From: Cancer Drug Discovery and Development: Death Receptors in Cancer Therapy Edited by: W. S. El-Deiry © Humana Press Inc., Totowa, NJ
355
21 Combination of Chemotherapy
and Death Ligands in Cancer Therapy
Simone Fulda, MD ,
and Klaus-Michael Debatin, MD
SUMMARY
Killing of cancer cells by diverse cytotoxic therapies including chemotherapy, a-irradia- tion, or immunotherapy is predominantly mediated by induction of apoptosis, the cell’s intrinsic death program. Failure to undergo apoptosis in response to anticancer therapy may result in resistance. Signaling through death receptors has been implicated to con- tribute to the efficacy of cancer therapy. Importantly, combined treatment with chemo- therapy together with death ligands resulted in enhanced antitumor activity and may even overcome some forms of resistance. Understanding the molecular events that regulate apoptosis induced by anticancer therapy or by death ligands may provide new opportu- nities for drug development. Thus, novel strategies targeting tumor cell resistance will be based on further insights into the molecular mechanisms of cell death.
INTRODUCTION
Apoptosis, a distinct, intrinsic cell-death program, occurs in various physiological and
pathological situations and has an important regulatory function in tissue homeostasis
and immune regulation (1,2). Apoptosis is characterized by typical morphological and
biochemical hallmarks including cell shrinkage, nuclear DNA fragmentation, and mem-
brane blebbing. Various stimuli can trigger an apoptosis response, e.g., withdrawal of
growth factors or stimulation of cell-surface receptors (1,2). Also, killing of tumor cells
by diverse cytotoxic strategies, such as anticancer drugs, a-irradiation, or immunotherapy,
has been shown to involve induction of apoptosis in target cells (3–9). The mechanisms
responsible for triggering apoptosis in response to cytotoxic treatments may differ for
different stimuli, and have not exactly been delineated. However, damage to DNA or to
other critical molecules is considered to be a common primary event, which initiates a
cellular stress response (10). Various stress-inducible molecules, including JNK, MAPK/
ERK, NF gB, or ceramide, have been implicated in the regulation of apoptosis (10–13).
Also, T-cells or natural killer (NK) cells may release cytotoxic compounds such as granzyme B that can directly initiate apoptosis effector pathways inside the cell (2).
Proteolytic enzymes called caspases are important effector molecules of different forms of cell death (14,15). Apoptosis pathways are tightly controlled by a number of inhibitory and promoting factors (16). The antiapoptotic mechanisms regulating apoptotic cell death have also been implicated in conferring drug resistance to tumor cells (4). Impor- tantly, combinations of anticancer agents together with death-inducing ligands have been shown to synergize in triggering apoptosis in cancer cells and may even overcome some forms of drug resistance (3–7). Further insights into the mechanisms controlling tumor cell death in response to cytotoxic therapies or death receptor ligation will provide a molecular basis for novel strategies targeting death pathways in apoptosis-resistant forms of cancer.
APOPTOSIS SIGNALING PATHWAYS IN CANCER THERAPY In most cases, anticancer therapies eventually result in activation of caspases, a family of cysteine proteases that act as common death effector molecules in various forms of cell death (14,15). Caspases are synthesized as inactive proforms, and upon activation, they cleave next to aspartate residues (14,15). The fact that caspases can activate each other by cleavage at identical sequences results in amplification of caspase activity through a protease cascade (14,15).
Caspases cleave a number of different substrates in the cytoplasm or nucleus, leading to many of the morphologic features of apoptotic cell death (14,15). For example, polynucleosomal DNA fragmentation is mediated by cleavage of inhibitor of caspase- activated DNase (ICAD), the inhibitor of the endonuclease caspase-activated DNase (CAD) that cleaves DNA into the characteristic oligomeric fragments (17). Likewise, proteoly- sis of several cytoskeletal proteins such as actin or fodrin leads to loss of overall cell shape, while degradation of lamin results in nuclear shrinking (1).
Activation of caspases can be initiated from different angles, e.g., at the plasma mem- brane upon ligation of death receptor (receptor pathway) or at the mitochondria (mito- chondrial pathway) (18). Stimulation of death receptors of the tumor necrosis factor (TNF) receptor superfamily, such as CD95 (APO-1/Fas) or TNF-related apoptosis- inducing ligand (TRAIL) receptors, results in activation of the initiator caspase-8, which can propagate the apoptosis signal by direct cleavage of downstream effector caspases such as caspase-3 (19). The mitochondrial pathway is initiated by the release of apoptogenic factors such as cytochrome c, apoptosis inducing factor (AIF), Smac/
DIABLO, Omi/HtrA2, endonuclease G, caspase-2, or caspase-9 from the mitochondrial intermembrane space (20). The release of cytochrome c into the cytosol triggers caspase- 3 activation through formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex, while Smac/DIABLO and Omi/HtrA2 promote caspase activation through neutralizing the inhibitory effects to IAPs (20).
Links between the receptor and the mitochondrial pathway exist at different levels
(21,22). Upon death receptor triggering, activation of caspase-8 may result in cleavage
of Bid, a Bcl-2 family protein with a BH3 domain only, which in turn translocates to
mitochondria to release cytochrome c, thereby initiating a mitochondrial amplification
loop (21). In addition, cleavage of caspase-6 downstream of mitochondria may feed back
to the receptor pathway by cleaving caspase-8 (22).
DEATH RECEPTORS AND THEIR LIGANDS
Death receptors belong to the TNF/nerve growth factor (NGF) superfamily of cell- surface receptors, which is defined by several similar, cysteine-rich extracellular domains (2,19,23–25). They also contain a homologous cytoplasmic protein motif, termed the death domain, which typically enables death receptors to engage the cell’s apoptotic machinery (23–25). Death receptors play an essential role in cell turnover and tissue homeostasis through the regulation of apoptosis (23–25). Currently, six different death receptors have been identified, including CD95 (APO-1/Fas), TNF receptor type I (TNFRI), death recep- tor (DR)3 (TRAMP), TRAIL-R1 (DR4), TRAIL-R2 (DR5), and DR6 (23–25). The CD95, TNF, and TRAIL receptors have been studied extensively, while the role of DR3 and DR6 has not exactly been defined (23–25).
Death receptors are activated upon binding by their cognate ligands (2,19,23–25).
Death receptor ligands include CD95L, TRAIL, TNF-_ and lymphotoxin_ (both binding to TNFRI), and TWEAK, which activates DR3 (23–25). One common feature of death- receptor ligands is that all ligands except lymphotoxin-_ are type II transmembrane proteins that are synthesized as membrane-bound molecules and can be cleaved by spe- cific metalloprotesases to generate soluble ligands (2,23–25).
CD95
The CD95 receptor/CD95 ligand system has been described as a key signaling path- way involved in the regulation of apoptosis in several different cell types (2,19). CD95, a 48-kDa type I transmembrane receptor, is expressed in activated lymphocytes, in a variety of tissues of lymphoid or nonlymphoid origin, as well as in tumor cells (2,19).
CD95L, a 40-kDa type II transmembrane molecule, occurs in a membrane-bound and in a soluble form generated through cleavage by specific metalloprotesases (2). CD95L is produced by activated T-cells and plays a crucial role in the regulation of the immune system by triggering autocrine suicide or paracrine death in neighboring lymphocytes or other target cells (2). Also, constitutive expression of CD95L on cancer cells has been implicated as a mechanism of immune escape of tumors (26,27). By constitutive expres- sion of death receptor ligands such as CD95L on the cell’s surface, tumors may adopt a killing mechanism from cytotoxic lymphocytes to delete the attacking antitumor T-cells through induction of apoptosis via CD95/CD95L interaction (27). However, this model of tumor counterattack has also been challenged, since no study has so far conclusively demonstrated that tumor counterattack by CD95L expression is a relevant immune escape mechanism in vivo (27).
TRAIL
TRAIL is constitutively expressed in many tissues, as are the agonistic TRAIL recep-
tors TRAIL-R1 and TRAIL-R2 (19,28–30). Similar to CD95L, TRAIL rapidly triggers
apoptosis in many tumor cell lines. The TRAIL ligand and its signaling pathway is of
special interest for cancer therapy, since TRAIL has been shown to predominantly kill
cancer cells, while sparing normal cells (19,28–30). The underlying mechanisms for this
differential sensitivity of malignant versus non-malignant cells have not exactly been
defined. One possible mechanism of protection of normal tissues may be based on a set
of antagonistic decoy receptors, which compete with the agonistic TRAIL receptors
TRAIL-R1 and TRAIL-R2 for binding to TRAIL (28,29). However, screening of various
tumor cell types and normal cells did not reveal a consistent association between TRAIL sensitivity and TRAIL receptor expression (28,29). Therefore, susceptibility for TRAIL- induced cytotoxicity has been suggested to be regulated intracellularly by distinct pat- terns of pro- and antiapoptotic molecules.
DEATH RECEPTOR SIGNALING
Ligation of death receptors such as CD95 or TRAIL receptors by their cognate ligands or agonistic antibodies results in receptor trimerization, clustering of the receptors’ death domains, and recruitment of adaptor molecules such as Fas-associated death domain protein (FADD) through homophilic interaction mediated by the death domain (2,19,23–
25,28–31). FADD in turn recruits caspase-8 to the activated CD95 receptor to form the CD95 death-inducing signaling complex (DISC) (24). Oligomerization of caspase-8 upon DISC formation drives its activation through self-cleavage (24). Caspase-8 then activates downstream effector caspases such as caspase-3. In addition to activation at the DISC, caspase-8 can also be activated downstream of mitochondria, e.g., by caspase-6, depending on the cell type and/or apoptotic stimulus (24). For the CD95 signaling path- way, two distinct prototypic cell types have been identified (32). In type I cells, caspase- 8 is activated upon death receptor ligation at the DISC in quantities sufficient to directly activate downstream effector caspases such as caspase-3 (32). In type II cells, however, the amount of active caspase-8 generated at the DISC is insufficient to activate caspase- 3 (32). In these cells, a mitochondrial amplification loop is required for full activation of caspases, involving Bid, which translocates to mitochondria upon cleavage by caspase- 8 to trigger the release of apoptogenic proteins such as cytochrome c from mitochondria into the cytosol (32). Accordingly, CD95-induced apoptosis is blocked by overexpression of Bcl-2 or Bcl-X
L, which inhibit mitochondrial alterations only in type II, but not in type I cells (32).
DEATH RECEPTORS IN CANCER AND CANCER THERAPY The idea to specifically target death receptors to trigger apoptosis in tumor cells is attractive for cancer therapy, since death receptors have a direct link to the cell’s death machinery. In addition, apoptosis upon death receptor ligation has been reported to occur independent of the p53 tumor suppressor gene, which is deleted or inactivated in more than half of human tumors (30). However, the clinical application of CD95L or TNF_ is hampered by severe toxic side effects (23,25). Systemic administration of TNF-_ or CD95L causes a severe inflammatory response syndrome or massive liver-cell apoptosis, respectively. In contrast, TRAIL appears to be a relatively safe and promising death ligand for clinical application, although some concerns about potential toxic side effects on human hepatocytes or brain tissue have recently been raised (28,29).
CD95 and Cancer Therapy
The CD95 system has been implicated in chemotherapy-induced tumor cell death in
a number of studies (33–47). Expression of CD95L was found to be up-regulated upon
treatment with anticancer drugs (33–42,46). In turn, CD95L then triggered the CD95
pathway in an autocrine or paracrine manner by binding to its receptor, CD95. In support
of this concept, an increase of CD95L mRNA and protein expression was found in a
variety of different tumor cell lines derived from leukemia, neuroblastoma, malignant brain tumors, hepatoma, colon, breast, or small lung cell carcinoma cells in vitro (33–42).
Upregulation of CD95L upon chemotherapy was also observed ex vivo in primary, patient-derived tumor cells (33,36). Various anticancer agents with different primary intracellular targets have been used in these studies, including DNA-damaging agents such as doxorubicin, etoposide, cisplatin, or bleomycin (33–42). The increase in CD95L transcription and mRNA levels by cytotoxic drugs was related to activation of the tran- scription factors AP-1 and NF-gB (44,45). AP-1 and NF-gB binding sites were identified in the human CD95L promoter, which respond to DNA damage or inhibition of DNA metabolism by upregulating NF-gB activity (44,45). In addition, CD95 expression on the cell’s surface increased upon drug treatment, in particular in cells harboring wild-type p53 (41,42). Accordingly, p53-responsive elements were identified in the first intron of the CD95 gene, as well as three putative p53-binding sites within the CD95 promoter, which showed limited homology with the p53 consensus binding site (41). Also, rapid transport of presynthesized CD95 receptor molecules, which were stored in vesicles in the cytoplasm, to the cell membrane upon activation of p53 may contribute to upregulation of CD95 receptor surface expression in response to chemotherapy (48). Moreover, soluble antagonistic CD95 receptors, antagonistic CD95L antibodies, or DN-FADD were found to reduce drug-induced apoptosis under certain circumstances (33,34,42). Also, CD95L- independent activation of the CD95 pathway through CD95 receptor oligomerization has been reported, e.g., by ultraviolet (UV) irradiation or suicide gene therapy using the herpes simplex thymidine kinase (HSV/TK) system (49,50). Furthermore, resistance to CD95-triggered apoptosis has been associated with cross-resistance to various antican- cer agents in some leukemia and solid tumor cell lines (34,51). This indicates that cell- death signaling upon physiological stimuli such as CD95 triggering and chemotherapy required, at least in part, pathways similar to the CD95 system.
Despite the reproducibility of these findings in a variety of different model systems, other reports challenged the concept that CD95 signaling is involved in drug-mediated cell death (52–56). To that end, antagonistic antibodies against CD95L or CD95 did not confer protection against apoptosis induced by cytotoxic drugs in other cell lines (54–56).
Although splenocytes from lpr mice showed decreased sensitivity to a-irradiation, thy- mocytes of these mice did not show increased proliferation upon a-irradiation or cyto- toxic drugs (57). Enforced expression of FLICE-inhibitory protein (FLIP), DN-FADD, or the serpin crmA did not inhibit drug-induced apoptosis, although it inhibited caspase- 8 activation (54–56,58). Also, targeted disruption of genes involved in death receptor signaling conferred no protection against cytotoxic drug treatment, at least in nontransformed cells. FADD
–/–and caspase-8
–/–fibroblasts were sensitive to cytotoxic drugs, while they remained resistant to death receptor stimulation (59,60). In contrast, caspase-9
–/–embryonic stem cells and Apaf-1
–/–thymocytes were sensitive to death receptor triggering, but remained resistant to cytotoxic drugs (61,62).
How can these divergent findings be reconciled? The discrepancies in findings may
be related to the relative contribution of the death receptor vs the mitochondrial pathway
to drug-induced apoptosis, depending on the anticancer agent, dose, and kinetics or on
cell-type-specific differences. To this end, a cell-type-dependent signaling following
treatment with cytotoxic drugs has been described, similar to the cell-type-dependent
organization of the CD95 pathway (63). In type I cells, both the receptor and the mito-
chondrial pathway were activated upon drug treatment, since blockade of either the receptor pathway by overexpression of dominant-negative FADD (FADD-DN) or of the mitochondrial pathway by overexpression of Bcl-X
Lonly partially inhibited apoptosis (63). In contrast, in type II cells, apoptosis was predominantly controlled by mitochon- dria, since overexpression of Bcl-2 completely blocked drug-induced apoptosis, while overexpression of FADD-DN had no protective effect (63).
The discrepancies in data may also be explained by differences in blocking reagents used to inhibit CD95/CD95L interaction. The quality of CD95/CD95L blocking agents—
e.g., anti-CD95 antibody, anti-CD95L antibody, or soluble decoy CD95-Fc receptor constructs—may vary significantly depending on their origin and preparation. Impor- tantly, CD95/CD95L neutralizing agents may not be effective because they simply can- not gain access to their proposed targets. Experiments with adenoviral delivery of a CD95L-GFP construct showed that CD95 and CD95L are stored intracellularly and colocalize to the same intracellular compartments, e.g., the Golgi and/or endoplasmatic reticulum (64). An anti-CD95 blocking antibody did not inhibit CD95L-induced cell death, suggesting that CD95L may trigger CD95 within the same intracellular compart- ment and that these two molecules may already interact prior to surface presentation (64).
Under those circumstances, the lack of efficacy of CD95/CD95L neutralizing agents may be explained by the inaccessibility of their targets. Moreover, some studies that challenge an involvement of the CD95 system in chemotherapy of tumor cells are based on experi- ments performed in nontransformed cells, e.g., embryonic fibroblasts, but not in cancer cells. However, the mechanisms regulating apoptosis in non-malignant cells may vary considerably from those in malignant tumor cells, which is highlighted by the differential sensitivity of these cell types to various death stimuli.
Despite the involvement of the CD95 system in anticancer drug-induced apoptosis under certain circumstances, most cytotoxic drugs are considered to primarily initiate cell death by triggering a cytochrome c/Apaf-1/caspase-9-dependent pathway linked to mito- chondria (5). Collectively, these data point to a crucial role of the mitochondrial pathway in drug-induced apoptosis, while the CD95 system may amplify drug-induced apoptosis under certain conditions. Importantly, this amplification of the chemoresponse may have important clinical implications, since it may critically affect the time required for execu- tion of the death program (65). However, the model of mitochondria being the key initiator to integrate stress stimuli into an apoptotic response has also been challenged by recent evidence demonstrating that a functional apoptosome is dispensable for stress- induced apoptosis (66,67). Remarkably, activation of caspases, e.g., caspase-2, follow- ing cellular stress, was found to be required for mitochondrial perturbation rather than vice versa (66). In addition, Bcl-2 was shown to regulate a caspase activation program that did not depend on the cytochrome c/caspase-9/Apaf-1-containing apoptosome com- plex (67). These recent studies indicate that mitochondria may act as amplifiers rather than initiators of death upon cellular stress. Thus, key components of apoptosis pathways may yet have to be reconsidered.
In addition to the CD95 system being involved in chemotherapy-triggered cell death,
numerous studies have shown that anticancer agents or irradiation can synergize with
CD95L or agonistic CD95 antibodies to signal through CD95 (68–71). Importantly,
chemotherapy or irradiation can even sensitize resistant tumor cells for CD95 triggering
by modulating several components of the CD95 signaling pathway. Multiple molecular
mechanisms may underlie defects in the CD95 system (16,46,72). For example, death receptor expression may vary among different cell types and can be downregulated or absent in resistant tumor cells, which has been assumed to contribute to immune escape of tumor cells from negative growth control (2,27). Drug-resistant leukemia or neuro- blastoma cells showed strong downregulation of CD95 expression, suggesting that criti- cal levels of CD95 expression may have an impact on drug sensitivity (51). Impaired transmembrane expression of CD95 may be caused by promoter hypermethylation, histone acetylation, transcriptional repression, inactivating CD95 mutations, or alternative mRNA splicing to generate soluble CD95 receptors lacking a transmembrane anchor (72,73).
Also, drug-resistant tumor cells were found to be deficient in upregulation of CD95L in response to treatment with cytotoxic drugs that were involved in drug response in chemosensitive tumor cells (51). CD95 signaling can also be negatively regulated by proteins that associate with their cytoplasmic domains, e.g., FLIP, which prevents the interaction between the adaptor molecule FADD and procaspase-8 (74). Importantly, several mechanisms may account for the synergistic interaction between chemotherapy and the CD95 system. For example, upregulation of CD95 or CD95L upon treatment with DNA-damaging drugs occurred in a p53-dependent manner (41,42). As described above, wild-type p53 can transcriptionally activate CD95L or CD95 and/or can stimulate the translocation of presynthesized molecules from intracellular stores, e.g., the Golgi com- partment, to the cell membrane (41,42,48). Also, treatment with cytotoxic drugs has been reported to modulate expression of anti- and proapoptotic components of the CD95 DISC (75). Upregulation of FADD and procaspase-8 by cisplatin or doxorubicin was observed in colon carcinoma cells (75). In addition, enhanced recruitment of FADD and caspase- 8 to form the CD95 DISC was found upon treatment with cytotoxic drugs in type I cells (63). Treatment with actinomycin D or cisplatin sensitized neuroblastoma or osteosar- coma cells for CD95 triggering by downregulating FLIP expression (76,77). However, the impact of FLIP on apoptosis sensitivity towards cytotoxic drugs may vary among cell types, since overexpression of FLIP did not confer protection against cytotoxic drugs in T-cell leukemia cells (58). A recent study has provided further evidence that CD95/CD95 ligand interactions are important in the control of tumor growth and treatment response (78). In tumors with epigenetically silenced CD95, restoration of CD95 expression by histone deacetylase inhibitors resulted in suppression of tumor growth and restoration of chemosensitivity in an NK cell-dependent manner (78). These findings demonstrated that the level of CD95 expression and the intact function of the CD95 system has an important impact on chemosensitivity and on immune surveillance by CD95L-express- ing NK cells.
TRAIL and Cancer Therapy
Although most tumor cells express both agonistic TRAIL receptors, many resistant
tumors remain refractory towards treatment with TRAIL, which has been related to the
dominance of anti-apoptotic signals, e.g., those delivered by NF- gB, AKT, or by inhibi-
tor of apoptosis proteins (IAPs) (28,29). Importantly, numerous studies have shown that
TRAIL together with cytotoxic drugs or a-irradiation strongly synergized to achieve
antitumor activity in various cancers, including malignant glioma, melanoma, leukemia,
breast, colon, or prostate carcinoma (79–82). Remarkably, TRAIL and anticancer agents
also cooperated to suppress tumor growth in different mouse models of human cancers
(83,84). In addition, agonistic antibodies targeting TRAIL-R1 or TRAIL-R2 showed promising antitumor activity, alone or in combination with anticancer agents (85,86).
The molecular mechanisms that account for this synergistic interaction may include transcriptional up-regulation of the agonistic TRAIL receptors TRAIL-R1 and –R2, which occurred in a p53-dependent or p53-independent manner (28–30,79–83). Also, downregulation of anti-apoptotic proteins such as Bcl-2, Bcl-X
L, or FLIP, as well as upregulation of pro-apoptotic molecules including FADD or caspase-8 upon treatment with cytotoxic drugs, may sensitize tumor cells for TRAIL. In addition, biological response modifiers such as interferon (IFN)-a strongly enhanced the cytotoxic activity of TRAIL by upregulating caspase-8 expression in a STAT-1-dependent manner (87) Transcriptional upregulation of caspase-8 or caspase-3 upon cytotoxic drug treatment was also reported to occur independently of STAT-1 (88). Since caspase-8 expression is frequently impaired by hypermethylation in several tumors, including neuroblastoma, Ewing tumors, malig- nant brain tumors, or melanoma, administration of TRAIL together with IFNa may overcome some forms of resistance towards TRAIL, e.g., in tumors with impaired caspase-8 expression (89,90). Also, small molecules may serve as molecular targeted therapeutics to enhance TRAIL sensitivity of resistant cancers (91). To this end, Smac agonists have recently been reported to potentiate the efficacy of TRAIL treatment by antagonizing the inhibitory effect of IAPs, which are overexpressed in many tumors (91).
Importantly, Smac peptides synergized with TRAIL to eradicate malignant glioma in an orthotopic mouse model, without any detectable toxicities to the normal brain tissue (91).
Furthermore, there is mounting evidence for an important role of TRAIL in tumor surveillance (92–94). To this end, tumor formation induced by carcinogens was found to be enhanced in the presence of antagonistic TRAIL antibodies (92). Also, TRAIL-defi- cient mice were more susceptible to tumor metastasis than wild-type mice (93). These data are in accordance with studies showing an important role of NK cells, which con- stitutively express TRAIL, in the control of tumor metastasis (94). Together, these find- ings point to a critical role of TRAIL in NK cell-dependent tumor surveillance.
CONCLUSIONS
Numerous reports over the last years have provided evidence that anticancer therapies primarily act by triggering apoptosis in cancer cells. Key elements of the basic apoptosis machinery, including death receptors and their ligands or mitochondria, have been inferred to play a critical role in cancer therapy as well as in surveillance of tumor formation. In addition, studies on the regulation of apoptosis signaling pathways upon cancer therapy gave substantial insights into the molecular mechanisms regulating the response of cancer cells towards current treatment regimens. Defects in apoptosis pro- grams may result in treatment resistance of cancers. Remarkably, synergistic interaction between cytotoxic therapies such as anticancer drugs or a-irradiation and death ligands to trigger tumor cell death has been reported in a variety of cancers. Most importantly, combined treatment with chemotherapy together with death ligands was found to be even effective against resistant tumors, and may thus overcome some forms of resistance.
However, detailed studies of the therapeutic potential of death ligands combined with
chemotherapy in preclinical in vivo mouse models and in primary patient-derived tumor
cells are far from being complete. This approach may hopefully lead to novel therapeutic
strategies targeting apoptosis-resistant forms of cancers.
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