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
Document Version
Final published version
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.
and DNA damage-induced apoptosis
Chiel Maas
This research was financially supported by the Dutch Cancer Society (grant NKI 2008-4110 to J.Borst and M.Verheij) Cover: The Deadvlei in Namibia, photo C.Maas
Printed by: Gildeprint Drukkerijen - Enschede, the Netherlands ISBN/EAN: 9789461080738
Printing of this thesis was financially supported by the Dutch Cancer Society (KWF), the Jurriaanse Stichting and the Netherlands Cancer Institute (NKI)
death receptor- and DNA damage-induced apoptosis
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus prof. dr. D.C. van den Boom
ten overstaan van een door het college voor promoties ingestelde commissie,
in het openbaar te verdedigen in de Agnietenkapel op vrijdag 10 september 2010, te 14.00 uur
door Chiel Maas geboren te Apeldoorn
Promotor: Prof. dr. J. Borst Overige leden: Prof. dr. R. Bernards
Dr. E. Eldering Prof. dr. J.P. Medema Prof. dr. J.J. Neefjes Prof. dr. M. Verheij
Chapter 1 General Introduction
Chapter 2 Smac/DIABLO release from the mitochondria and XIAP inhibition are essential to limit clonogenicity of Type I tumor cells after TRAIL receptor stimulation
Cell Death and Differentiation 2010; in press. Chapter 3 Apoptosis induction by Bid requires unconventional
ubiquitination and degradation of its N-terminal fragment
The Journal of Cell Biology 2007; 179(7): 1453-1466. Chapter 4 Bid is required for DNA damage-induced apoptosis in
p53-deficient cells
In revision for Oncogene.
Chapter 5 Radiation and anticancer drugs can facilitate mitochondrial bypass by CD95/Fas via c-FLIP downregulation
Cell Death and Differentiation 2010; 17(3): 551-561. Chapter 6 Identification of SRP72 as an essential regulator of
TRAIL-induced apoptosis and clonogenic elimination
Manuscript in preparation.
Chapter 7 Summarizing Discussion
Nederlandse samenvatting List of Publications Curriculum Vitae Dankwoord 10 22 42 64 80 102 118 128 131 132 133
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.
Chapter
2
Cell Death and Differentiation 2010, in press
Chiel Maas, Inge Verbrugge, Evert de Vries, Gleb Savich , Lambertus W. van de Kooij, Stephen W.G. Tait and Jannie Borst
Smac/DIABLO release from the mitochondria
and XIAP inhibition are essential to limit
clonogenicity of Type I tumor cells after
TRAIL receptor stimulation
Death receptors such as Fas/CD95 and the TRAIL receptors activate the extrinsic apoptotic pathway. This involves assembly of the death-inducing signaling complex (DISC) at their cytoplasmic tail, which results in Caspase-8 and/ or -10 activation [1]. These inducer caspases subsequently cleave and activate effector caspases and the BH3 domain-only Bcl-2 family member Bid [2]. The effector caspases can in principle directly dismantle the cell to generate apoptotic bodies. Bid, however, connects death receptors to the intrinsic pathway for effector
caspase activation. Upon Bid cleavage, its carboxy-terminal fragment translocates to the mitochondria, where it triggers mitochondrial outer membrane permeability (MOMP) via the Bcl-2 family members Bak and Bax [2,3]. This releases apoptogenic factors, including Cytochrome c, Smac/DIABLO and HtrA2/Omi into the cytosol. There, Cytochrome c binds to Apaf-1, creating the apoptosome, which is a platform for recruitment and activation of Caspase-9. This inducer caspase in turn cleaves and activates effector caspases [3].
Smac/DIABLO release from the mitochondria and XIAP
inhibition are essential to limit clonogenicity of Type I
tumor cells after TRAIL receptor stimulation
Chiel Maas1, Inge Verbrugge1,2 Evert de Vries1, Gleb Savich1,3, Lambertus W. van de Kooij1, Stephen W.G. Tait1,4 and Jannie Borst1,5
1Division of Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The
Netherlands
2Current address: Cancer Therapeutics Program, The Peter MacCallum Cancer Centre, East
Melbourne Victoria, Australia
3Current address: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel
Hill, NC 27599, USA
4Current address: Department of Immunology, St Jude Children’s Research Hospital, Memphis,
TN 38105, USA
5Correspondance to Jannie Borst: J.Borst@nki.nl
Death receptors such as Fas/CD95 and TRAIL receptors engage the extrinsic pathway for caspase activation, but also couple to the intrinsic mitochondrial route. In so-called Type II cells, death receptors require the mitochondrial pathway for apoptotic execution, whereas in Type I cells they reportedly do not. For established tumor cell lines, the Type I/Type II distinction is based on short-term apoptosis assays. We report here that the mitochondrial pathway is essential for apoptotic execution of Type I tumor cells by death receptors, when long-term clonogenicity is taken into account. A blockade of the mitochondrial pathway in Type I tumor cells - by RNA interference for Bid or Bcl-2 overexpression - reduced effector Caspase activity and mediated significant clonogenic resistance to TRAIL. Downstream from the mitochondria, Caspase-9 did not contribute to clonogenic death of TRAIL-treated Type I cells. Rather, the release of Smac/DIABLO and the inhibition of XIAP activity proved to be crucial for full effector caspase activity and clonogenic execution. Thus, in Type I cells the intrinsic pathway downstream from death receptors is not redundant, but limits clonogenicity by virtue of Smac/DIABLO release and XIAP inhibition. This finding is relevant for cancer therapy with
death receptor agonists.
Introduction
Given these scenarios, the mitochondrial pathway is generally viewed as a signal amplifier for effector caspase activation after death receptor stimulation. Moreover, different cell types proved more or less reliant on the mitochondrial pathway for effector caspase activation. In so-called Type I cells, inhibition of MOMP by Bcl-2 overexpression did not block death receptor-induced apoptosis, whereas it did so in Type II cells [4]. This distinction was valid for long-term established tumor cell lines in vitro, but also for primary cells in vivo. Specifically, hepatocytes are classified as Type II cells [5], whereas thymocytes and peripheral T cells are classified as Type I cells [6-9]. The efficiency of Caspase-8/10 activation in response to death receptor stimulation is greater in Type I cells than in Type II cells [4]. Therefore, it is generally assumed that Type I cells do not require the mitochondrial amplification loop for effector caspase activation, whereas Type II cells do [10]. Amongst solid tumor cell lines, Type I and Type II cells have distinctions which are connected to the mode of death receptor signaling: Type I cells express mesenchymal-like genes, while Type II cells express epithelium-like markers [11]. In Type I cells, but not in Type II cells, Fas receptors aggregate upon ligand binding and efficiently form a DISC with the aid of filamentous actin [10].
These earlier studies focused on the strength of the Caspase-8/10 signal in Type I versus Type II cells and essentially viewed the mitochondria as signal amplifier for effector caspase cleavage, by engaging Caspase-9. Recent studies, however, have highlighted another control point in apoptotic execution by death receptors, which is that imposed by XIAP [7,12]. XIAP is an Inhibitor of Apoptosis Protein (IAP) that controls the catalytic activities of Caspase-9, Caspase-3- and Caspase-7 after their initial activation. XIAP uses a Baculovirus Inhibitor Repeat (BIR) domain to bind to an IAP binding motif (IBM) in the small subunit of these caspases, which is exposed upon their proteolytic cleavage. In addition, XIAP shields the substrate binding site of the effector
caspases with a peptide strand preceding the BIR domain [13].Upon their release from the mitochondria, Smac/DIABLO and HtrA2/Omi bind to XIAP via their own IBM and thereby free the caspases from inhibition [14-17]. The caspases can subsequently process themselves into fully active, non-XIAP inhibitable fragments and propagate the apoptotic signal [18].By XIAP neutralization, mitochondria make a unique contribution to effector caspase activation, which is not offered by the extrinsic pathway. In Type II cells, the mitochondrial pathway makes two potential contributions to effector caspase activation downstream from death receptors: Caspase-9 activation for initial effector caspase cleavage and XIAP elimination for full effector caspase activity [19].It was recently shown that loss of XIAP rendered hepatocytes independent of Bid for apoptosis induction, i.e. allowed for an apparent switch from Type II to Type I signaling [7]. This suggests that in Type II cells, death receptors need the mitochondrial pathway for XIAP neutralization rather than for Caspase-9 activity.
In Type I cells, Caspase-8 activity also leads to Bid cleavage, MOMP and the consequent release of Smac/DIABLO and HtrA2/Omi, but this is reportedly not essential for the apoptotic response. We surmised that in Type I cells these factors play a role in death receptor-induced apoptosis that had previously been overlooked. Therefore, we examined the impact of the mitochondrial pathway on clonogenic death of Type I tumor cells. We found that blockade of the mitochondrial pathway by Bid RNA interference (RNAi) or Bcl-2 overexpression mediated significant clonogenic resistance to death receptor ligand TRAIL. Upon close examination, the mitochondrial pathway did appear to contribute to effector caspase activation. Smac/DIABLO function, but not Caspase-9 activity was decisive for full effector caspase activation and clonogenic execution of Type I cells. Downregulation of XIAP by RNAi significantly reduced clonogenicity of TRAIL-treated Type I cells, indicating that the inhibition of XIAP activity is an important function of Smac/DIABLO. We conclude that
the mitochondrial pathway limits clonogenicity of Type I cells after death receptor stimulation by enabling full effector caspase activation, not with the aid of the apoptosome, but by virtue of IAP antagonism.
Results
Identification of Bid as a regulator of clonogenic death in Type I tumor cells
We performed a RNAi screen in MCF-7 breast carcinoma cells to identify novel regulators of TRAIL-induced apoptosis. MCF-7 cells were chosen because they proved an excellent model system for studying cell biological aspects of death receptor signaling [20]. MCF-7 cells lack Caspase-3, but behave as Type I cells when Caspase-3 has been reconstituted [4]. Upon stable reconstitution of Caspase-3, only 1 in 5000 MCF-7Casp-3 cells proved spontaneously
resistant to isoleucine zippered (IZ)-TRAIL [21] in a clonogenic assay (results not shown). The screening approach was validated by the selection of a Caspase-8-targeting shRNA from the retroviral library of about 24,000 shRNAs directed at 8000 genes [22]. In addition, a Bid-targeting shRNA emerged from the screen. Since the mitochondrial pathway is thought to be redundant for death receptor induced apoptosis in Type I cells, this was unexpected. Therefore, we decided to look further into the contribution of the mitochondrial pathway to TRAIL-induced apoptosis in MCF-7Casp-3 cells.
A stable Bid RNAi MCF-7Casp-3 cell line was made
with the Bid-targeting shRNA (Bid shRNA1) identified in the RNAi screen. In addition, a stable Caspase-8 RNAi version of this cell line was made. The same batch of MCF-7Casp-3 cells
was transduced in parallel with empty vector (EV), shRNA for Caspase-8, or shRNA for Bid to exclude background effects. High efficiency of retroviral transduction furthermore excluded clonal selection of transduced cells. Both Bid and Caspase-8 expression were efficiently silenced by their targeting shRNAs (Figure 1a,b). To examine the Type I nature of
MCF-7Casp-3 cells, we monitored TRAIL
receptor-induced Caspase-3 cleavage in control and
Bid RNAi versions of this cell line. Cells were stimulated with 25, 50 or 100 ng/ml IZ-TRAIL for 3 h and Caspase-3 cleavage was monitored by flow cytometry using an antibody that recognizes all cleaved forms of Caspase-3, but not the proform. This analysis validated the effect of the shRNA for Caspase-8, since cleavage of its substrate Caspase-3 was significantly impaired at all IZ-TRAIL doses (Figure 1c). In case of Bid RNAi, Caspase-3 Figure 1. MCF-7Casp-3 cells are of a Type I nature.
MCF-7Casp-3 cells were retrovirally transduced with an empty RNAi
vector (EV) or vectors encoding shRNAs targeting Bid or Caspase-8. (a,b) Bid protein expression (a) and Caspase-8 protein expression (b) in cells transduced (Td) with shRNA are reduced as compared to that in EV control cells, as demonstrated by immunoblotting on total cell lysates. Actin served as loading control. (c) The same cells as character-ized in (a,b) were treated (TX) with 0, 25, 50 or 100 ng/ ml of IZ-TRAIL for 3 h. The percentage of cells with cleaved Caspase-3 was determined by flow cytometry after intra-cellular staining with an antibody that specifically recognizes cleaved Caspase-3. Data presented are expressed as mean of 3 independent experiments + S.D. Statistically significant differences between values of EV and Casp-8 shRNA samples are indicated for *P<0.05 and **P<0.01. (d) MCF-7Casp-3 cells
stably expressing EV or Bid shRNA1 were treated with 0, 25, 50 or 100 ng/ml of IZ-TRAIL for 16 h. The percentage (%) of dead cells was determined by flow cytometry after staining with FITC Annexin-V and PI.
0 20 40 60 80 100 0 25 50 100 ng/ml TRAIL % de ad ce lls EV Bid shRNA Bid Actin 17 EV Blot: Td: Bid shRNA1 MCF-7Casp-3 Casp-8 Actin Casp-8 shRNA 49 63 Td: Blot: EV MCF-7Casp-3 0 10 20 30 40 50 60 70 0 25 50 100 0 25 50 100 0 25 50100 ng/ml TRAIL EV Bid shRNA1 % cell s w ith cl ea ve d Ca sp-3 Casp-8 shRNA * ** ** TX: Cells: a b c d
cleavage was not significantly altered (Figure 1c). Also, (apoptotic) cell death, as read out after 16 hours by membrane permeability and phosphatidyl serine (PS) exposure was not evidently affected by Bid RNAi (Figure 1d). These data confirm that MCF-7 cells
reconstituted with Caspase-3 are of a Type I nature. However, the recovery of Bid-targeted shRNA from the screen suggested that in these cells, the mitochondrial route contributes to cellular execution by TRAIL.
Figure 2. Type I cells require the mitochondrial apoptosis pathway for TRAIL-induced clonogenic cell death. (a,b)
MCF-7Casp-3 cells stably expressing EV, Bid shRNA1 or Caspase-8 shRNA were plated at 50,000 or 10,000 cells per 10 cm dish for
colony visualization (a) or counting purposes (b), respectively, and treated with 50 ng/ml IZ-TRAIL for 15 days. Resistant colonies were visualized with Coomassie-blue fixing solution and counted. Plates (a) and colony numbers (b) are representative of 2 inde-pendent experiments. The plating efficiency of untreated cells was 100% (results not shown). (c-g) MCF-7Casp-3 cells and SKW6.4
B-lymphoma cells were transduced to stably express an empty vector (EV) or a vector encoding Bcl-2. (c,e) Bcl-2 overexpression in MCF-7Casp-3 and SKW6.4 cells was confirmed by immunoblotting on total cell lysates, where Actin served as a loading control.
(d) Control (EV) or Bcl-2 overexpressing MCF-7Casp-3 cells were plated for the colony assay at 50,000 cells per 10 cm dish and left
untreated (-) or treated (TX) with 50 ng/ml IZ-TRAIL for 15 days. Resistant colonies were visualized with Coomassie-blue fixing solution. (f) SKW6.4 cells are of a Type I nature. Control (EV) and Bcl-2 overexpressing SKW6.4 cells were treated with 0, 12,5, 25 or 50 ng/ml of IZ-TRAIL for 3 h. The percentage of cells with cleaved Caspase-3 was determined by flow cytometry after in-tracellular staining with an antibody that specifically recognizes cleaved Caspase-3. Data presented are expressed as mean of 3 independent experiments + S.D. (g) Control (EV) and Bcl-2 overexpressing SKW6.4 cells were plated in 96-well plates at 1 cell/ well and were treated with 50 ng/ml IZ-TRAIL for 15 days. Resistant colonies were scored by visual inspection. The percentages of colony-forming cells are depicted, corrected for plating efficiencies. Data are means of two independent experiments + S.D.
MCF-7Casp-3
EV Bid shRNA1 Casp-8 shRNA Cells: 0 100 200 300 400 500 EV Bid shRNA1 Casp-8 shRNA
# of c ol on ie s Cells: _ EV Bcl-2 TRAIL MCF-7Casp-3 Cells: TX: Bcl-2 EV Bcl-2 Actin 28 MCF-7 Casp-3 Blot: Td: EV Bcl-2 Bcl-2 Actin 28 SKW6.4 Blot: Td: % cell s w ith cl ea ve d Ca sp-3 0 20 40 60 80 0 12,5 25 50 EV Bcl-2 ng/ml TRAIL % c ol on y-fo rm in g cel ls 0 2 4 6 8 10 12 EV Bcl-2 Cells: a b c d e f g
The mitochondrial pathway limits clonogenicity of Type I tumor cells after TRAIL treatment
Since the Bid RNAi phenotype was revealed at the level of clonogenicity, subsequent assays were performed using this readout. For this purpose, MCF-7Casp-3 cells were treated with 50
ng/ml IZ-TRAIL and examined for clonogenic outgrowth at day 15. RNAi for Caspase-8 gave rise to clonogenic resistance to TRAIL as compared to EV-transduced cells, serving as a positive control for the assay (Figure 2a,b). In repeated assays, Bid RNAi mediated significant clonogenic resistance to TRAIL (Figure 2a,b, also see Figure 4c,d).
To further address the relevance of the mitochondrial apoptosis signaling pathway, it was blocked by Bcl-2 overexpression. MCF-7Casp-3
cells were retrovirally transduced with a Bcl-2-encoding vector or with EV as control (Figure
2c). Bcl-2 overexpression conferred resistance to TRAIL, confirming that the mitochondrial apoptosis pathway contributed to clonogenic death of MCF-7Casp-3 cells after TRAIL treatment
(Figure 2d, also see Figure 6b,c). To examine whether this phenotype could be observed in other Type I cells, we used the B-lymphoma cell line SKW6.4 and the breast carcinoma cell line T47D [11]. SKW6.4 cells were transduced with EV or a vector encoding Bcl-2 and T47D cells with EV or a Bid-targeting shRNA (Bid shRNA2). Immunoblotting demonstrated effective Bcl-2 overexpression in SKW6.4 cells (Figure 2e) and silencing of Bid protein expression in T47D cells (Supplementary Figure 1a). Bcl-2 overexpression in SKW6.4 cells did not significantly affect TRAIL-induced Caspase-3 cleavage, in line with the Type I classification [11], but it did produce clonogenic resistance to TRAIL (Figure 2f,g). Likewise, Bid
Figure 3. Caspase-9 is not required for clonogenic elimination of Type I cells by TRAIL. MCF-7Casp-3 cells were transduced
(Td) to stably express an empty vector (EV) or dominant-negative Caspase-9 mutant (dnCasp-9). (a) Overexpression of dnCasp-9 protein was confirmed by immunoblotting on total cell lysates, where Actin served as a loading control. (b,c) EV- and dnCasp-9 ex-pressing cells were exposed to the indicated doses of UV light (b) or IZ-TRAIL (c) and percentages of cells with cleaved Caspase-3 were determined by flow cytometry after 16 h or 3 h, respectively. Data are expressed as mean of 3 independent experiments + S.D. Statistically significant differences between values of EV and dnCasp-9 samples are indicated for *P<0.05 and ***P<0.001. (d,e) EV- and dnCasp-9 expressing cells were plated at 50,000 or 10,000 cells per 10 cm dish for colony visualization (d) and counting (e) purposes, respectively, and left untreated (-) or treated with 50 ng/ml IZ-TRAIL for 15 days. Resistant colonies were visualized with Coomassie-blue fixing solution and counted. Colony numbers in (e) are means + S.D. of 3 independent experi-ments and plates in (d) are representative of these. Differences were statistically non-significant (NS).
% c el ls w ith c le av ed C as p-3 0 20 40 60 0 4 8 12 J/m2UV EV dnCasp-9 *** *** *** dnCasp-9 EV _ TRAIL Cells: TX: MCF-7Casp-3 ng/ml TRAIL 0 10 20 30 40 50 0 25 50 100 % c el ls w ith cl ea ve d C as p-3 * EV dnCasp-9 0 10 20 30 40 50 EV dnCasp9 # of c ol on ie s Cells: NS Casp-9 Actin EV dnCasp-9 49 Blot: Td: MCF-7Casp-3 a b c d e
