UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
Molecular regulation of death receptor- and DNA damage-induced apoptosis
Maas, C.
Publication date
2010
Link to publication
Citation for published version (APA):
Maas, C. (2010). Molecular regulation of death receptor- and DNA damage-induced
apoptosis.
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s)
and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open
content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please
let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material
inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter
to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You
will be contacted as soon as possible.
Chapter
1
Apoptosis and cancer
In order to eliminate aged, damaged or infected cells, metazoans have developed the highly coordinated cell death program termed apoptosis. This genetic program is essential for embryogenesis, maintenance of adult tissue homeostasis and proper functioning of the immune system. Typical morphological and biochemical features characterize apoptosis, including cell shrinkage, nuclear fragmentation, chromatin condensation, DNA degradation and membrane blebbing, discerning it from a more pathological form of cell death termed necrosis [1,2]. Finally, cells undergo orderly dismantlement into apoptotic bodies, facilitating their removal by the immune system. By exposing phosphatidyl serine on the outer leaflets of their membranes, apoptotic bodies send out ‘eat-me’ signals for phagocytes, which engulf them upon recognition. Hereby, the release of toxic cellular content into the environment and concomitant induction of an inflammatory response, characteristic of necrosis, are prevented and damage to surrounding tissue is avoided. Every single day the human body removes about 60 billion cells by apoptosis and replaces them with new ones. In a year, this amounts to the elimination and proliferation of a mass of cells equal to an individual’s body weight [3]. Understandably, defects in apoptosis can disturb this delicate balance and can contribute to the development of various diseases. Whereas excessive apoptosis can cause hypotrophy, manifesting in neuro-degenerative diseases, insufficient apoptosis can lead to hypertrophy, as seen in autoimmune diseases and cancer [4].
Defects in apoptosis pathways are also a cause of tumor cell resistance to conventional anti-cancer regimens [5]. However, because the core apoptosis machinery remains intact in tumor cells, they are often still vulnerable to cytotoxic signals. Indeed, paradoxically, as a result of several aberrations acquired during oncogenic transformation, e.g. in cell cycle progression and DNA damage repair, tumor cells are often even more sensitive to apoptosis-inducing stimuli than normal untransformed cells [6].
Hence, promoting apoptosis appears to be an attractive anti-cancer strategy, which might be more tumor-specific than conventional therapeutics and mediate only low level toxicity to normal tissue.
Apoptosis signaling pathways
Apoptosis is tightly regulated by Caspases, a family of highly conserved aspartate-specific cysteine proteases. Caspases are synthesized as inactive pro-forms and require either dimerization or proteolytic cleavage to become activated. Apoptotic Caspases can be subdivided into two classes: inducer Caspases and effector Caspases. Inducer Caspases (Caspases-2, -8, -9 and -10) initiate apoptosis by cleaving and activating the effector Caspases (Caspase-3, -6 and -7), which in turn execute apoptosis by cleaving a large subset of other proteins [7,8]. Two Caspase-activating apoptosis pathways can be distinguished in mammalian cells: the intrinsic pathway and the extrinsic pathway. The intrinsic pathway is induced by different endogenous stress signals, such as DNA-damage or shortage of growth factor support, while the extrinsic pathway is engaged upon ligation of death receptor ligands to their receptors on the cell surface[9,10].
Intrinsic pathway of apoptosis
The intrinsic pathway proceeds via the mitochondria and is regulated by the Bcl-2 family of proteins, which comprises the pro-apoptotic BH3-only and Bax/Bak-type proteins and the anti-apoptotic Bcl-2 proteins (Figure
1). The Bcl-2 proteins are characterized by the
presence of one or multiple Bcl-2 Homology (BH) domains. BH3-only proteins (Bid, Bim, Bad, Noxa, Puma, Bik, Bmf and Hrk) contain just one BH domain; the BH3 domain. Bax/ Bak-type proteins (Bax, Bak and Bok) contain three BH domains, BH1-3, while the anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL, Mcl-1, A1 and Bcl-w) contain four BH domains, BH1-4 [11]. Different cellular stressors mediate the induction of distinct BH3-only proteins, which subsequently activate Bax/Bak. For instance, several DNA damaging anti-cancer regimens
(e.g. ionizing radiation and etoposide) induce double strand DNA breaks, which is sensed by ATM and CHK2 kinases, that phosphorylate and activate p53 [12,13]. Subsequently, p53 transcriptionally activates BH3-only proteins Puma and Noxa, which in turn activate Bax/ Bak (Figure 2) [14,15]. Upon activation, Bax/ Bax homomultimerize and form permeabilizing pores in the outer mitochondrial membrane, mediating the release of several apoptogenic factors including Cytochrome c, Smac/DIABLO
and HtrA2/Omi [16]. Cytochrome c binds Apaf-1, inducing the formation of a heptameric complex called the apoptosome, which subsequently binds and activates inducer Caspase-9. In turn, Caspase-9 cleaves and activates the effector Caspases-3, -6 and -7 [17]. Smac/DIABLO and HtrA2/Omi neutralize inhibitor of apoptosis (IAP) proteins (e.g. XIAP), which can inhibit partially activated initiator and effector Caspases [18-20]. Upon IAP inhibition by Smac/DIABLO and HtrA2/Omi, the effector Caspases are fully activated and subsequently irreversibly induce cell death. The anti-apoptotic Bcl-2 proteins sequester their pro-apoptotic relatives and hereby inhibit Bax/Bak activation and mitochondrial
permeabilization [11]. The exact mechanism of Bax/Bak activation is not yet fully understood; two distinct models have been proposed. The ‘direct activation’ model postulates that the BH3-only proteins can be subdivided into ‘direct activators’ and ‘sensitizers’. In this model, the direct activators (tBid, Bim and potentially Puma) directly bind to Bax/Bak and promote their activation. The sensitizers (Bad, Noxa, Bik, Hrk and Bmf) displace the direct activators from the anti-apoptotic Bcl-2
proteins, allowing them to activate Bax/Bak [21]. The ‘indirect activation’ model poses that all the BH3-only proteins indirectly activate Bax and/or Bak by neutralizing the inhibitory Bcl-2 proteins [22,23].
Extrinsic pathway of apoptosis
Death receptor ligands of the TNF cytokine superfamily tumor necrosis factor (TNFα), Fas/CD95 ligand and TNFα-related apoptosis inducing ligand (TRAIL) initiate the extrinsic pathway of apoptosis (Figure 2). TNFα and Fas ligand induce apoptosis via their receptors TNFR1 and Fas, respectively. For TRAIL, four different membrane-bound receptors have been identified: death receptor 4 (DR4), death Figure 1. Diagrammatic representation of the family of Bcl-2 proteins. TM = transmembrane domain. BH = Bcl-2 homology
do-main TM Bcl-2 TM Bcl-xL TM Bcl-w TM A1 TM Mcl-1 Anti-apoptotic Pro-apoptotic BH3-only proteins Bad Bid Noxa TM TM Bim Bik TM Hrk Puma TM Bax TM Bok TM
Bak Bax/Bak-type proteins
BH1 BH2 BH3 BH4
receptor 5 (DR5), decoy receptor 1 (DcR1) and decoy receptor 2 (DcR2). In addition, TRAIL can bind to the soluble receptor osteoprotegerin (OPG) with low affinity. Whereas DR4 and DR5 signal apoptosis, DcR1 and DcR2 are non-apoptotic and behave as decoy receptors [9]. Binding of death receptor ligands to their pro-apoptotic receptors induces receptor trimerization and mediates formation of the death inducing signaling complex (DISC) through recruitment of (at least) FADD and inducer Caspases-8 and -10.
Induced proximity of pairs of Caspases-8 or -10 molecules mediates their dimerization and autocatalytic activation and subsequent release into the cytoplasm, where they cleave and activate the effector Caspases [24-26]. In addition, Caspases-8 and -10 cleave BH3-only protein Bid to engage the mitochondrial pathway of effector Caspase activation, thereby connecting the extrinsic pathway with the intrinsic pathway. In a positive feedback loop, activated effector Caspases further enhance apoptosis by activating more Caspase-8 or -10
[27]. Paradoxically, death receptor ligands can also activate pro-surival pathways at the same time, e.g. the NF-κB pathway [28]. Whether a cell dies or survives upon death receptor activation appears to depend on the balance between pro- and anti-apoptotic signaling. Cells are distinguished into two types on basis of their dependence on the mitochondrial pathway for death receptor-induced apoptosis; Type I and Type II cells. Type I cells can die independently of the mitochondria in response to death ligands, because of their ability to
activate sufficient levels of Caspase-8 and -10 at the DISC to directly activate the effector Caspases. In contrast, Type II cells require the mitochondria for death ligand-induced apoptosis because of their incapacity to robustly activate Caspases-8/-10 [29]. They rely on the concerted action of Cytochrome c and IAP antagonists Smac/DIABLO and HtrA2/Omi for effector Caspase activation. Although generally believed to be unimportant for apoptosis, Bid cleavage and mitochondrial permeabilisation also occurs in Type I cells. Figure 2. Schematic representation of the extrinsic and intrinsic pathways of apoptosis. Death receptor ligands activate the
extrin-sic pathway, whereas endogenous stress signals mediate the activation of the intrinextrin-sic pathway.
Death receptor
(e.g. Fas, DR4, DR5)
Death receptor ligand
(e.g. Fas ligand, TRAIL)
Smac/DIABLO Cytochrome c XIAP Active Caspase-8/-10 Mitochondrion Bid HtrA2/Omi Pro Caspase-8/10 FADD DISC c-FLIP Bax Bak Bcl2 Puma Noxa DNA damage P53 Extrinsic pathway
Death receptor ligands
Intrinsic pathway Stress signals Apaf-1 Apoptosome Apoptosis Caspase-9 Caspase-3/6/7
The distinction between Type I and Type II cells can be made both in vitro, for long-term established cell lines and in vivo, for primary cell types. In vivo, thymocytes and activated T cells are regarded as typical Type I cells and hepatocytes as Type II cells [30-34].
Death receptor ligands as
anti-cancer therapeutics
Since their discovery, the death receptor ligands have attracted much attention as potential anti-cancer reagents. The tumor-killing properties of TNFα, Fas ligand and TRAIL have been evaluated extensively, both in vitro and in vivo. Both TNFα and Fas ligand can kill tumor cells, but their systemic administration causes severe toxicity in vivo. Whereas TNFα mediates a strong inflammatory response, Fas ligand induces apoptosis of liver cells resulting in severe hepatitis [35-38]. Clearly, this has tempered their potential as anti-cancer therapeutics. In contrast, TRAIL has been found to selectively induce apoptosis in tumor cells and leave most normal cells unharmed. Systemic administration of TRAIL has been shown to induce significant regression of human xeno-transplanted tumors in mice and to mediate no apparent side effects in both mice and primates [39,40]. Why tumor cells are sensitive to TRAIL and normal cells are not, is not yet clear. However, possible explanations begin to emerge, as will be discussed. Importantly, TRAIL triggers apoptosis in tumor cells irrespective of their p53 status. This is a powerful feature, since inactivating mutations in p53, which are present in approximately 50% of all tumors, do convey resistance to many conventional anti-cancer regimens, particularly to those that engage the intrinsic pathway of apoptosis [41]. This ability, together with its tumor selectivity, makes TRAIL a potentially powerful anti-cancer therapeutic. Particularly, the combination with conventional regimens that can synergize with TRAIL, due to engagement of the (partially) distinct intrinsic apoptosis pathway, holds great promise. Recently, different clinical trials have been initiated with TRAIL and with specific agonistic antibodies to
its receptors DR4 or DR5, as monotherapies and in combination with other anti-cancer regimens. The results of these trials need to be awaited to see whether TRAIL can meet the expectations. A new formulation of Fas ligand, APO010 (or MegaFasligand), expected to be more tumor-specific and therefore less toxic to normal tissue than formerly tested variants, is currently being studied as an alternative to TRAIL.
Regulation of death
receptor-induced apoptosis
Obtaining detailed knowledge of factors modulating the apoptotic response to death receptor ligands is of great importance for their future clinical application as anti-cancer therapeutics. Importantly, it will help to identify ‘biomarkers’ of sensitivity and resistance to death receptor ligands. On basis of the levels of such biomarkers present in tumors, patients suitable for treatment with death receptor ligands can be rationally selected. In addition, it will aid the development of new anti-cancer regimens that can enhance the response to death receptor ligands. Over the years, different factors regulating death receptor-induced apoptosis have been identified and much knowledge has been gained on their mechanisms of action.
Obviously, the availability of pro-apoptotic death receptors at the plasma membrane is crucial for the response to death receptor ligands. Loss of membrane expression of death receptors can be a cause of tumor cell resistance to death receptor ligands. In TRAIL-selected colon carcinoma cells, defects in the transport machinery of DR4 and DR5 have been shown to abrogate their membrane expression and mediate resistance to TRAIL [42]. Loss of general expression of DR4 and/ or DR5, caused by chromosomal aberrations or single nucleotide polymorphisms, is frequently observed in human tumors and might also mediate TRAIL-resistance [43]. Decoy receptors DcR1 and DcR2 can specifically influence the response to TRAIL, by competing for TRAIL binding with DR4 and DR5. Indeed,
overexpression of DcR1 or DcR2 mediates resistance to TRAIL. Low expression levels of decoy receptors have been suggested to render tumor cells sensitive to TRAIL and, vice versa, high expression levels to render normal cells resistant [44-46]. Although possible, a clear correlation between decoy receptor expression and TRAIL sensitivity in healthy and tumor cells has not been found [47]. Interestingly, a recent study has found that tumor cells might be more sensitive to TRAIL as a result of higher expression of O-glycosylation enzymes. The level of O-glycosylation of DR4 and DR5 was found to determine their capacity to cluster and mediate apoptosis and expression levels of O-glycosyltransferase GALNT14 were found to correlate with TRAIL sensitivity in different tumor cell types [48]. In another study, elevated expression of oncoprotein c-Myc was demonstrated to render tumor cells more sensitive to TRAIL than normal cells [49]. Together, these findings might explain, at least partially, why tumor cells are sensitive to TRAIL and normal cells are not.
Yet, various other factors have also been found to influence the sensitivity to death receptor ligands and might be differentially expressed in normal and tumor cells. For instance, c-FLIP can regulate the formation of the DISC and Caspase-8/-10 activation. The best-characterized isoforms are c-FLIPSand c-FLIPL. Both closely resemble Caspase-8 and -10 but lack a functional catalytic domain. FLIPS can interfere with the recruitment and activation of Caspase-8/-10 in the DISC. When present at high levels in the DISC, FLIPL prevents Caspase-8/-10 activation by competing for binding with FADD. However, at low levels, FLIPL behaves pro-apoptotically by heterodimerizing with Caspase-8/-10 and enhancing their activation [50,51]. Nevertheless, high FLIP levels have been shown to confer resistance to Fas ligand in neuroblastoma and prostate carcinoma cells and to TRAIL in colon carcinoma cells [52-54].
Conversely, downregulation of both c-FLIP isoforms by RNAi sensitizes melanoma cells to both Fas ligand and TRAIL [55]. Thus, c-FLIP plays an important role in the modulation of
death receptor-induced apoptosis.
In Type II cells, which are dependent on the mitochondrial pathway for death receptor-induced apoptosis, members of the Bcl-2 family of proteins also control the response to death receptor ligands. As mentioned, Bcl-2 proteins control the permeabilization of the mitochondria and thereby the release of several apoptogenic factors that mediate effector Caspase activation. Elevated expression of anti-apoptotic Bcl-2 proteins such as Bcl-2, Bcl-xL and Mcl-1 has been shown to cause resistance to Fas ligand and TRAIL in different Type II cell types [29,56-58].
Modulation of Bid activity can also significantly affect the response to death receptor ligands in Type II cells. For instance, phosphorylation by Casein Kinases inhibits Bid cleavage by Caspase-8 and induction of Fas ligand-induced apoptosis in Hela cervical carcinoma cells [59]. Also, myristoylation of tBid-C, the active Bid fragment that is generated upon cleavage by Caspase-8/-10, has been reported to facilitate its mitochondrial targeting and hereby enhance its pro-apoptotic activity [60]. At the start of this thesis research, there were indications that tBid-C activity is in addition regulated via another mechanism. Under non-apoptotic conditions, the N-terminal domain of Bid restrains the pro-apoptotic BH3 domain of tBid-C. Different reports suggested that, after cleavage, tBid-N can still inhibit tBid-C activity and might need to be actively removed. In solution, the separate tBid-N and tBid-C fragments generated upon Caspase-8 cleavage have been shown to remain physically associated. The complex hereby formed did not undergo an apparent conformational change [60-62]. Moreover, free tBid-N has been shown to inhibit tBid-C from permeabilizing mitochondria when overexpressed [62]. This suggested that tBid-N needs to be degraded upon cleavage to allow tBid-C to expose its BH3 domain and engage in apoptosis.
Inhibitor of apoptosis proteins (IAP) constitute another class of apoptosis regulators. XIAP binds partially processed Caspases-3, -7 and -9, thereby preventing their full activation.
Whereas cIAP-1 and c-IAP-2 can also do so, their main function appears to lie in the activation of NF-κB [63]. NF-κB can upregulate different anti-apoptotic proteins, including c-FLIP and several anti-apoptotic Bcl-2 proteins [64]. In addition, IAPs can function as ubiquitin ligases and, as such, can promote the proteasomal degradation of Caspases, IAP antagonists Smac/DIABLO and HtrA2/Omi and the IAPs themselves [65]. Targeting of IAPs with Smac/ DIABLO mimetics has been shown to enhance the sensitivity of different tumor cell types to TRAIL both in vitro and in vivo, implicating an important role for IAPs in regulating death receptor-induced apoptosis [66,67].
Combination strategies with
death receptor ligands
Although many tumor cell types are sensitive to death receptor ligands, others have developed resistance through mechanisms as just described. Combination treatments with other anti-cancer therapeutics might be very effective in breaking resistance in these cells. Different treatment strategies have been employed in mouse tumor models and often additive or even synergistic responses have been obtained. For instance, death receptor ligands can be effectively combined with chemotherapy or radiotherapy. Different DNA-damaging regimens, such as cisplatin, doxorubicin, etoposide and ionizing radiation, have been shown to strongly enhance death receptor-induced apoptosis. Diverse mechanisms have been shown to underlie the effects of these regimens, e.g. augmentation of DISC formation through up-regulation or enhanced recruitment of death receptors, Caspase-8 or FADD or down-modulation of IAPs or anti-apoptotic Bcl-2 proteins [68-72]. Several combination treatments with agonistic antibodies against DR4 (Mapatumumab/HGS-ETR1) or DR5 (Lexatumumab/HGS-ERT2) and chemotherapeutics have entered early phase clinical trials [73].
Another promising strategy is the combined use of death receptor ligands and Smac/DIABLO mimetics. Like endogenous Smac/DIABLO, the
mimetics inhibit XIAP from sequestering semi-activated Caspases-3, -7 and -9 and hereby can enhance apoptosis. In addition, recent studies have shown that Smac/DIABLO mimetics can also promote c-IAP1 degradation, thereby inducing NF-κB activation and subsequent TNFα upregulation and Caspase-8 activation [74-76]. Particularly, Type II tumor cells with elevated Bcl-2 and/or XIAP expression are suitable for this combination treatment. In these cells, activation of effector Caspases can be hampered by a Bcl-2 mediated mitochondrial blockade, which prevents Smac/DIABLO release, and by high XIAP expression. As has been shown, combined activation of the death receptor pathway with TRAIL and targeting of XIAP with a Smac/DIABLO mimetic allows for a bypass of the mitochondrial pathway in Bcl-2 overexpressing cells and restores effector Caspase activation and apoptosis [66]. The BH3 mimetic ABT-737 also seems a very suitable therapeutic to combine with death receptor ligands in the treatment of Bcl-2-, and also Bcl-xL- and Bcl-w-, overexpressing cells. ABT737 neutralizes Bcl-2, Bcl-xL and Bcl-w and hereby lowers the threshold for mitochondrial permeabilization [77]. Hereby, ABT-737 has been shown to potentiate the cytotoxicity of various apoptosis-inducing compounds, including TRAIL [78,79]. An advantage of using a BH3 mimetic such as ABT-737 is that it enables the release of the entire mitochondrial arsenal of pro-apoptotic factors, not just Smac/ DIABLO, and might therefore be more effective than Smac/DIABLO mimetics. In addition, several other promising combination strategies with recombinant TRAIL or agonistic antibodies against DR4 or DR5 are currently being tested in mouse tumor models, including combinations with the immuno-modulatory anti-CD40 and anti-CD137 mAbs (TrimAb), HDAC inhibitors, NF-κB inhibitors and PI3K-Akt inhibitors [80].
Scope of this thesis
Induction of apoptosis is an attractive anti-cancer therapy, as tumor cells are often very sensitive to apoptosis-inducing regimens because of various abberations acquired
during malignant transformation. However, as previously discussed, tumor cells also commonly acquire defects in apoptosis pathways during their oncogenic evolution, hampering optimal responses to anti-cancer therapeutics. Insight in the molecular regulation of apoptosis can help to develop new therapeutic strategies that can break resistance in these cells. Moreover, identification of novel apoptosis-regulating factors that can serve as biomarkers for sensitivity can aid in the selection of patients suitable for a specific treatment. The discovery of RNA interference (RNAi) has made it possible to silence the expression of individual genes and perform large ‘loss-of-function’ RNAi screens, enabling the identification of novel regulatory proteins. Death receptor ligands, particularly TRAIL, are promising new anti-cancer therapeutics. Unlike most conventional therapeutics, death receptor ligands induce apoptosis independently of the p53 status. In addition, TRAIL has the unique capacity to selectively kill tumor cells and spare normal, untransformed cells. To identify new regulators of TRAIL-induced apoptosis, we performed a clonogenicity-based RNAi screen in MCF-7 breast carcinoma cells using the retroviral NKI shRNA library, which targets 7.914 different genes with 23.742 shRNA constructs. MCF-7 cells lack Caspase-3 but, when recomplemented with Caspase-3, behave as Type I cells [29] and thus, unlike Type II cells, reportedly do not require the mitochondrial pathway for death receptor-induced apoptosis. Therefore, we were surprised to identify Bid as an important regulator of clonogenic execution of a significant proportion of MCF-7Casp-3 cells upon
TRAIL treatment. Subsequently, we found that Type I cells generally require the mitochondrial pathway for full TRAIL-mediated clonogenic execution. The release of Smac/DIABLO and the inhibition of XIAP were found to be essential (chapter 2) [81]. Next, we studied the molecular regulation of Bid activation during death receptor-induced apoptosis. We found that, following cleavage, the N-terminal Bid fragment (tBid-N) needs to be ubiquitinated and degraded by the proteasome to allow the
C-terminal Bid fragment (tBid-C) to become activated and mediate apoptosis via the mitochondria. The ubiquitination of tBid-N was unconventional, as it did not involve any lysine residues or the N-terminus (chapter 3) [82]. In addition to its role in death receptor-induced apoptosis, Bid also appears to play a role in DNA-damage induced apoptosis. However, there is no general consensus on this issue yet. Different studies have found Bid to be important for DNA-damage induced apoptosis [83,84], whereas others have found Bid to be irrelevant [85-87]. Indeed, Puma has been shown to be the main BH3-only protein required for DNA-damage induced apoptosis in p53-proficient cells [88]. However, we previously found that Bid is essential for the apoptotic responses to etoposide and ionizing radiation in p53-mutant Jurkat T cell lymphoma cells [89]. We here show that the p53 status determines the level of contribution of Bid to DNA-damage induced apoptosis, a finding that might reconcile previous conflicting observations. In mouse embryonic fibroblasts, Bid can participate in DNA-damage induced apoptosis in presence of p53, but is only fully required in absence of p53. Bid activation during DNA-damage induced apoptosis was found to be unconventional, as it did not require cleavage in the unstructured loop of Bid (chapter 4).
As mentioned, tumor cells often inactivate p53 and/or elevate their expression of anti-apoptotic Bcl-2 proteins to escape from apoptosis induced via the intrinsic pathway. As a result, they can become resistant to different conventional anti-cancer therapeutics. By combining conventional therapeutics with death receptor ligands, resistance imposed by such blockades might be overcome. We combined different conventional regimens with a new formulation of Fas ligand, APO010, and found that, together, they efficiently kill Bcl-2 overexpressing p53-mutant Jurkat cells. All regimens were found to sensitize to APO010 by downregulating c-FLIP levels, rather than by breaking the Bcl-2-imposed mitochondrial block (chapter 5) [90].
signal recognition particle 72 (SRP72) as a regulator of TRAIL-induced apoptosis. SRP72 is part of the larger signal recognition particle, which transports membrane and secretory proteins emerging from the ribosome to the endoplasmic reticulum (ER) and hereby controls their membrane transport and secretion, respectively. SRP72 depletion by RNAi caused resistance to TRAIL-induced
Caspase-3 activation and clonogenic execution. SRP72-deficiency mediated a strong reduction in membrane expression of DR4 and not DR5, but this was found not to be the cause of TRAIL resistance. Rather, SRP72 RNAi was found to inhibit the apoptotic response to TRAIL through an additional effect on DR5-mediated apoptosis (chapter 6). The implications of our findings are discussed in chapter 7.
References
[1] Hengartner, M.O. (2000) The biochemistry of apoptosis. Nature 407, 770-776.
[2] Kerr, J.F., Wyllie, A.H. and Currie, A.R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26, 239-257. [3] Cotter, T.G. (2009) Apoptosis and cancer: the genesis of
a research field. Nat Rev Cancer 9, 501-507.
[4] Hanahan, D., Weinberg, R.A. (2000) The hallmarks of cancer. Cell 100, 57-70.
[5] Johnstone, R.W., Ruefli, A.A. and Lowe, S.W. (2002) Apoptosis: a link between cancer genetics and chemotherapy. Cell 108, 153-164.
[6] Adams, J.M., Cory, S. (2007) The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 26, 1324-1337.
[7] Nicholson, D.W. (1999) Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 6, 1028-1042.
[8] Thornberry, N.A., Lazebnik, Y. (1998) Caspases: enemies within. Science 281, 1312-1316.
[9] Ashkenazi, A. (2002) Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2, 420-430.
[10] Strasser, A., O’Connor, L. and Dixit, V.M. (2000) Apoptosis signaling. Annu Rev Biochem 69, 217-245. [11] Youle, R.J., Strasser, A. (2008) The BCL-2 protein family:
opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9, 47-59.
[12] Canman, C.E., Lim, D.S., Cimprich, K.A., et al. (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281, 1677-1679. [13] Takai, H., Naka, K., Okada, Y., et al. (2002)
Chk2-deficient mice exhibit radioresistance and defective p53-mediated transcription. EMBO J 21, 5195-5205. [14] Nakano, K., Vousden, K.H. (2001) PUMA, a novel
proapoptotic gene, is induced by p53. Mol Cell 7, 683-694.
[15] Oda, E., Ohki, R., Murasawa, H., et al. (2000) Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288, 1053-1058.
[16] Newmeyer, D.D., Ferguson-Miller, S. (2003) Mitochondria: releasing power for life and unleashing the machineries
of death. Cell 112, 481-490.
[17] Wang, X. (2001) The expanding role of mitochondria in apoptosis. Genes Dev 15, 2922-2933.
[18] Du, C., Fang, M., Li, Y., Li, L. and Wang, X. (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33-42.
[19] Suzuki, Y., Imai, Y., Nakayama, H., et al. (2001) A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell 8, 613-621.
[20] Verhagen, A.M., Ekert, P.G., Pakusch, M., et al. (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102, 43-53.
[21] Letai, A., Bassik, M.C., Walensky, L.D., et al. (2002) Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2, 183-192.
[22] Willis, S.N., Chen, L., Dewson, G., et al. (2005) Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev 19, 1294-1305.
[23] Willis, S.N., Fletcher, J.I., Kaufmann, T., et al. (2007) Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science 315, 856-859. [24] Medema, J.P., Scaffidi, C., Kischkel, F.C., et al. (1997)
FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J 16, 2794-2804.
[25] Sprick, M.R., Rieser, E., Stahl, H., et al. (2002) 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. EMBO J 21, 4520-4530.
[26] Hughes, M.A., Harper, N., Butterworth, M., et al. (2009) Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival. Mol Cell 35, 265-279. [27] Slee, E.A., Harte, M.T., Kluck, R.M., et al. (1999)
Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 144,
281-292.
[28] Legembre, P., Barnhart, B.C. and Peter, M.E. (2004) The relevance of NF-kappaB for CD95 signaling in tumor cells. Cell Cycle 3, 1235-1239.
[29] Scaffidi, C., Fulda, S., Srinivasan, A., et al. (1998) Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17, 1675-1687.
[30] Huang, D.C., Hahne, M., Schroeter, M., et al. (1999) Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-x(L). Proc Natl Acad Sci U S A 96, 14871-14876.
[31] Jost, P.J., Grabow, S., Gray, D., et al. (2009) XIAP discriminates between type I and type II FAS-induced apoptosis. Nature 460, 1035-1039.
[32] Lacronique, V., Mignon, A., Fabre, M., et al. (1996) Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice. Nat Med 2, 80-86.
[33] Strasser, A., Harris, A.W., Huang, D.C., Krammer, P.H. and Cory, S. (1995) Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J 14, 6136-6147.
[34] Yin, X.M., Wang, K., Gross, A., et al. (1999) Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400, 886-891.
[35] Creaven, P.J., Plager, J.E., Dupere, S., et al. (1987) Phase I clinical trial of recombinant human tumor necrosis factor. Cancer Chemother Pharmacol 20, 137-144. [36] Hersh, E.M., Metch, B.S., Muggia, F.M., et al. (1991)
Phase II studies of recombinant human tumor necrosis factor alpha in patients with malignant disease: a summary of the Southwest Oncology Group experience. J Immunother (1991. ) 10, 426-431.
[37] Kondo, T., Suda, T., Fukuyama, H., Adachi, M. and Nagata, S. (1997) Essential roles of the Fas ligand in the development of hepatitis. Nat Med 3, 409-413. [38] Ogasawara, J., Watanabe-Fukunaga, R., Adachi, M., et
al. (1993) Lethal effect of the anti-Fas antibody in mice. Nature 364, 806-809.
[39] Ashkenazi, A., Pai, R.C., Fong, S., et al. (1999) Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 104, 155-162.
[40] Walczak, H., Miller, R.E., Ariail, K., et al. (1999) Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 5, 157-163. [41] Harris, C.C. (1996) p53 tumor suppressor gene: from
the basic research laboratory to the clinic--an abridged historical perspective. Carcinogenesis 17, 1187-1198. [42] Jin, Z., McDonald, E.R., III, Dicker, D.T. and El Deiry,
W.S. (2004) 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. J Biol Chem 279, 35829-35839.
[43] Pennarun, B., Meijer, A., de Vries, E.G., et al. (2010) Playing the DISC: Turning on TRAIL death receptor-mediated apoptosis in cancer. Biochim Biophys Acta 1805, 123-140.
[44] Degli-Esposti, M.A., Dougall, W.C., Smolak, P.J., et al.
(1997) The novel receptor TRAIL-R4 induces NF-kappaB and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity 7, 813-820.
[45] Degli-Esposti, M.A., Smolak, P.J., Walczak, H., et al. (1997) Cloning and characterization of TRAIL-R3, a novel member of the emerging TRAIL receptor family. J Exp Med 186, 1165-1170.
[46] Merino, D., Lalaoui, N., Morizot, A., et al. (2006) Differential inhibition of TRAIL-mediated DR5-DISC formation by decoy receptors 1 and 2. Mol Cell Biol 26, 7046-7055.
[47] Spierings, D.C., de Vries, E.G., Vellenga, E., et al. (2004) Tissue distribution of the death ligand TRAIL and its receptors. J Histochem Cytochem 52, 821-831. [48] Wagner, K.W., Punnoose, E.A., Januario, T., et al. (2007)
Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL. Nat Med 13, 1070-1077.
[49] Wang, Y., Engels, I.H., Knee, D.A., et al. (2004) Synthetic lethal targeting of MYC by activation of the DR5 death receptor pathway. Cancer Cell 5, 501-512.
[50] Peter, M.E. (2004) The flip side of FLIP. Biochem J 382, e1-e3.
[51] Scaffidi, C., Schmitz, I., Krammer, P.H. and Peter, M.E. (1999) The role of c-FLIP in modulation of CD95-induced apoptosis. J Biol Chem 274, 1541-1548.
[52] Burns, T.F., El Deiry, W.S. (2001) Identification of inhibitors of induced death (ITIDs) in the TRAIL-sensitive colon carcinoma cell line SW480 using a genetic approach. J Biol Chem 276, 37879-37886.
[53] Fulda, S., Meyer, E. and Debatin, K.M. (2000) Metabolic inhibitors sensitize for CD95 (APO-1/Fas)-induced apoptosis by down-regulating Fas-associated death domain-like interleukin 1-converting enzyme inhibitory protein expression. Cancer Res 60, 3947-3956. [54] Hyer, M.L., Sudarshan, S., Kim, Y., et al. (2002)
Downregulation of c-FLIP sensitizes DU145 prostate cancer cells to Fas-mediated apoptosis. Cancer Biol Ther 1, 401-406.
[55] Geserick, P., Drewniok, C., Hupe, M., et al. (2008) Suppression of cFLIP is sufficient to sensitize human melanoma cells to T. Oncogene 27, 3211-3220. [56] Fulda, S., Meyer, E. and Debatin, K.M. (2002) Inhibition
of TRAIL-induced apoptosis by Bcl-2 overexpression. Oncogene 21, 2283-2294.
[57] Hinz, S., Trauzold, A., Boenicke, L., et al. (2000) Bcl-XL protects pancreatic adenocarcinoma cells against. Oncogene 19, 5477-5486.
[58] Taniai, M., Grambihler, A., Higuchi, H., et al. (2004) Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells. Cancer Res 64, 3517-3524.
[59] Desagher, S., Osen-Sand, A., Montessuit, S., et al. (2001) Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8. Mol Cell 8, 601-611.
S.J. (2000) Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. Science 290, 1761-1765.
[61] Chou, J.J., Li, H., Salvesen, G.S., Yuan, J. and Wagner, G. (1999) Solution structure of BID, an intracellular amplifier of apoptotic signaling. Cell 96, 615-624. [62] Kudla, G., Montessuit, S., Eskes, R., et al. (2000) The
destabilization of lipid membranes induced by the C-terminal fragment of caspase 8-cleaved bid is inhibited by the N-terminal fragment. J Biol Chem 275, 22713-22718.
[63] Varfolomeev, E., Goncharov, T., Fedorova, A.V., et al. (2008) c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. J Biol Chem 283, 24295-24299.
[64] Kucharczak, J., Simmons, M.J., Fan, Y. and Gelinas, C. (2003) To be, or not to be: NF-kappaB is the answer--role of Rel/NF-kappaB in the regulation of apoptosis. Oncogene 22, 8961-8982.
[65] Vaux, D.L., Silke, J. (2005) IAPs, RINGs and ubiquitylation. Nat Rev Mol Cell Biol 6, 287-297. [66] Fulda, S., Wick, W., Weller, M. and Debatin, K.M. (2002)
Smac agonists sensitize for Apo2L/T. Nat Med 8, 808-815.
[67] Li, L., Thomas, R.M., Suzuki, H., et al. (2004) A small molecule Smac mimic potentiates T. Science 305, 1471-1474.
[68] Belyanskaya, L.L., Marti, T.M., Hopkins-Donaldson, S., et al. (2007) Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin. Mol Cancer 6, 66.
[69] El Zawahry, A., McKillop, J. and Voelkel-Johnson, C. (2005) Doxorubicin increases the effectiveness of Apo2L/TRAIL for tumor growth inhibition of prostate cancer xenografts. BMC Cancer 5, 2.
[70] Ray, S., Almasan, A. (2003) Apoptosis induction in prostate cancer cells and xenografts by combined treatment with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand and CPT-11. Cancer Res 63, 4713-4723.
[71] Shankar, S., Singh, T.R. and Srivastava, R.K. (2004) Ionizing radiation enhances the therapeutic potential of TRAIL in prostate cancer in vitro and in vivo: Intracellular mechanisms. Prostate 61, 35-49.
[72] Verbrugge, I., de Vries, E., Tait, S.W., et al. (2008) Ionizing radiation modulates the TRAIL death-inducing signaling complex, allowing bypass of the mitochondrial apoptosis pathway. Oncogene 27, 574-584.
[73] Ashkenazi, A., Herbst, R.S. (2008) To kill a tumor cell: the potential of proapoptotic receptor agonists. J Clin Invest 118, 1979-1990.
[74] Petersen, S.L., Wang, L., Yalcin-Chin, A., et al. (2007) Autocrine TNFalpha signaling renders human cancer cells susceptible to Smac-mimetic-induced apoptosis. Cancer Cell 12, 445-456.
[75] Varfolomeev, E., Blankenship, J.W., Wayson, S.M., et al. (2007) IAP antagonists induce autoubiquitination of
c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell 131, 669-681.
[76] Vince, J.E., Wong, W.W., Khan, N., et al. (2007) IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis. Cell 131, 682-693.
[77] Oltersdorf, T., Elmore, S.W., Shoemaker, A.R., et al. (2005) An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, 677-681. [78] Huang, S., Sinicrope, F.A. (2008) BH3 mimetic
ABT-737 potentiates TRAIL-mediated apoptotic signaling by unsequestering Bim and Bak in human pancreatic cancer cells. Cancer Res 68, 2944-2951.
[79] Labi, V., Grespi, F., Baumgartner, F. and Villunger, A. (2008) Targeting the Bcl-2-regulated apoptosis pathway by BH3 mimetics: a breakthrough in anticancer therapy? Cell Death Differ 15, 977-987.
[80] Johnstone, R.W., Frew, A.J. and Smyth, M.J. (2008) The TRAIL apoptotic pathway in cancer onset, progression and therapy. Nat Rev Cancer 8, 782-798.
[81] Maas, C., Verbrugge, I., de Vries, E., et al. (2010) Smac/ DIABLO release from mitochondria and XIAP inhibition are essential to limit clonogenicity of Type I tumor cells after TRAIL receptor stimulation. Cell Death Differ, in press.
[82] Tait, S.W., de Vries, E., Maas, C., et al. (2007) Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its N-terminal fragment. J Cell Biol 179, 1453-1466.
[83] Sarig, R., Zaltsman, Y., Marcellus, R.C., et al. (2003) BID-D59A is a potent inducer of apoptosis in primary embryonic fibroblasts. J Biol Chem 278, 10707-10715. [84] Sax, J.K., Fei, P., Murphy, M.E., et al. (2002) BID
regulation by p53 contributes to chemosensitivity. Nat Cell Biol 4, 842-849.
[85] Kaufmann, T., Tai, L., Ekert, P.G., et al. (2007) The BH3-only protein bid is dispensable for DNA damage- and replicative stress-induced apoptosis or cell-cycle arrest. Cell 129, 423-433.
[86] Wei, M.C., Zong, W.X., Cheng, E.H., et al. (2001) Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292, 727-730.
[87] Zinkel, S.S., Hurov, K.E., Ong, C., et al. (2005) A role for proapoptotic BID in the DNA-damage response. Cell 122, 579-591.
[88] Michalak, E.M., Villunger, A., Adams, J.M. and Strasser, A. (2008) In several cell types tumour suppressor p53 induces apoptosis largely via Puma but Noxa can contribute. Cell Death Differ 15, 1019-1029.
[89] Werner, A.B., Tait, S.W., de Vries, E., Eldering, E. and Borst, J. (2004) Requirement for aspartate-cleaved bid in apoptosis signaling by DNA-damaging anti-cancer regimens. J Biol Chem 279, 28771-28780.
[90] Verbrugge, I., Maas, C., Heijkoop, M., Verheij, M. and Borst, J. (2010) Radiation and anticancer drugs can facilitate mitochondrial bypass by CD95/Fas via c-FLIP downregulation. Cell Death Differ 17, 551-561.
