedifferentiate, cancer cell lines not uncommonly originate from a mixture of different cell types. All these circumstances complicate the choice for normal reference cell populations from patients, if a comparison of normal versus tumour cells is envisaged. In this work we aimed to understand how a normal cell gains TRAIL sensitivity through the process of transformation. Due to beforehand mentioned disadvantages and limits of patient--derived cell lines, we chose to exploit the stepwise transformation system of Hahn and Weinberg[START_REF] Hahn | Creation of human tumour cells with defined genetic elements[END_REF] as a model, which provides transformed cells with defined genetic alterations as well as normal cells with isogenic background. DISC analyses require considerable (10 7 ) cell numbers and normal BJ fibroblasts proliferated better than normal HEK or HMEC cells. Therefore, all of the following work was performed in normal foreskin fibroblasts (BJ) and their HTERT, SV40ER and HRASG12V--transformed counterparts (BJELR).Normal and transformed cells of thestepwise transformation model differ in cell culture characteristics. When grown in vitro normal BJ cells have a flat morphology, form one or several cellular protrusions (Figure 23, left image) and appear as large cells that rapidly and firmly attach to the cell culture vessel. Upon transformation fibroblasts change their morphology drastically, they become elevated, spindle--shaped and their margins display dynamic membrane blebbing (Figure 23, right image), a common phenomenon in cells during apoptosis

Figure 23 :

 23 Figure 23: Morphology of normal (BJ) and HTERT, SV40ER and HRASG12V--transformed (BJELR) human foreskin fibroblasts. Images were taken from living cells by differential interference contrast microscopy (DIC). The size bar represents 30µm (left image) and 19µm, respectively (right image).

  BJ and BJELR WCLs by Western blot, we found no obvious difference in total protein expression levels of DR5, DR4, FADD, CASP8, cFLIP, DcR1 and DcR2 (Figure24, C).Experiments in which cell viability of rhTRAIL--stimulated BJ and BJERL cells was investigated subsequent to the depletion of either DR5 or DR4 by siRNA--mediated knockdown (KD), identified DR5 as the main TRAIL--apoptosis signalling receptor in the BJ stepwise transformation model (unpublished data). Hence, we assessed relative DR5 surface levels by direct immunofluorescence and FACS analysis of mean fluorescence intensities (MFI) and observed no difference in DR5 surface levels between BJ and BJELR cells (Figure24, D). The diverging TRAIL sensitivity of normal and transformed cells therefore cannot be explained by a divergence in DISC protein expression levels or DR5 surface levels.Having eliminated the possibility that altered DISC protein levels or DR5 surface levels account for the different TRAIL--response, we aimed to isolate the signalling level at which the TRAIL apoptotic signal does not propagate and/or is potentially blocked in normal cells. Therefore we stimulated normal and transformed cells with 1µg mL --1 rhTRAIL for 1h and compared total levels of cleaved CASP8, CASP3 and PARP, which were reduced in BJ and enhanced in BJELR cells (Figure24, E). Thus, TRAIL--resistance in normal BJ cells is accompanied by an inefficient apoptotic cascade activation that originates from impaired CASP8 recruitment or CASP8 cleavage at the DISC.The decreased apoptotic cascade activation observed in normal BJ cells after 1h of rhTRAIL treatment (Figure24, E) prompted the question whether CASP8 and/or downstream signalling molecules could be activated at later timepoints in normal than in transformed cells. Therefore we performed time course experiments treating normal and transformed cells for diverse times with rhTRAIL. Subsequently, we investigated the WCLs for CASP8 and CASP3 cleavage. Even though on average only 11% of BJ cells are dying upon 16h of rhTRAIL exposure (Figure24, B), cleavage of CASP8 and CASP3 into the active p18, p19 and p17 fragments was detectable in BJ cells already 30 min after rhTRAIL addition. The onset of active CASP8 p18 fragment generation in WCL of normal cells occurred simultaneously with CASP3 cleavage into its active p19 fragment (Figure24, F, left panel). With regard to the timing of rhTRAIL--induced CASP8 p18 and CASP3 p19 appearance, normal BJ cells do not differ from their transformed BJELR analogues (Figure24, F, right panel). However, since total CASP8 p18 and CASP3 p19/p17 after 1h of rhTRAIL were clearly lower in normal than transformed cells (Figure24, E), we concluded that at all times total CASP cleavage levels in the sum of analysed normal cells do not reach the ones in transformed cells.Activation of CASP8 and/or CASP10 marks the initial step of TRAIL apoptotic signalling. Contrary, CASP3 cleavage can be carried out by initiator CASP8 and/or CASP10 (type I cells) or by effector CASP9 (type II cells), which is activated at the apoptosome. Apoptosome formation requires mitochondrial perturbation initiated by CASP8--facilitated cleavage of BID to tBID (Introduction, Figure3). Regarding the fact that the onset of CASP8 or CASP3 processing in normal BJ versus transformed BJELR cells was detectable at equal moments in time course experiments (after 30min of rhTRAIL treatment) (Figure24, F), we assumed that BJ and BJELR cells do not differ in their type of TRAIL signal propagation. Thus, events of the apoptosis cascade occurring after CASP8 and prior to CASP3 activation were analysed in BJELR cells only.

  and 7A6 exposure on the mitochondrial membrane requires insertion of tBID into the mitochondrial membrane [Nagahara et al 2007], BID cleavage must occur after 4h of rhTRAIL treatment. Notably, BID phosphorylation inhibits cleavage by CASP8 [Desagher et al 2001, Degli--Esposti et al 2003] and CASP8 activation and BID cleavage are temporally uncoupled events in type II HeLa cells [Hellwig et al 2010]. Thus, CASP3 cleavage by CASP8 could precede BID cleavage in the stepwise system cells and BID cleavage then could be mediated by CASP3

  populations and high CASP8 processing in populations of transformed cells substantiated the underlying hypothesis that TRAIL resistance in normal cells has its origin in the DISC formation and/or composition itself. Analyses of DISC compositions of cells of the stepwise tumorigenesis revealed that comparative levels of DR5 were immunoprecipitated from normal BJ and transformed BJELR cells. Moreover, similar signals of co--immunorecipitated rhTRAIL indicated that at the moment of rhTRAIL treatment, comparable levels of DR5 and DR4 molecules were exposed to the cell surface of BJ and BJELR cells (Figure24, G), which confirmed results of DR5 surface level measurements (Figure24, D). In the absence of the ligand no pre--formed DR5/DR4 heterocomplexes were detected, which is evidenced by the observation that in absence of rhTRAIL no association between DR4 and DR5 is observed in IP assays. In transformed BJELR cells rhTRAIL stimulation induced the formation of DR5/DR4 heterocomplexes, recruitment of FADD and cleavage of CASP8. Interestingly we found that normal cells are indeed capable of forming a DISC, as displayed by comparable rhTRAIL--induced DR4 recruitment to DR5. However, even if comparable amounts of DR5 can be immunoprecipitated from normal and transformed cells and rhTRAIL induced similar microaggregation with DR4, lower FADD recruitment and reduced CASP8 cleavage and/or recruitment occurs at the DISC of normal cells. Thus, rhTRAIL-resistance in normal cells is associated with poor CASP8 activation at the DISC. Importantly, reduced CASP8 cleavage at the DISC was not associated with a co-immunoprecipitation of DcR1 or DcR2 (Figure24, G). Thus, recruitment of DcR2 into the TRAIL--DISC does not account for the inhibition of CASP8 activation[START_REF] Merino | Differential inhibition of TRAIL--mediated DR5--DISC formation by decoy receptors 1 and 2[END_REF].

Figure 24 :

 24 Figure 24: Characterisation of TRAIL--signalling features in normal BJ and transformed BJELR cells. (A) Receptor saturation of HTERT, SV40ER and HRASG12V--transformed (BJELR) cells for 2 different lots of recombinant human TRAIL (rhTRAIL) preparations. BJELR cells were stimulated with the indicated concentrations of rhTRAIL and the percentage of apoptotic cells was quantified as 7A6 positivity in FACS analysis after 16h of treatment. Mean values were generated from 2 independent experiments. (B) Apoptotic response of normal (BJ) and BJELR cells treated with 1µg mL --1 purified rhTRAIL. Apoptosis was quantified as described in (A). Values derive from 3 to 5 independent experiments. Significance was calculated using unpaired, 2--tailed Student's t--test. (C) Representative Western blots demonstrating average DISC protein expression levels in whole cell lysates (WCL) of BJ and BJELR cells.* (D) DR5 surface levels of BJAB cells monoallelic for DR5 (BJAB DR5-/+ ), BJAB DR5 knockout (BJAB DR5--/--), BJ and BJELR stepwise system cells. Cells were left unlabelled (no AB), or were immunolabelled with phycoerythrine-conjugated antibodies against DR5 or IgG. Overlays depict fluorescence intensities as assessed by FACS.*

Figure

  Figure 24 continued: (E) Western blots presenting caspase 8 (CASP8), caspase 3 (CASP3) and Poly--[ADP--ribose]--polymerase (PARP) cleavage in BJ and BJELR cells treated with 1µg mL --1 rhTRAIL for 1h.* (F) Caspase cleavage in BJ and BJELR after addition of 1µg mL --1 rhTRAIL for the indicated times.** (G) DR5--DISC analysis of equal numbers of BJ and BJELR cells incubated with 1µg mL --1 rhTRAIL for 1h. Left 4 lanes served as loading control and contain equal volumes of pre--cleared WCL (inputs) that were used in the immunoprecipitation (IP) experiment. The 5 lanes on the right contain equal volumes of DR5--IP eluates originating either from the indicated cell line, or from the DR5/beads mixture (B) utilised in the experiment.*** Depicted are Western blots for 1 of *3, **2 or ***4 independent experiments.

  , D). It is well established that 2 NMHC molecules homodimerise and bind 2 ELC and 2 MLC2 [Vicente--Manzanares et al 2009]. This hecameric complex forms one NMII molecule (Introduction, Figure 14, A). Due to the fact that MLC2 are critical for NMII conformation, activity and filament formation capacity (Introduction, Figure 15, B) [Vicente--Manzanares et al 2009], we analysed and confirmed the association of MLC2 with the DISC--IPs (Supplementary, Figure 31).

Figure 25 :

 25 Figure 25: Identification of novel TRAIL--DISC interactors immunoprecipitating DR5 in normal BJ and transformed BJELR fibroblasts followed by on--membrane digestion of differential bands and mass spectrometry. (A) DR5 was isolated from normal (BJ) and HTERT, SV40ER and HRASG12V--transformed (BJELR) cells that were left unstimulated (--) or that were stimulated for 1h with 1µg mL --1 rhTRAIL (+) by immunoprecipitation (IP). IP eluates were split into two fractions of which both were separated by SDS--PAGE and blotted onto nitrocellulose membranes. (B) One nitrocellulose membrane onto which one portion of the DR5--DISC IP was blotted was stained with Ponceau S and differential bands were cut out, digested and given for mass--spectrometrical peptide identification. (C) Mass spectrometry results obtained from the single cutouts generated in (B). (D) Another nitrocellulose membrane onto which the other portion of the DR5--DISC IP was blotted was subjected to regular Western blot analysis to validate the mass--spectrometric results obtained from (B, C) (upper panel). Western blot analysis of a DR4--IP (lower panel).

Figure 26 :

 26 Figure 26: Cytoskeletal protein expression levels in normal BJ and transformed BJELR cells. The expression levels of non--muscle myosin heavy chain IIa and IIb (NMHCIIa, NMHCIIb), myosin regulatory light chain 2 (MLC2) and beta--actin (ßACT) was assessed in whole cell lysates (WCL) of normal (BJ) and HTERT, SV40ER and HRASG12V-transformed (BJELR) cells. Transcription intermediary factor 1--alpha (TIF1a) served as loading control. Depicted are representative Western blots of 3 independent experiments.

  was additionally co--purifed. Intriguingly, whilst MS and Western blot detection yielded more peptides and strong detection signals for the DR5--IP of unstimulated and rhTRAIL-treated normal BJ cells, DR5--IP of transformed BJELR cells brought about less peptides and weak Western blot signals that declined even more upon rhTRAIL addition.Importantly, exact same interaction patterns were found for DR4--IP of BJ and BJELR cells and for DR5--IP of transformed HA1ER cells. Unfortunate poor growth of normal HEK cells complicated an analysis of the DR5 interaction hitherto. However, detecting the NMII/DR and ßACT/DR interaction in transformed cells of two different transformation models (BJELR and HA1ER) underscores the biological significance of interaction.

  . As actin cytoskeleton constituent, NMII are implicated to function in central processes such as cell adhesion and migration [reviewed by Vicente--Manzanares et al 2009], cellspreading[START_REF] Hirata | Diphosphorylation of regulatory light chain of myosin IIA is responsible for proper cell spreading[END_REF], Golgi vesicle budding and transport[START_REF] Stow | Budding roles for myosin II on the Golgi[END_REF][START_REF] Lin | Nonmuscle myosin IIA facilitates vesicle trafficking for MG53--mediated cell membrane repair[END_REF], exocytosis[START_REF] Ludowyke | Phosphorylation of nonmuscle myosin heavy chain IIA on Ser1917 is mediated by protein kinase C beta II and coincides with the onset of stimulated degranulation of RBL--2H3 mast cells[END_REF], ligand--induced receptor capping[START_REF] Lorimer | Myosin II -Physiological properties[END_REF] De Lanerolle 1996, Komatsu et al 2000], redistribution of membrane proteins[START_REF] Kuo | Analysis of the myosin--II--responsive focal adhesion proteome reveals a role for beta--Pix in negative regulation of focal adhesion maturation[END_REF] and receptor endocytosis[START_REF] Rey | Myosin IIA is involved in the endocytosis of CXCR4 induced by SDF--1alpha[END_REF]. Interestingly, not only some of these processes have been implicated in TRAIL signalling and/or sensitisation (adhesion/spreading[START_REF] Goldberg | Global effects of anchorage on gene expression during mammary carcinoma cell growth reveal role of tumor necrosis factor--related apoptosis--inducing ligand in anoikis[END_REF][START_REF] Algeciras--Schimnich | Molecular ordering of the initial signaling events of CD95[END_REF][START_REF] Kurenova | Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor--interacting protein[END_REF][START_REF] Laguinge | DR5 receptor mediates anoikis in human colorectal carcinoma cell lines[END_REF][START_REF] Phipps | Targeting cell spreading: a method of sensitizing metastatic tumor cells to TRAIL--induced apoptosis[END_REF], transport [reviewed by[START_REF] Weinlich | Control of death receptor ligand activity by posttranslational modifications[END_REF], redistribution[START_REF] Song | Lipid rafts and nonrafts mediate tumor necrosis factor related apoptosis--inducing ligand induced apoptotic and nonapoptotic signals in non small cell lung carcinoma cells[END_REF][START_REF] Rossin | Palmitoylation of the TRAIL receptor DR4 confers an efficient TRAIL--induced cell death signalling[END_REF], endocytosis[START_REF] Jin | Deficient tumor necrosis factor-related apoptosis--inducing ligand (TRAIL) death receptor transport to the cell surface in human colon cancer cells selected for resistance to TRAIL--induced apoptosis[END_REF][START_REF] Cheng | Multiple mechanisms underlie resistance of leukemia cells to Apo2 Ligand/TRAIL[END_REF], Zhang et al 2008]) but also kinases known to modulate TRAIL sensitivity and/or signalling in tumour cells (CK2[START_REF] Ravi | Sensitization of tumor cells to Apo2 ligand/TRAIL--induced apoptosis by inhibition of casein kinase II[END_REF][START_REF] Izeradjene | Influence of casein kinase II in tumor necrosis factor--related apoptosis--inducing ligand--induced apoptosis in human rhabdomyosarcoma cells[END_REF][START_REF] Izeradjene | Casein kinase II (CK2) enhances death--inducing signaling complex (DISC) activity in TRAIL--induced apoptosis in human colon carcinoma cell lines[END_REF][START_REF] Shin | Caspase--2 primes cancer cells for TRAIL--mediated apoptosis by processing procaspase--8[END_REF][START_REF] Wang | Role of protein kinase CK2 in the regulation of tumor necrosis factor--related apoptosis inducing ligand--induced apoptosis in prostate cancer cells[END_REF][START_REF] Llobet | CK2 controls TRAIL and Fas sensitivity by regulating FLIP levels in endometrial carcinoma cells[END_REF], Pallares et al 2008], TRPM7[START_REF] Liu | Inhibition of transient receptor potential melastain 7 channel increases HSCs apoptosis induced by TRAIL[END_REF], PKCβ[START_REF] Kaunisto | PKC--mediated phosphorylation regulates c--FLIP ubiquitylation and stability[END_REF][START_REF] Meng | Protein kinase Cbeta modulates ligand--induced cell surface death receptor accumulation: a mechanistic basis for enzastaurin--death ligand synergy[END_REF], ROCK[START_REF] Hoogwater | Oncogenic K--Ras turns death receptors into metastasis--promoting receptors in human and mouse colorectal cancer cells[END_REF][START_REF] Ehrenschwender | Mutant PIK3CA licenses TRAIL and CD95L to induce non--apoptotic caspase--8--mediated ROCK activation[END_REF], PAK1[START_REF] Wei | Tumor necrosis factor (TNF)--related apoptosis-inducing ligand (TRAIL) induces chemotactic migration of monocytes via a death receptor 4--mediated RhoGTPase pathway[END_REF]) are key NMHCIIa and IIb regulators (see'Introduction' and [Vicente--Manzanares et al 2009]).Therefore we investigated whether the prominent NMHCII/DR interaction in normal cells is of functional relevance for TRAIL resistance and / or apoptosis.Chemical inhibition of NMII Mg2+-ATPase activity decreased NMHCIIa,NMHCIIb and MLC2 association with DR5 and DR4 and increased rhTRAIL-induced apoptosis in normal BJ cells. The non--competitive inhibitor blebbistatin specifically acts on skeletal muscle and non--muscle myosin II isoforms[START_REF] Limouze | Specificity of blebbistatin, an inhibitor of myosin II[END_REF] blocking the actin--activated Mg 2+ --ATPase activity housed in the head domain.Blebbistatin preferentially binds to the myosin II head when ADP and phosphate are still bound at the active site of the Mg 2+ --ATPase domain, whereby it blocks the phosphate release process that is required for the binding of NMII to actin (Figure27, A)[START_REF] Kovacs | Mechanism of blebbistatin inhibition of myosin II[END_REF][START_REF] Allingham | The structural basis of blebbistatin inhibition and specificity for myosin II[END_REF][START_REF] Sandquist | The C--terminal tail region of nonmuscle myosin II directs isoform--specific distribution in migrating cells[END_REF]. Treatment of normal BJ cells with 25µM blebbistatin for 30 min sufficed to diminish NMHCIIa, NMHCIIb and MLC2 co--immunoprecipitation with DR5 and DR4. Of note, ßACT binding to DR5 and DR4 was unaffected by blebbistatin treatment (Figure27, B), suggesting that ßACT binds to DR5 and DR4 independently of NMIIa and NMIIb. Normal cells pre--incubated for 30 min with increasing blebbistatin concentrations and successively treated with blebbistatin and 1µg mL --1 rhTRAIL for 16h, demonstrated an elevated dose--dependent response to rhTRAIL--apoptosis as compared to vehicle and TRAIL treated cells. Cells pre--treated with 25µM blebbistatin yielded 52% ±3 apoptotic cells, which was significantly more than obtained in cells pre--incubated with equal concentrations of solvent (DMSO) or without pre--treatment (19% ±5, p=0.02 and 13% ±4, respectively) (Figure27, C). Given that blebbistatin blocks NMII in an actin--detached state (Figure27, A)[START_REF] Kovacs | Mechanism of blebbistatin inhibition of myosin II[END_REF] and blebbistatin removes NMII but not ßACT from DR, we assume that ßACT binding to DR does not require NMII Mg 2+ --ATPase activity. Contrary, the magnitude of NMIIa and NMIIb co--purification depends on NMII Mg 2+ --ATPase activity.Preceding blebbistatin experiments revealed that inhibiting NMII Mg 2+ --ATPase activity lead to a weakening of NMII/DR interaction and an increase in rhTRAIL-apoptosis in normal cells. Aiming to reinforce these data we anticipated another chemical approach to interfere with NMII Mg 2+ --ATPase activity. Reportedly NMIIs are highly regulated by phosphorylation events amongst which MLC2 phosphorylation at serine 19 (Ser19) and threonine 18 (Thr18) is of utmost importance for NMIIs to display active conformation and to enable NMII/protein interaction (Figure27, D). Importantly, MLC2 phosphorylation at Ser19 and Thr18 increases the actin--activated Mg 2+ --ATPase function of NMII [Vicente--Manzanares et al 2009]. Even though MLCK is not the only kinase capable to phosphorylate MLC2 at these residues (Introduction, Figure 17) [please see Vicente--Manzanares et al 2009 for detailed information], MLCK has the advantage of having a limited substrate reservoir. In fact, MLC2 so far is the only accepted MLCK substrate for that in vivo data exist [phosphosite.org]. Aiming to minimize effects arising from impaired phosphorylation of secondary proteins, we therefore decided to interfere with NMII Mg 2+ --ATPase activity by blocking MLCK-mediated MLC2 phosphorylation.

  NMHCIIa and MLC2 binding to DR5. In contrast to the results achieved with blebbistatin, ML7 treatment additionally decreased ßACT--co--purification with DR5 (Figure27, E), indicating a requirement of MLC2 phosphorylation for the association of MLC2 and ßACT to NMHCII or DR5. Pre--incubation for 30 min with varying ML7 concentrations followed by subsequent additon of 1µg mL --1 rhTRAIL for 16h increased the percentages of apoptotic cells in dosage--dependent manner. Co--treatment of BJ cells with 50µM ML7 and 1µg mL --1 rhTRAIL resulted in 29% ±0.3 apoptotic cells, which was significantly more than what was acquired for cells co--incubated with equal concentrations of vehicle (ethanol/water mix at a ratio 1:1) (7% (±3), p=0.0002) (Figure27, F). Notably, increasing ML7 concentrations to 100µM or 200µM resulted in detachment of BJ cells from the surface and apoptosis induction in absence of rhTRAIL (43% (±0.3), respectively 81 (

Figure 27 :

 27 Figure 27: Effects of NMII inhibition on NMHCIIa, NMHCIIb interaction with DR5 and DR4 and TRAIL-sensitivity in normal BJ cells. (A) During the NMII crossbridge cycle blebbistatin preferentially binds to the NMII head with ADP and phosphate bound at the active site of the Mg 2+ --ATPase domain. Since it slows down phosphate release, NMII does not bind to actin. (B) NMHCII/DR5 and NMHCII/DR4 interaction upon inhibition of NMII ATPase activity. Normal (BJ) cells were incubated for 30 minutes with vehicle DMSO (--) or 25µM NMII ATPase inhbitor blebbistatin (+) and then collected the cells to immunoprecipitate (IP) death receptor 5 and 4 (DR5, DR4). The IP--eluates were analysed for the interaction with non--muscle myosin heavy chains IIa and IIb (NMHCIIa, NMHCIIb), the myosin regulatory light chain (MLC2) and beta--actin (ßACT).* (C) rhTRAIL--induced apoptosis under the inhibition of NMII ATPase activity. BJ cells were pre--treated for 30 minutes with the indicated concentrations of solvent or blebbistatin and then co--treated with 1µg mL --1 rhTRAIL (+). Apoptosis was assessed as 7A6 positivity 16 hours after the addition of rhTRAIL.** (D) ML7 binds at the ATP--binding site of MLCK inhibiting its catalytic activity. In the absence of MLC2 phosphorylation the NMII molecule has a circular, inactive and assembly--incompetent conformation (left), cannot open (middle) or spontaneously assemble into myosin filaments (right). The figure is based on the idea of [Vicente--Manzanares et al 2009, Book: Alberts et al Molecular Biology of the Cell (Garland Science)]. (E) NMHCII/DR5 interaction upon inhibition of NMII activity. Normal BJ cells were incubated for 30 minutes with vehicle ethanol:water (1:1) (--) or 50µM MLC2--phosphorylating kinase (MLCK) inhibitor ML7 (+) and then collected to IP DR5. The IP--eluates were analysed as described in (A).*** (F) rhTRAIL--induced apoptosis under the inhibition of the MLC2-phosphorylating kinase (MLCK). BJ cells were pre--treated for 30 minutes with the indicated concentrations of solvent or ML7 and then co--treated with 1µg mL --1 rhTRAIL (+). Apoptosis was assessed as in (B).**** Depicted are Western blots for 1 of *3, respectively ***2 independent experiments. Values derive from **3 to 4, respectively ****2 to 5 independent experiments. Significances were calculated using unpaired, 2--tailed Student's t--test.

Figure 28 :

 28 Figure 28: Differences in MLC2 phosporylation and kinase expression levels in normal BJ and transformed BJELR cells. (A) Normal (BJ) and HTERT, SV40ER and HRASG12V--transformed transformed (BJELR) cells were lysed and whole cell lysates (WCL) were subjected to Western blot analysis to explore the relative expression levels of the myosin regulatory light chain (MLC2). (B) BJ and BJELR cells were left untreated (--) or were treated for 15min with 1µg mL --1 rhTRAIL (+) and analysed for protein expression levels of the MLC2 phosphorylating kinases myosin light chain kinase (MLCK), Rho--associated kinase 1 (ROCK1) and the MLCK phosphorylating and inactivating p21--activated protein kinase 1 (PAK1). PAK1 activity was assessed immuno--blotting for phosphorylated BAD. Representative Western blots for 1 of 2 independent experiments.

  NMHCIIb--KD (21% ±10) or SRC--control BJ cells (14% ±5) (Figure29, C). Questioning whether the increased rhTRAIL apoptosis was mediated at the level of the DISC, we performed Western blot analysis of KD cells treated with TRAIL and revealed blots for CASP8 cleavage. We found stronger CASP8 cleavage product signals in NMHCIIa--(Figure29, D) but not in NMHCIIb--KD cells (not shown) corresponding to the fact that upon NMHCIIa--KD more normal cells respond to rhTRAIL--induced apoptosis. Given that CASP8 ativation was increased upon NMHCIIa--KD, we performed DISC analyses in NMHCIIa--KD BJ cells immunoprecipitating DR5. Importantly, NMHCIIa--KD did not alter total expression levels of DISC protein members (Figure29, E). We found that upon NMHCIIa--KD and rhTRAIL--induction DR4 recruitment to DR5 was lowered, whereas CASP8 cleavage an/or recruitment was increased and in turn cFLIP(L) processing and/or recruitment was reduced (Figure29, E). Thus, NMHCII depletion sensitises normal BJ cells to rhTRAIL--induced apoptosis reducing anti--apoptotic cFLIP(L) levels and increasing active CASP8 levels at the DISC. Likewise we have observed in blebbistatin experiments (Figure27, B), NMHCIIa--depletion did not abrogate the interaction of ßACT with DR5, supporting the hypothesis that ßACT binding to DR5 occurs independent of NMIIa.Attempts to measure DR5 and DR4 surface levels upon NMHCIIa--KD in normal BJ cells were not finished at the time this manuscript was generated, and hence were incorporated into the section 'Work in Progress'.

Figure 29 :

 29 Figure 29: Effects of NMHCIIa-- and NMHCIIa siRNA knockdown on cell proliferation, rhTRAIL apoptosis, caspase cleavage and DISC formation in normal BJ cells. Normal (BJ) cells were transfected with 15nM siRNA against non--muscle myosin heavy chain IIa or IIb (IIa, IIb), scramble control siRNA (S) or were mock--transfected with lipofectamine (M) for 4d. Cell culture medium was renewed 1d and 3d after start of transfection. (A) Whole cell lysates (WCL) of BJ cells were analysed for NMHCIIa and NMHCIIb expression levels upon 4d knockdown (KD) by Western blot.* (B) Total BJ cell numbers were counted at 4d of KD. Values are means of 3 independent experiments. (C) TRAIL--induced apoptosis upon NMHCIIa and NMHCIIb siRNA KD. Four days after start of KD BJ cells were left untreated (--) or were treated with 1µg mL --1 rhTRAIL (+). Apoptosis was assessed as 7A6 positivity 16 hours after the addition of rhTRAIL. Values represent mean values of 4 independent experiments. (D) BJ cells were treated with 1µg mL --1 rhTRAIL for the indicated time 4 days after start of KD. Whole cell lysates were analysed for NMHCIIa knockdown efficiency and caspase--8 and --3 (CASP8, CASP3) cleavage by Western blot.** (E) TRAIL--induced DISC formation upon NMHCIIa siRNA KD. BJ cells were left untreated (--) or were treated with 1µg mL --1 rhTRAIL (+) for 1h 4 days after start of KD. Death receptor 5 (DR5) was immunoprecipitated and analysed for co-purified proteins by Western blot. Pre--cleared cell lysates (inputs) served as loading controls and as indicators for KD--mediated alterations in protein expression.** Depicted are Western blots for 1 out of *3 and **2 independent experiments. Significances were calculated using unpaired, 2--tailed Student's t--test.

  at the DISC by cleaved cFLIP(L)p43 [Kataoka et al 2000, Park et al 2001]. RAF1 phosphorylates and activates MEK1 [Zheng et al 1996], it regulates apoptosis targeting FAS, apoptosis signal--regulating kinase 1 (ASK1), mammalian STE20--like protein kinase 2 (MST2), and ROCK2 [Piazzolla et al 2005, Matallanas et al 2011] and it inhibits MYPT1

Figure 30 :

 30 Figure 30: GRB2 and RAF1 binding to DR5 and effects of GRB2 siRNA knockdown on cell proliferation, TRAIL apoptosis and TRAIL--induced caspase cleavage in normal BJ cells. (A) Amino acid sequences of tumor necrosis factor receptor 1 (TNFR1) and death receptor 5 and 4 (DR5, DR4). Highlighted in yellow are the cytoplasmic domains and boxed in red are SH3 protein binding motifs. (B) Normal (BJ) fibroblasts were left untreated (--) or were treated with 1µg mL --1 rhTRAIL and the DISC isolated by immunoprecipitation (IP) targeting DR5. Equal volumes of pre--cleared lysates (inputs) or DR5--IP eluates

Figure 33 :

 33 Figure33: Identification of novel TRAIL DR5--DISC interactors by in--solution digestion of whole DR5--DISC--IP eluates of normal BJ and transformed BJELR cells followed by mass spectrometry. (A) The TRAIL-induced DISC was isolated immunoprecipitating (IP) DR5 from normal (BJ) and transformed (BJELR) cells that were left unstimulated (--T) or that were stimulated for 1h with 1µg mL --1 rhTRAIL (+T). DR5 and co-purified proteins were eluted from beads, digested in--solution and given for mass--spectrometric analysis. Parallel control--IPs using a non--specific immunoglobulin G (IgG)--antibody (AB) were performed and processed as DR5--IPs. Eluates of utilised IP--AB--coupled beads were analysed to determine levels of contamination. (B) Mass spectrometry results for non--muscle myosin heavy chain IIa and IIb (NMHCIIa, NMHCIIb) and beta--actin (ßACT) obtained from DR5--IP indicated cells and condition and of control DR5--IP--AB--coupled beads (B). (C) Showcase mass spectrometry results acquired from parallel DR5--and IgG--IP of unstimulated BJELR cells. Indicated are total numbers of identified proteins per IP (total # proteins) and the position of NMHCIIa and DR5 in each IP that were ranked according to the number of identified peptides (rank (# peptides)). A protein was considered 'identified' if at least 2 peptides were specified.

Figure 34 :

 34 Figure 34: Apoptosis induction at high levels of myosin light chain kinase (MLCK) inhibitor ML7 in normal BJ and transformed BJELR cells. Apoptotic response of normal (BJ) and HTERT, SV40ER and HRASG12V--transformed (BJELR) cells were assessed in paralleled experiments. Cells were pre--incubated with indicated concentrations of ML7 or solvent (ethanol/water mix at a 1:1 ratio) for 30 min and then co-treated with 1µg mL --1 purified rhTRAIL. Apoptosis was quantified as 7A6 positivity in FACS analysis after 16h of treatment. Values derive from 2 independent experiments.

Figure 35 :

 35 Figure 35: Actin stress fiber formation in normal BJ and transformed BJELR cells. Normal (BJ) and HTERT, SV40ER and HRASG12V--transformed (BJELR) human foreskin fibroblasts were fixed and stained with rhodamine--phalloidin and Hoechst. Images were taken by differential interference contrast microscopy (DIC) and fluorescence microscopy. Size bar in the upper image represents 30µm and in the lower image 19µm.

(

  Figure24, B&G). Caspase--facilitated cleavage of cytoskeleton proteins during apoptosis progression is well documented (actin filaments[START_REF] Chen | Activation of actin--cleavable interleukin 1beta--converting enzyme (ICE) family protease CPP--32 during chemotherapeutic agent--induced apoptosis in ovarian carcinoma cells[END_REF][START_REF] Van Damme | Caspase--specific and nonspecific in vivo protein processing during Fas--induced apoptosis[END_REF], intermediate filaments[START_REF] Oshima | Apoptosis and keratin intermediate filaments[END_REF], microtubules[START_REF] Gerner | The Fas--induced apoptosis analyzed by high throughput proteome analysis[END_REF], microtubule motors[START_REF] Lane | Apoptotic cleavage of cytoplasmic dynein intermediate chain and p150(Glued) stops dynein--dependent membrane motility[END_REF][START_REF] Dix | Global mapping of the topography and magnitude of proteolytic events in apoptosis[END_REF]). More specifically, during FAS-induced apoptosis cleavage of NMHCIIa, IIb, MLC2 and ßACT by CASP8, 3 and 7, respectively was reported[START_REF] Fischer | Many cuts to ruin: a comprehensive update of caspase substrates[END_REF][START_REF] Van Damme | Caspase--specific and nonspecific in vivo protein processing during Fas--induced apoptosis[END_REF]. Since proteolytic cleavage can modulate protein interactions, we asked whether cleavage of DR-interacting actin cytoskeleton proteins can be observed during TRAIL--apoptosis. As expected, in transformed BJELR cells that were treated with 1µg mL --1 rhTRAIL for 16h we observed a decline of full--length NMHCIIa and NMHCIIb protein signals as well as the appearance of small--molecular weight fragments below the full--length signals of NMHCIIa, NMHCIIb and ßACT (Figure36, A). Parallel experiments in normal BJ cells gave no cleavage of cytoskeleton proteins (not shown). Thus, NMHCIIa, NMHCIIb and ßACT are broken up during TRAIL--apoptosis. We then asked if the NMII dissociation from DR5 and DR4 observed at 1h of rhTRAIL (Results, Figure24, D, Supplementary,

  and in this way could inhibit NMII Mg 2+ --ATPase activity leading to the disassembly of NMII from DR. At present, nothing can be stated of whether rhTRAIL--stimulated PAK2 cleavage in transformed cells occurs, but will be investigated in future experiments. At least, evidence for the occurrence of an increase in MLC2 phosphorylation levels upon rhTRAIL in transformed cells is low since the observed an rhTRAIL--mediated induction of MLCK expression was weak in transformed BJELR and robust in normal BJ cells. Moreover, the ROCK1 full--length protein signals remained unaltered upon rhTRAIL stimulation in BJELR cells (Results, Figure28, B).This suggests that a CASP--mediated activating cleavage of MLCK or ROCK1 do not play a role in the early TRAIL apoptosis signalling steps of BJELR cells.

Figure 36 :

 36 Figure 36: Fragmentation of NMHCIIa, NMHCIIb and ßACT during rhTRAIL apoptosis and NMII/DR5 interaction in transformed BJELR cells with pan--caspase inhibition. (A) Transformed (BJELR) cells were

Figure 37 :

 37 Figure 37: Caldesmon 1 and calmodulin co--purify with DR5 in normal BJ and transformed BJELR fibroblasts. (A) Normal (BJ) and hTERT, SV40ER and HRASG12V--transformed (BJELR) cells that were left unstimulated (--) or that were stimulated with 1µg mL --1 rhTRAIL (+) for 1h by immunoprecipitation (IP). IP eluates were split into two fractions of which the death receptors 5 or 4 (DR5, DR4) were immunopurified. DR5--and DR4--IP--eluates were separated by SDS--PAGE, blotted onto nitrocellulose membranes and investigated for caldesmon 1 (CALD1) signals.* (B) DR5 was purified and analysed as in (A), except that cells were stimulated for 45min with rhTRAIL and that blots were explored for calmodulin (CALM).** Indicated are Western blots for 1 of *1, respectively of **2 independent experiments.

  minutes and subsequently assessed DR5 surface levels by direct immuno--staining and analysis of alterations (Δ) in mean or median fluorescence intensities (MFI) by FACS. We found that rhTRAIL--induced DR5 internalisation was less pronounced in normal BJ than in transformed BJELR cells (Figure38, A). This suggested that NMII activity and NMII/DR5 interaction prevents rather than promotes internalisation of DR5. On the other hand, if NMII activity and NMII/DR5 interaction were involved in the DR5 transport to the cell surface, lower TRAIL--induced DR5 internalisation in normal BJ cells could result from enhanced transport processes in normal cells.Probing whether inhibition of NMII activity and NMII/DR5 interaction would increase the level of DR internalisation observed in normal BJ cells, we treated cells with 25µM blebbistatin for 30min before assessing rhTRAIL--induced DR5 internalisation. We found that FACS--assisted immunostaining of DR5 is inappropriate to assess rhTRAIL-induced DR5 internalisation under the influence of blebbistatin, because single blebbistatin treatment lead to a decline in DR5 surface levels over time. Whilst DR5 surface levels were equal to vehicle treated cells after 30 min of treatment with agent, 90 and 210 min after agent addition DR5 surface levels were decreased (Figure38, B). This result implies a functional role of NMII activity in the cell surface transport of DR5 transport in normal BJ cells, which contrasts previously mentioned absence of correlation between NMII/DR5 interaction intensity and NMII activity and general DR5 surface levels in normal BJ and transformed BJELR cells. Attempts to measure DR5 surface levels upon inhibition of NMII activity by siRNA--mediated KD of NMHCIIa protein revealed that the assessment of DR5 surface levels by immunostaining and FACS analysis appears to be inappropriate, since the normalisation of mean fluorescence intensities (MFI) to autofluorescence causes a bias. We observed that 4d NMHCIIa KD significantly increased normal BJ cell autofluorescence as compared to scramble control cells (NMHCIIa=15.6 (±0.4) (p=0.03), SCR=13.3 (±0.7)) (Figure 38, C, central plot). Whilst absolute measured MFI of DR5 revealed no difference in NMHCIIa KD and control cells (Figure 38, C, upper left plot), after normalisation to autofluorescence values it appears as if NMHCIIa KD would be associated with decreased DR5 surface levels (Figure 38, C, upper right plot). This result would confirm data obtained in blebbistatin experiments. However, to the current state we refrain from drafting a final conclusion about the function of NMII regarding DR5 surface transport and rather work on direct immunofluorescence analyses to verify beforehand mentioned data.

Figure 38 :

 38 Figure 38: DR5 internalisation and DR5 surface levels upon blebbistatin treatment and NMHCIIa siRNA knockdown in normal BJ cells. (A) TRAIL--mediated DR5 internalisation in normal (BJ) and transformed (BJELR) cells. Graphs depict the decline of the mean DR5 fluorescence intensity (MFI) in dependence of the time of rhTRAIL incubation [minutes]. MFIs were normalised to the mean autofluorescence intensity of each cell line and treatment, respectively. To enable a comparison between cell lines and treatments, normalised MFIs at 0 min of rhTRAIL were set to 100% and all normalised MFIs measured during rhTRAIL incubation were referred to this value. Left plot depicts mean fluorescence values [%], right plot shows median fluorescence intensities [%]. (B) DR5 surface levels upon blebbistatin--mediated inhibition of NMII activity in BJ cells. Graph depicts the decline of the mean DR5 MFI in dependence of the time of 25µM blebbistatin or vehicle (DMSO) incubation (in minutes). (C) DR5 surface levels upon NMHCIIa siRNA KD in BJ cells. Plots depict the decline of the mean DR5 MFI after 4d of transfection with lipofectamin (M), scambled siRNA (S) or NMHCIIa (IIa). Prior to DR5 surface level assessment cell culture medium was changed 1d and 3d after start of transfection. Values of each of the presented plots derive from 3 independent experiments.

Figure 39 :

 39 Figure 39: NMII/DR5 interaction in pre--transformed BJEL and transformed BJELR cells. Unstimulated (--) HTERT and SV40ER pre--transformed cells (BJEL) and hTERT, SV40ER and HRASG12V--transformed (BJELR) cells were subjected to immunoprecipitation (IP) targeting death receptor 5 (DR5). IP eluates were separated by SDS--PAGE, blotted onto nitrocellulose membranes and investigated for non--muscle myosin heavy chain IIa and IIb (NMHCIIa, NMHCIIb) signals. Shown are the Western blot results of a single experiment.

  NMII filaments might inhibit TRAIL apoptosis and limit DR--mediated CASP8 activation also in malignant cells, mechanically inhibiting CASP8 recruitment by compartmentalisation. Like the actin cytoskeleton protein spectrin [reviewed by[START_REF] Sheetz | Continuous membrane--cytoskeleton adhesion requires continuous accommodation to lipid and cytoskeleton dynamics[END_REF], NMIIa--tethered fibers could form a barrier to membrane protein lateral diffusion. Consistent with the refined DISC model (Introduction, Figure6)[START_REF] Schleich | Stoichiometry of the CD95 Death--Inducing Signaling Complex: Experimental and Modeling Evidence for a Death Effector Domain Chain Model[END_REF][START_REF] Dickens | A Death Effector Domain Chain DISC Model Reveals a Crucial Role for Caspase--8 Chain Assembly in Mediating Apoptotic Cell Death[END_REF], NMIIa activity may even determine CASP8 chain lengths by compartmentalisation. Verification of this aspect will require the DISC characterisation in NMHCIIa KD cells under inhibition of CASP activation (please see also 'Work in Progress', p131), as it will demonstrate whether increased levels of CASP8 cleavage products are caused by enhanced CASP8 recruitment or CASP8 cleavage. Finally, we observed reduced cFLIP(L)p43 levels in the DISC of NMHCIIa KD normal BJ cells as compared to SCR control cells, whilst cFLIP(L) protein levels were unaltered (Results, Figure29, E) and normal cell adhesion was reduced. As NMII function in integrin--mediated cell adhesion (Introduction, Figure16) [reviewed by[START_REF] Vicente--Manzanares | Non--muscle myosin II takes centre stage in cell adhesion and migration[END_REF], these data provide an important link to studies on FAS signalling, which demonstrated that the cytosolic solubility and availability of cFLIP(L) for binding to FADD in the DISC and for inhibiting CASP8 activation decreased upon disruption of β1 integrin--mediated adhesion to fibronectin[START_REF] Shain | Adhesion--mediated intracellular redistribution of c--Fas--associated death domain--like IL--1--converting enzyme--like inhibitory protein--long confers resistance to CD95--induced apoptosis in hematopoietic cancer cell lines[END_REF]. Notably, a transcriptional or translational regulation of cFLIP(L) by integrin--mediated adhesion was excluded and the adhesion--induced increase of cytosolic cFLIP(L) originated from pre--existing cFLIP(L) that was associated to the membrane fraction. Thus, during adhesion NMIIa activity may control intracellular cFLIP(L) localisation, positively regulating the availability of cFLIP(L) to bind to the DISC.NMIIa-facilitated TRAIL resistance in suspension cells. Given the prominent role of NMII in cell adhesion, it remains to be investigated if an NMII/DR interaction in TRAIL--resistant suspension cells at all occurs and, if so, whether NMIIa likewise confers TRAIL--resistance. Investigating a potential NMII/DR interaction and function in suspension cells is particularly interesting since aplidin--induced apoptosis in Jurkat cells depended on the clustering of FAS in lipid rafts, an event that required an intact actin cytoskeleton and that was accompanied by the co--recruitment of RHOA and RHO--GDI into lipid rafts (see 'Introduction', p36)[START_REF] Gajate | Cytoskeleton--mediated death receptor and ligand concentration in lipid rafts forms apoptosis--promoting clusters in cancer chemotherapy[END_REF]. Since RHO--GDI sequesters inactive (GDP--bound) RHOA, preventing RHOA--and ultimately ROCK/NMII activation, NMII activity is most probably not involved in the formation of those FAS caps. Contrary, since RHO--GDI recruitment can be strongly inhibited by NMII Mg 2+ ATPase activity[START_REF] Kuo | Analysis of the myosin--II--responsive focal adhesion proteome reveals a role for beta--Pix in negative regulation of focal adhesion maturation[END_REF], it seems more likely that the disruption of NMII activity leads to a 'misorganisation' of plasma membrane proteins. Notably, whilst in most cases we could detect at least some residual NMII signals at the DR of BJELR cells, no signal at all was discovered at the DR of BJAB cells. If this is due to the higher TRAIL sensitivity of BJAB cells, or due to their growth in suspension, remains to be elucidated. If and how NMII could compartmentalise suspension cells is difficult to predict. Even if suspension cells are capable to form fibers upon surface attachment [Rosengart et al 2002, Nakamura et al 2005], there is nothing known about the existence of actomyosin fibers in cells while being in suspension. Isoform specificity of observed effects on TRAIL sensitivity. We found that only NMHCIIa KD, but not NMHCIIb KD lead to a decrease in cell adhesion and a robust release of the TRAIL resistance block in normal BJ cells. Contrary, loss of NMHCIIb strongly reduced normal BJ cell proliferation, whilst cell adhesion and TRAIL--sensitivity were left unaltered (Results, Figure 29, C). This finding to a certain degree reflects the importance of the NMHCIIa and IIb isoforms for in vivo functions. Whilst gene depletion of NMHCIIa results in a complete failure of organogenesis in mice, NMHCIIb diminuition primarily affects the morphogenesis of heart and brain. Thus, NMHCIIa appears to have a more generalised role in the cell adhesion processes and differentiation than NMHCIIb. Since NMHCIIa in vivo can at least partially replace NMHCIIb gene function, we cannot rule out that observed consistent TRAIL--resistance upon NMHCIIb KD is due to compensation by NMHCIIa. Unfortunately, no in vivo data exist where NMHCIIb was substituted for NMHCIIa. Thus, it is unknown if NMHCIIb can functionally replace NMHCIIa to a similar degree. Finally, since the biochemical properties of NMHCIIc and NMHCIIb more closely resemble than NMHCIIc and NMHCIIa (see 'Introduction', p64), NMHCIIc could compensate NMHCIIb but not NMHCIIa, which could be the underlying reason for the differential effects of isoform KD on TRAIL--sensitivity [Golomb et al 2004]. The NMHCIIc isoform has not at all been considered in this study, as it was never detected in mass--spectrometrical analyses. Nevertheless, we cannot exclude that it might impact on TRAIL signalling. Mode of NMII/DR interaction. To the current state we can only speculate about how NMII may interact with DR5 and DR4. Like it has been reported for other membrane receptors [Rey et al 2002, Timmermann et al 2004, Rey et al 2007], in a direct binding situation NMII theoretically could bind to DR via the NMHCII tail, via the NMHCII head domain, via the region N--terminal of the head (SH3--like fold), or via MLC2 (Introduction, Figure 14, A) [Amparan et al 2005, Bajaj et al 2009]. Interestingly, MLC2 binding to N--methyl--D--aspartate receptor subunits occurred via the recognition of an incomplete IQ2--motif (GXXXR) [Bajaj et al 2009], a sequence pattern which can be found in the intracellular region, just upstream the DD of DR5(L), DR5(S) and DR4 and in the intracellular region of DcR2, but not in the DcR1 sequence. If SH3--like folds, such as the one located N--terminal of the NMII head, indeed behave like SH3 domains [D'Aquino and Ringe 2003], then SH3--like folds preferentially bind to PXXP (and RXXK) motifs [Cohen et al 1995, Berry et al 2002]. All TRAIL receptors except DR4 contain a PXXP sequence in their protein sequence. Thus, at least 2 different direct binding options are possible. In an indirect binding setting one or several proteins could act as bridge. Whilst we found that ßACT binds to the DR independently of NMII (Results, Figure 27, B) and NMIIa and NMIIb bind independently of each other (Results, Figure 27, E), we cannot exclude that ßACT mediates the interaction between NMIIa and DR since we have not created a situation in which NMIIa remained at the DR and ßACT was lost. To verify this, we will deplete ezrin and moesin in normal BJ cells, immunoprecipitate DR5 and control the co--purification of ßACT and NMIIa. Apart from ßACT, NMII could bind to DR5 and DR4 via a yet unidentified linker protein.

  normal and transformed cells. Comparing MLC2 phosphorylation levels and MLC2 regulatory kinase expression levels in normal BJ and transformed BJELR cells (Results, Figure 28, B&C) we concluded that general NMII Mg 2+ --ATPase activity in transformed cells must be low. Indeed, comparison with pre--existing transcriptomic data of the stepwise tumourigenesis system cells revealed that in transformed BJELR and HA1ER cells transcript levels of ROCK or MLCK are decreased. Contrary, PAK1 transcripts were unaltered, indicating a post--translational regulation of PAK1, which is often overexpressed in multiple cancer types [reviewed by Molli et al 2009, Dummler et al 2009, Eswaran et al 2009]. Interestingly, PAK1 protein accumulation upon TNFα--or IL2--stimulation in human dermal fibroblasts and keratinocytes was also concluded to be regulated at post--translational level, based on the evidence that suppression of protein synthesis did not inhibit PAK1 accumulation

  Interestingly, interference with DR signalling in myoblasts by different means (pan--CASP or CASP8 inhibition, expression of dominant--negative FADD or DR5 constructs), decreased mRNA and protein levels of the transcription factor myoblast determination protein 1 (MYOD) [Freer--Prokop et al 2009]. Binding sites for MYOD can be found in the gene promoter of smMLC2, but not nmMLC2 or nmlMLC2. Moreover, MYOD levels are downregulated by calpains [Stuelsatz et al 2010], which in turn are activated by ERK at focal adhesions [Glading et al 2000, Glading et al 2004]. In transformed BJELR cells the whole RHOA signalling pathway required for focal adhesion formation might have collapsed due to their SV40ER background. As a result, BJELR cells most likely have few

  Figure 24, G), raising the question of what mediates cFLIP(L) processing if not CASP8?With respect to the refined DISC model (Introduction, Figure6) it could be that the initiator CASP and cFLIP 'chains' binding to one FADD molecule in normal BJ cells primarily consist of cFLIP(L) molecules. In this case, few activated CASP8 molecules would process many cFLIP(L) proteins.

Figure 40 :

 40 Figure 40: Proposed model of the NMIIa/DR interaction regulating the cFLIP(L)--mediated switch between morphogenic and apoptotic TRAIL signalling in normal (N) and transformed (T) cells. In normal cells NMHCIIa positively regulates cell adhesion and cytoplasmic cFLIP(L) levels whilst it blocks cell migration. During the process of transformation NMIIa activity is reduced to enable detachment/reattachment cycles during enhanced proliferation and to facilitate migration putatively mediated by NMIIb. Therefore NMIIa is lost from the DR of transformed cells which leads to decreased cFLIP(L) availablility and hence increased CASP8 cleavage. In normal cells that have high NMIIa activity and a strong NMIIa/DR interaction, TRAIL stimulation induces robust cFLIP(L) cleavage. Thus, the NMIIa/cFLIP(L) axis in normal cells promotes TRAIL--induced morphogenesis.

Constitutive

  GRB2 and RAF1 interactions with DR5 and DR4 in normal BJ cells strongly point to a pro--survival signalling function of the DR. Indeed, observed equal DR expression levels and similar rhTRAIL--induced DR clustering in normal BJ and transformed BJELR cells, but absent apoptosis induction in normal cells implied the question about the function of TRAIL signalling in normal cells. Why should normal cells express DR at high levels if they were of no function? The notion that DR via RAF1 may signal towards survival in normal cells is supported by the fact that all attempts to generate constitutive DR5 or DR4 knockout cells failed as yet either because of cell death or poor cell growth. Contribution of this study to the understanding of TRAIL resistance mechanisms in transformed cells and possible therapeutic implications. This study provides direct evidence that TRAIL apoptosis signalling requires a disrupted (NMIIa--) cytoskeleton. Unlike normal cells, cancer cells do not undergo apoptosis upon cytoskeletal changes. This is most probably due to activated survival pathways (XYZ) counterbalancing the cytoskeleton--mediated apoptosis signal allowing the cancer cell to survive (or to an inactivated cytoskeleton/apoptosis pathway). If TRAIL is added to transformed cells, this balance is challenged/abolished and moved towards apoptosis.Contrary, in normal cells we find regular cytoskeleton architecture, therefore TRAIL does not induce apoptosis. Inherently TRAIL--resistant cancer cells might escape from TRAIL--apoptosis either because they have maintained their NMIIa/DR interaction, or in these cells the XYZ--pathway(s) signal(s) stronger than in TRAIL--sensitive cancer cells and thus counterbalance TRAIL--apoptotic challenge. Future studies investigating the NMIIa/DR interaction in TRAIL--insensitive (solid) cancer cells will have to elucidate this assumption. For TRAIL--based cancer therapies one can only hope that the second situation is the case since a safe cancer cell--specific rupture of the NMIIa/DR interaction is unlikely to be achieved by the combined use of cytoskeleton inhibitors and rhTRAIL preparations. If TRAIL--resistance in cancer cells is based on hyperactive XYZ--survival pathway(s) at least those could be blocked in targeted therapies and a combo--therapy of those cancers with XYZ--inhibitors and TRAIL were imaginable.Findings presented in this study might help to calculate the risk for an enhanced migratory cancer phenotype in TRAIL--therapy. At least in cancer cells with inactivated NMII activity, through for instance down--regulated RHOA and/or increased RAC1 or CDC42 activity, TRAIL will most probably not enhance migration, but will signal to apoptosis.Concluding remarks. Plenty remains to be learnt about the initial steps of TRAIL signalling. Certainly, the DISCome is not yet fully defined and the complete understanding of its single compounds stays a challenge. In future DISC studies extra care should be taken on the cell context, the model cells' genetic background as well as on the stimulus applied. The development of technologies enabling quantitative analyses of single DISC formation events would help to advance our understanding of TRAIL apoptotic signalling.

  . The death receptors 5 and 4 (DR5, DR4) contain a death domain (DD) that serves for the assembly of signaling complexes[MacFarlane et al 1997, Pan et al 1997a, Pan et al 1997b[START_REF] Schneider | TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD--dependent apoptosis and activate NF--kappaB[END_REF][START_REF] Degli--Esposti | Cloning and characterization of TRAIL--R3, a novel member of the emerging TRAIL receptor family[END_REF]. The other 2 TRAIL receptors, the decoy receptors (DcR1, DcR2), possess no or a truncated DD and do not transduce apoptotic signals [MacFarlane et al 1997, Pan et al 1997b, Schneider et al 1997, Degli--Esposti et al 1997]. Oligomerisation of DR upon TRAIL binding induces interaction with adaptor molecules such as Fas--associated death domain (FADD) that in turn recruits initiator caspase--8 (CASP8) and/or 10 (CASP10) [Chaudhary et al 1997, MacFarlane et al 1997, Bodmer et al 2000, Kischkel et al 2000, Sprick et al 2000; Kischkel et al 2001, Wang et al 2001, Sprick et al 2002]. Similarly, anti-apoptotic cellular FLICE--like inhibitory protein (cFLIP) is recruited to the DR competing with CASP8 for FADD binding and triggering survival pathways [Thome et al 1997, Burns and El--Deiry 2001, Krueger et al 2001, Chang et al 2002, Jin et al 2004]. The complex comprising TRAIL--receptors, FADD and initiator caspases constitutes the death--inducing signaling complex (DISC) (Figure 1) [Bodmer et al 2000]. Whilst DISC formation in transformed cells is extensively studied, almost no information is available about native DISC composition in normal cells.

Figure 1 :

 1 Figure1: TRAIL DISC formation and apoptotic signalling. TNFalpha--related--apoptosis inducing ligand (TRAIL) binding induces the formation of death receptor 5 and 4 (DR5, DR4) microaggregates. Receptor aggregation leads to the recruitment of the adaptor molecule Fas-associated death domain (FADD) that co--recruits initiator pro--caspase 8, 10 (CASP8, CASP10) or the anti--apoptotic cellular Flice--like inhibitory protein (cFLIP), which lacks protease activity. Resulting close proximity of CASP molecules and/or cFLIP at the DR results in their mutual cleavage and activation. The complex minimally comprising DR, FADD, CAP8/10 and/or cFLIP is called the death inducing signalling complex (DISC) (yellow). Once activated at the DISC, CASP8/10 can directly cleave and activate effector caspases 3 and 7 (CASP3, CASP7) and hence fulfil the so--called extrinsic apoptosis pathway. In certain cells the apoptotic signal is propagated and enhanced via the intrinsic apoptotic cascade. Therefore DISC--activated CASP8/10 proteolytically convert BH3--interacting domain death agonist (BID) to the active, truncated form (tBID) that induces BCL2--antogonist/killer (BAK) and BCL2--associated X protein (BAX) at the mitochondria and in turn results in permeabilisation of the outer mitochondrial membrane and release of proteins such as cytochrome c, second mitochondria--derived activator of caspases (SMAC)/direct inhibitor of apoptosis protein (IAP)--binding protein with low pI (DIABLO). Cytochrome c binds to adaptor molecule apoptotic protease--activating factor 1 (APAF1) that binds to initiator procaspase 9 (CASP9) to form the apoptosome. At the apoptosome, CASP9 is activated leading to processing of CASP3/7 and eventually cell death. The figure is based on the idea of[START_REF] White--Gilbertson | Transformation, translation and TRAIL: an unexpected intersection[END_REF][START_REF] Newsom--Davis | Is TRAIL the holy grail of cancer therapy?[END_REF].

  , therefore representing an attractive therapeutic option for cancer therapy [Russo et al 2010, Gerspach et al 2011, Pavet et al 2011, Yerbes et al 2011, Martinez--Lostao et al 2012]. Although TRAIL promotes cell death in a plethora of cancer cells, some tumour cells are either insensitive or acquire resistance upon TRAIL exposure [Gerspach et al 2011, Yerbes et al 2011]. Albeit multiple modulators of TRAIL signalling are known [Newsom--Davis et al 2009, Gonzalvez and Ashkenazi 2010, Gerspach et al 2011, Shirley et al 2011, Martinez--Lostao et al 2012], the molecular mechanisms underlying the lack of TRAIL sensitivity in normal cells remain obscure. Understanding the mechanistic basis of normal cells being refractory to TRAIL stimulation will be important to decipher how normal cells loose their TRAIL resistance upon transformation. The present thesis work therefore focused on the central question how normal cells that are originally TRAIL-resistant gain TRAIL--sensitivity by the process of tumourigenic transformation.

Figure 2 :

 2 Figure 2: Scheme of Hahn and Weinberg's stepwise transformation model. Normal cells (here: human foreskin fibroblasts (BJ) or human embryonic kidney (HEK)) were gradually transformed by introducing the human telomerase catalytic subunit (HTERT), simian virus 40 early region (SV40ER) and a constitutively active HRAS mutant (HRASG12V).Whilst hTERT maintains telomere length and confers replicative immortality to BJ cells, HEK cells require additional SV40ER--encoded elements to reach immortality. Obtained transformed cells (here: BJELR or HA1ER) display cancer cell features like unlimited proliferation, anchorage--independent growth and tumour formation in vivo and are sensitive to TRAIL--induced apoptosis.

  Indeed, using mass spectrometry of differential protein bands co--purifying with DR-immunoprecipitations in normal and transformed cells and DR--immunoprecipitations followed by Western blot analyses, the cytoskeleton proteins nonmuscle myosin heavy chain IIa and IIb (NMHCIIa, NMHCIIb), myosin regulatory light chain 2 (MLC2) (not shown) and beta--actin (ßACT) were identified as novel DR--interactors in DISC immunoprecipitates (Figure4, A&B). Importantly, normal cells displayed higher levels of NMHCIIa and IIb co--immunoprecipitating with DR5 and DR4 than transformed cells.Upon TRAIL stimulus, NMHCIIa and IIb interaction with DR was sustained in normal cells, whilst they were largely lost from the DISC formed in transformed cells (Figure4, B).

Figure 4 :

 4 Figure 4: Identification of novel TRAIL--DISC interactors in normal BJ and transformed BJELR fibroblasts.The scheme indicates the experimental set--up. After immunoprecipitation (IP), proteins were eluted and eluates were split into 2 fractions. One aliquot was separated on SDS--PAGE gels and transferred onto nitrocellulose membranes that were stained with Ponceau S. Observed differential bands were excised from the membranes, proteins were digested and analysed by mass spectrometry (MS). The other aliquot was separated on SDS--PAGE gels, blotted onto nitrocellulose membranes and analysed for protein interactions using specific antibodies (AB). In this way (A) death receptor 5 (DR5) was purified from lysates of BJ and BJELR cells that were left unstimulated (--) or that were stimulated for 1h with 1µg mL --1 recombinant human TRAIL (rhTRAIL) (+). Differential bands visualised with Ponceau S were cut out, digested and given for mass--spectrometrical peptide identification. (B) Western blot analysis of DR5--IP eluates to validate the mass--spectrometric results obtained in (A) (upper panel). Death receptor 4 (DR4) was purified under similar experimental conditions and analysed by Western blot (lower panel). Depicted

Figure 6 :

 6 Figure 6: Effects of NMHCIIa--and NMHCIIa siRNA knockdown on TRAIL apoptosis and DISC formation in normal BJ cells. BJ cells were transfected with 15nM siRNA against non--muscle myosin heavy chain IIa or IIb (IIa, IIb), scramble control siRNA (S) or were mock--transfected with lipofectamine (M) for 4d. Cell culture medium was renewed 1d and 3d after start of transfection. (A) Whole cell lysates (WCL) of BJ cells were analysed for NMHCIIa and NMHCIIb expression levels upon 4d knockdown (KD) by Western blot analysis.* (B) TRAIL--induced apoptosis upon NMHCIIa and NMHCIIb siRNA KD. Four days after start of KD BJ cells were left untreated (--) or were treated with 1µg mL --1 rhTRAIL (+). Apoptosis was assessed as 7A6 positivity 16 hours after the addition of rhTRAIL. Values represent mean values of 4 independent experiments. (C) TRAIL--induced DISC formation upon NMHCIIa siRNA KD. BJ cells were left untreated (--) or were treated with 1µg mL --1 rhTRAIL (+) for 1h 4 days after start of KD. Death receptor 5 (DR5) was immunoprecipitated and analysed for co--purified proteins by Western blot. Pre--cleared cell lysates (inputs) served as loading controls and as indicators for KD--mediated alterations in protein expression. Depicted are Western blots for 1 out of *3, independent experiments. Significances were calculated using unpaired, 2--tailed Student's t--test.

Figure 7 :

 7 Figure 7: Myosin regulatory light chain (MLC2) Ser18/Thr19 phosphorylation and kinase expression in normal BJ and hTERT, SV40ER and H--Ras transformed BJELR cells. (A) Complexity of MLC2 Thr18/Ser19 phosphorylation. Myosin regulatory light chain (MLC2) is activated by phosphorylation of Thr18/Ser19. Major phosphorylation site is Ser19 that allows non--muscle myosin II (NMII) to form NMII filaments, to interact with actin, to assemble actomyosin complexes and to initiate contraction. Di--phosphorylation at both Ser19 and Thr18 further promotes filament assembly. Many kinases known to phosphorylate and activate MLC2 Thr18/Ser19 (red) also phosphorylate and inhibit (red lines) MLC2 inactivating myosin phosphatase (blue). The figure is based on [Matsumura 2005] (B) Difference in MLC2 phosporylation levels. Whole cell lysates (WCL) of BJ and BJELR cells were analysed by Western blot for the relative phosphorylation and expression levels of MLC2. (C) BJ and BJELR cells were left untreated (--) or were treated with 1µg mL --1 rhTRAIL for 15min (+) and analysed for protein expression levels of MLCK, ROCK and PAK1. PAK1 activity was assessed immuno--blotting for phosphorylated BAD. Imaged were representative Western blots for 1 of 2 independent experiments. (D) Apoptosis induction at high levels of MLCK inhibitor ML7. Apoptotic response of BJ and BJELR cells were assessed in paralleled experiments.Cells were pre--incubated with indicated concentrations of ML7 or solvent (ethanol/water mix at a 1:1 ratio) for 30 min and then co--treated with 1µg mL --1 purified TRAIL. Apoptosis was quantified as 7A6 positivity in FACS analysis after 16h of treatment. Values derive from 2 independent experiments.

Figure 1 :

 1 Figure1: Formation de TRAIL--DISC et voie de signalisation apoptotique. La liaison de TRAIL (TNFalpha--related--apoptosis inducing ligand) induit la micro--agrégation des "récepteurs de mort" (DR ou "Death Receptors") DR4 et DR5. L'agrégation de ces récepteurs conduit au recrutement de la molécule adaptatrice FADD (Fas--associated death domain) qui co--recrute les pro--caspases initiatrices 8 et 10 (CASP8, CASP10), ou le facteur anti--apoptitique cFLIP (cellular Flice-like inhibitory protein), qui ne possède pas d'activité protéolytique. La promiscuité au niveau des DR des CASP et/ou de cFLIP permet leur activation par clivage. Le complexe minimal DR, FADD, CASP8/10 et/ou cFLIP est nommé DISC (pour "death inducing signalling complex", complexe de signalisation induisant la mort, en jaune dans la figure). Une fois activée dans DISC, les CASP8/10 peuvent directement cliver et activer les caspases effectrices 3 et 7 (CASP3, CASP7) et ainsi s'acquitter de la voie dite d'apoptose extrinsèque. Dans certaines cellules, le signal d'apoptose est propagé et amplifié via la voie dite d'apoptose intrinsèque. Ainsi, les CASP8/10 activées au sein de DISC protéolysent la protéine BID (BH3--interacting domain death agonist) en sa forme tronquée et active, tBID. A son tour, tBID active BAK et BAX (respectivement "BCL2-antogonist/killer" et "BCL2--associated X protein") à la mitochondrie, ce qui résulte en une perméabilisation de la membrane externe mitochondriale, et à la libération de facteurs tels le cytochrome C et SMAC/DIABLO [second mitochondria--derived activator of caspases (SMAC)/direct inhibitor of apoptosis protein (IAP)--binding protein with low pI (DIABLO)]. Le cytochrome C se lie à la molécule adaptatrice APAF1 (apoptotic protease--activating factor 1) qui se lie à la pro--caspase initiatrice 9 (CASP9) pour former l'apoptosome. A l'apoptosome, CASP9 est activée, ce qui entraîne le clivage de CASP3/7 et la mort cellulaire. La figure est basée sur les propositions de[START_REF] White--Gilbertson | Transformation, translation and TRAIL: an unexpected intersection[END_REF][START_REF] Newsom--Davis | Is TRAIL the holy grail of cancer therapy?[END_REF].

C

  'est pourquoi elle représente une option thérapeutique prometteuse pour le traitement du cancer [Russo et al 2010, Gerspach et al 2011, Pavet et al 2011, Yerbes et al 2011, Martinez--Lostao et al 2012]. Bien que TRAIL induise la mort cellulaire dans une pléthore de cellules cancéreuses, il existe certaines cellules tumorales qui y sont soit insensibles soit qui acquièrent une résistance à la suite d'une exposition à TRAIL [Gerspach et al 2011, Yerbes et al 2011]. Même si de nombreux modulateurs de la signalisation de TRAIL sont connus [Newsom--Davis et al 2009, Gonzalvez and Ashkenazi 2010, Gerspach et al 2011, Shirley et al 2011, Martinez--Lostao et al 2012], les mécanismes moléculaires qui sont à la base de l'absence de sensibilité à TRAIL dans les cellules normales restent obscurs. Comprendre quel est le mécanisme à la base de la résistance de cellules normales à la mort induite par TRAIL est important car il permettrait de découvrir la façon dont les cellules normales perdent leur résistance à TRAIL au cours de la transformation. Ce travail de thèse porte donc sur cette question centrale : comment une cellule normale qui est à l'origine résistante à TRAIL acquiert la sensibilité à TRAIL au cours du processus de transformation oncogénique.Etant donné que la formation du DISC est à l'origine de la signalisation de TRAIL (Figure1), nous avons émis l'hypothèse qu'il devait exister des partenaires encore inconnus dans le fonctionnement du DISC. Ces facteurs seraient recrutés, modifiés ou exprimés différemment au cours de la transformation oncogénique et entraineraient une différence dans la sensibilité à TRAIL. Par conséquent, nous avons entrepris l'identification de nouveaux facteurs du DISC qui sont recrutés de manière différente dans les cellules normales et tumorales.Nous avons utilisé un modèle cellulaire de transformation par étapes[START_REF] Hahn | Creation of human tumour cells with defined genetic elements[END_REF] qui permet une comparaison directe des cellules cancéreuses avec leurs cellules progénitrices normales isogéniques (Figure2). Une telle analyse comparative n'est pas possible entre des lignées de cellules cancéreuses et des lignées cellulaires soi--disant 'normales'. Alors que les cellules transformées issues de ce modèle par étapes présentent des caractéristiques tumorigéniques[START_REF] Hahn | Creation of human tumour cells with defined genetic elements[END_REF] et de sensibilité à TRAIL, les cellules humaines normales parentales y sont résistantes[START_REF] Nesterov | Oncogenic Ras sensitizes normal human cells to tumor necrosis factor--alpha--related apoptosis--inducing ligand-induced apoptosis[END_REF], Wang et al 2005].

Figure 2 :

 2 Figure 2: Schéma de Hahn et Weinberg expliquant le modèle de transformation progressive. Des cellules normales [ici fibroblastes de prépuce humain (BJ) ou cellules embryonnaires de rein humain (HEK)]sont progressivement transformées en introduisant la sous--unité catalytique télomérique HTERT, la séquence virale SV40ER (simian virus 40 early region) et HRASG12V, un mutant constitutivement actif de la protéine HRAS. Alors que hTERT permet de maintenir la longueur des télomères et de conférer l'immortalité réplicative aux cellules BJ, les cellules HEK requièrent des éléments additionnels encodés par SV40ER pour atteindre l'immortalité. Les cellules transformées ainsi obtenues (ici: BJELR ou HA1ER) présentent des propriétés de cellules cancéreuses: une prolifération illimitée, une croissance indépendante de leur adhésion, leur capacité de former des tumeurs in vivo, et leur sensibilité à l'apoptose après stimulation par TRAIL.

Figure 3 :

 3 Figure 3: Formation de TRAIL--DISC de cellules normales BJ et de cellules transformées BJELR. Un nombre équivalent de cellules BJ et BJELR sont incubées avec 1µg/ml de TRAIL humain recombinant (rhTRAIL) pendant 1h, puis lysées. Le récepteur de mort DR5 est immuno--précipité (IP) au moyen d'un anticorps dirigé contre la protéine endogène. Les protéines co--purifiées dans les éluats de l'IP sont analysées. Les 4 pistes de gauche, chargées par des volumes identiques d'extraits cellulaires (BJ ou BJELR) totaux pré-clarifiés (inputs) utilisés dans l'expérience d'IP, servent de contrôle. Les 5 pistes de droite sont chargées par des volumes identiques d'éluats d'IP de DR5 (IP:DR5), provenant soit de la lignée cellulaire indiquée (BJ ou BJELR), soit du mélange DR5/résine (B) utilisé dans l'expérience. Le résultat montré est une analyse par immuno--buvardage (western--blot).

Figure 4 :

 4 Figure 4: Identification de nouveaux partenaires de TRAIL--DISC dans des fibroblastes normaux (BJ) ou transformés (BJELR) stimulés ou non (+/--) par TRAIL. Le schéma (en haut) montre les étapes de l'expérience. Après immuno--précipitation (IP), les protéines sont éluées et les éluats séparés en 2 fractions. Un aliquot est séparé par électrophorèse en conditions dénaturantes (SDS--PAGE) et transféré sur membrane de nitrocellulose, qui est alors colorée au rouge Ponceau. Les bandes observées différant d'une condition à l'autre ont été excisées de la membrane, et leur contenu protéique digéré et analysé par spectrométrie de masse (MS). Ultérieurement, l'autre aliquot est ultérieurement séparé par SDS--PAGE, transféré sur membrane de nitrocellulose, et analysé par western--blot en utilisant des anticorps spécifiques (AB) des partenaires de la protéine immuno--précipitée nouvellement identifiés par MS, afin de confirmer leur identité. (A) Ainsi, DR5 est immuno--purifié à partir de lysats de cellules BJ ou BJELR, stimulées (+) ou non--stimulées (--) au préalable pendant 1h avec 1µg/ml de TRAIL humain recombinant (rhTRAIL). Les bandes différentielles visualisées par le rouge Ponceau sont coupées et analysées par MS afin d'identifier par leur signature peptidique les partenaires protéiques potentiels de DR5 présents dans ces bandes. (B) L'analyse par Western--Blot des DR5--IP (panneau du haut) permet de valider les partenaires identifiés en (A) par MS. Le récepteur de mort DR4 est analysé selon le même procédé expérimental, le panneau du bas montre l'analyse finale par western blot. Chaque western--blot présenté est représentatif de deux expériences menées indépendamment.

Figure 5 :

 5 Figure 5: Effets de l'inhibition de l'activité Mg2+--ATPase de NMHCII sur l'interaction NMHCII/DR dans les cellules normales. (A) Schéma représentant le cycle dit "crossbridge" régulé par l'activité Mg2+--ATPase associée au domaine "Head" de la chaîne lourde II non--musculaire de myosine (NMHCII), ainsi que le mécanisme d'action de l'inhibiteur blebbistatine. 1. la liaison de l'ATP à NMHCII--Head induit une conformation ne pouvant pas lier l'actine. 2. L'hydrolyse de l'ATP entraîne l'adoption par le domaine Head d'une conformation dite "chargée", dans laquelle ADP et Pi restent liés. Lorsque le Pi quitte NMHCII, le domaine Head se lie à l'actine, ce qui conduit à une libération subite d'énergie mécanique. 4. L'ADP est libéré et le cycle peut reprendre. A ce stade NMHCII est fortement lié à l'actine. La blebbistatine se fixe préférentiellement à NMHCII--Head dont le site actif Mg2+--ATPase est occupé par l'ADP et le Pi. La libération d'ADP est alors fortement ralentie et la liaison à l'actine est empêchée. (B) Interaction NMHCII/DR lors d'un traitement à la blebbistatine. Des cellules BJ normales sont incubées 30 minutes avec le solvant DMSO (--) ou la blebbistatine à 25µM dans du DMSO (+), puis collectées pour immuno--précipiter DR4 ou DR5. Les éluats d'IP sont analysés quant à l'interaction de NMHCIIa, NMHCIIb, de la chaîne légère de myosine regulatrice (MLC2) et de la beta-actine (ßACT) *. (C) Apoptose induite par TRAIL pendant traitement à la blebbistatine. Des cellules BJ sont pré-traitées pendant 30 minutes avec les concentrations indiquées de solvant ou de blebbistatine, puis co--traitées avec rhTRAIL (1µg/ml). ** (D) Schéma expliquant l'accroissement de l'activité Mg2+--ATPase par la phosphorylation de MLC2 et le mécanisme d'action de l'inhibiteur ML7. En absence de phosphorylation, NMHCII a une conformation circulaire, inactive et non--compétente pour assemblage (gauche). Après phosphorylation par MLC2 par la kinase de chaînes légères de myosine (MLCK), NMHCIIs adopte une conformation plus ouverte et active (centre) et s'assemble spontanément sous forme de filaments de myosine (droite). ML7 se lie au site de liaison à l'ATP de MLCK et inhibe son activité catalytique. La figure est inspirée de [Vicente--Manzanares et al 2009, Book: Alberts et al Molecular Biology of the Cell (Garland Science)]. (E) Interaction NMHCII/DR5b pendant traitement au ML7. Des cellules BJ normales sont incubées pendant 30 minutes avec le solvant éthanol:eau (1:1) (--) ou avec l'inhibiteur ML7 (MLC2--phosphorylating kinase (MLCK) inhibitor) à 50µM dans un solvant éthanol:eau (1:1) (+), puis collectées pour IP DR5. les éluats d'IP sont anaylysés comme décrit en (B). *** (F) Apoptose induite par TRAIL pendant traitement au ML7. Des cellules BJ sont pré--traitées pendant 30 minutes avec les concentrations indiquées de solvant ou de ML7, puis co--traitées avec rhTRAIL (1µg/ml). L'apoptose est estimée comme expliqué en (C). **** Les western--blots montrés sont représentatifs de respectivement 3 (*) et 2 (***) expériences indépendantes. Les valeurs moyennes sont calculées respectivement de 3 à 4 (**) et 2 à 5 (****) expériences indépendantes. Les significances statistiques sont calculées par test "unpaired, 2-tailed Student".

Figure 6 :

 6 Figure 6: Effets du "Knock--down" par siRNA de NMHCIIa et NMHCIIb sur l'apoptose liée à TRAIL et la formation de DISC dans des cellules normales BJ. Des cellules BJ sont transfectées en présence de lipofectamine par 15nM de siRNA dirigé contre NMHCIIa ou NMHCIIb (IIa, IIb), 15nM de siRNA contrôles "scramble" (S = non spécifiques), ou à vide (M = "mock") et cultivées pendant 4 jours. Le milieu de culture est changé 1 jour, puis 3 jours après transfection. (A) Les lysats totaux cellulaires (WCL) de cellules BJ ont été analysés quant au niveau d'expression de NMHCIIa et NMHCIIb après 4 jours de knockdown (KD) par western blot. * (B) Apoptose liée à TRAIL après Knock--Down de NMHCIIa et NMHCIIb par siRNA. Quatre jours après le début du KD, les cellules BJ sont non--traitées (--) ou traitées (+) avec rhTRAIL à 1µg/ml. L'apoptose est estimée 16h après addition de TRAIL par positivité à 7A6. Les valeurs indiquées représentent la moyenne de 4 expériences indépendantes. (C) Formation de DISC induite par TRAIL après siRNA--KD de NMHCIIa. Quatre jours après début du KD, des cellules BJ sont non--traitées (--) ou traitées (+) par rhTRAIL à 1µg/ml pendant 1h. DR5 est alors immuno--précipité et les protéines co--purifiées sont analysées par western blot. Des lysats cellulaires pré--clarifiés (inputs) servent de contrôles de chargement et sont indicateurs des altérations d'expression protéique liées au KD. Les western--blots montrés sont représentatifs de 3 expériences indépendantes (*). Les significances statistiques sont calculées par test "unpaired, 2--tailed Student".

Figure 7 :

 7 Figure 7: Phosphorylation de la chaîne légère régulatrice de myosine (MLC2) aux résidus Thr18/Ser19 et expression de kinase, dans des cellules normales BJ et dans des cellules BJELR transformées par hTERT, SV40ER et H--Ras. (A) Le patron de phosphorylation de MLC2 aux résidus Thr18/Ser19 est complexe. La chaîne légère régulatrice de myosine MLC2 est activée par phosphorylation de Thr18/Ser19. Le site majeur de phosphorylation est Ser19, qui permet à la myosine non--musculaire II (NMII) de former des filaments NMII, d'interagir avec l'actine, d'assembler des complexes d'actomyosin et d'initier la contraction. La double phosphorylation aux résidus Thr18/Ser19 stimule davantage l'assemblage en filaments. De nombreuses kinases connues pour phosphoryler et activer MLC2 Thr18/Ser19 (rouges) phosphorylent aussi et inhibent ainsi (lignes rouges) la phosphatase qui inactive MLC2 (bleue). La figure s'inspire de [Matsumara 2005]. (B) Différences de niveaux de phosphorylation de MLC2. Des extraits cellulaires totaux (WCL) de cellules BJ et BJELR sont analysés par western blot quant à la phosphorylation relative et aux niveaux d'expression de MLC2. (C) Des cellules BJ et BJELR sont soit non--traitées (--), soit traitées (+) par rhTRAIL à 1µg/ml pendant 15 minutes, et les niveaux d'expression des protéines MLCK, ROCK and PAK1 sont analysés. L'activité de PAK1 est estimée par western--blot au moyen d'un anticorps dirigé contre une forme phosphorylée de BAD. La figure montre un western blot représentatif de deux expériences indépendantes. (D) Forte induction d'apoptose pour des niveaux élevés de ML7, l'inhibiteur de MLCK. La réponse apoptotique de cellules normales BJ et tumorales BJELR est estimée dans des expériences menées en parallèle. Les cellules sont pré--incubées pendant 30 minutes avec les concentrations indiquées de ML7 ou de solvant (éthanol:eau au ratio 1:1), puis co--traitées avec TRAIL purifié à 1µg/ml. L'apoptose est quantifiée par la positivité à 7A6 par tri cellulaire (FACS) après 16h de traitement. Les valeurs données découlent de deux expériences indépendantes.

Table 1 :

 1 Examples for targeted cancer therapies directed against cancer hallmarks. Indicated are small molecule inhibitors and monoclonal antibodies, which are FDA--approved or currently tested in clinical trials [http://fda.gov, http://clinicaltrials.gov]. The primary mode of anti--estrogen ICI 182780 action is the rapid MYC expression[START_REF] Carroll | Mechanisms of growth arrest by c--myc antisense oligonucleotides in MCF--7 breast cancer cells: implications for the antiproliferative effects of antiestrogens[END_REF], reviewed by Hermeking 2003].

	cancer hallmark	cellular target	agent	name	product/company
	self--sufficiency in	epidermal growth	inhibitor	gefitinib	Iressa/AstraZeneca
	growth signalling	factor receptor (EGFR)
		tyrosine kinase--type	monoclonal	trastuzumab	Herceptin/Genentech
		cell surface receptor	antibody
		(HER2)
	insensitivity to	proto--oncogene MYC	inhibitor	ICI 182780	Fulvestrand/
	growth inhibitory	(via estrogen receptor)			AstraZeneca
	signals
	evasion from	tumour suppressor	inhibitor	JNJ--26854165,	JNJ--26854165/Janssen
	apoptosis	p53/ E3 ubiquitin--		RG7112	Research &
		protein ligase MDM2			Development,
		interaction			RG7112/Hoffmann--La
					Roche
	limitedless	telomerase	inhibitor	imetelstat	GRN163/Geron
	replicative
	potential
	sustained	vascular endothelial	monoclonal	bevacizumab	Avastin/Genentech/
	angiogenesis	growth factor (VEGF)	antibody		Roche
			inhibitor	/	Axitinib/Pfizer, Inc
	tissue invasion and	hepatocyte growth	inhibitor	tivantinib	ARQ197/ArQule
	metastasis	factor receptor (HGFR)
		cMET

Meinem Mann Thomas für die Weitsicht.

SUPPLEMENTARY

Figure 31: DR5--, DR4--and CASP8--DISC formation of normal BJ and transformed BJELR cells and the interaction with NMHCIIa and NMHCIIb. Equal numbers of normal (BJ) and HTERT, SV40ER and HRASG12V--transformed (BJELR) cells were left unstimulated (--) or were incubated with 1µg mL --1 rhTRAIL for 1h. Cell lysates were pre--cleared (inputs) and subsequently split into 3 fractions of which death receptor 5 or 4 (DR5, DR4) or caspase--8 (CASP8) were immunoprecipitated. Identical volumes of inputs, and immunoprecipitation (IP) eluates were separated by SDS--PAGE gel--electrophoresis and surveyed for DISC and cytoskeletal proteins by Western blot. The control lane (B) contains the eluate of IP--antibody coupled beads that were utilised in this experiment. Shown are Western blots of 1 full experiment out of 2 independent attempts.