Proapoptotic DR4 and DR5 signaling in cancer cells: toward clinical translation

Available online at www.sciencedirect.com

Proapoptotic DR4 and DR5 signaling in cancer cells: towardclinical translationAnnie Yang1, Nicholas S Wilson1 and Avi Ashkenazi

Proapoptotic receptor agonists (PARAs) targeting death

receptors (DRs) 4 and 5 hold promise for cancer therapy based

on their selective ability to kill malignant versus healthy cells.

Emerging clinical results have confirmed that DR4/5 PARAs are

relatively well-tolerated and suitable for further investigation.

Given that some cancer cell lines and models are not sensitive

to PARAs, it is important to develop strategies to identify what

specific types of tumor cells may be most responsive to PARA-

based therapy and how to overcome apoptosis resistance

mechanisms in tumors. Here we review the molecular and

biological determinants of responsiveness to PARAs in cancer

cells, and discuss the potential for predictive biomarkers and

drug combination strategies to maximize the anti-tumor activity

of these agents.

Address

Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA

Corresponding author: Ashkenazi, Avi ([email protected])1 Both these authors contributed equally to this paper.

Current Opinion in Cell Biology 2010, 22:837–844

This review comes from a themed issue on

Cell division, growth and death

Edited by Frank Uhlmann and Guy Salvesen

Available online 31st August 2010

0955-0674/$ – see front matter

# 2010 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.ceb.2010.08.001

BackgroundApoptosis plays an essential role in development, homeo-

stasis, and tumor suppression. In mammalian cells, two

major pathways, often referred to as the extrinsic and

intrinsic pathways, contribute to apoptotic signaling.

Both require activation of distinct initiator caspases that

target downstream effector caspases, which cleave

numerous cellular substrates culminating in the hallmark

features of apoptosis: plasma membrane blebbing,

nuclear condensation and DNA fragmentation. The

intrinsic, or mitochondrial, pathway is triggered by

DNA damage and other cellular stresses, upon which

the release of mitochondrial proteins such as cytochrome

C and Second Mitochondria-Derived Activator of Cas-

pases (Smac) activates the apoptosis initiating protease

caspase-9 and effector proteases caspase-3 and caspase-7

(Figure 1). This process is regulated by interplay be-

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tween pro apoptotic and anti apoptotic members of

the Bcl-2 family of proteins [1], as well as by inhibitor

of apoptosis proteins (IAPs) that can act directly on

caspases [2].

DRs are proteins within the tumor necrosis factor re-

ceptor (TNFR) superfamily that possess a cytoplasmic

death domain (DD) [3,4]. Binding of homotrimeric apop-

tosis ligand 2/TNF-related apoptosis-inducing ligand

(Apo2L/TRAIL) to DR4 or DR5 (also known as

TNFRSF10A and TNFRSF10B), promotes DD-de-

pendent association with the proximal adaptor Fas-

Associated Death Domain (FADD). This adaptor

recruits the apoptosis initiating proteases caspase-8

and caspase-10, thereby forming the Death-Inducing

Signaling Complex (DISC). The DISC facilitates auto-

catalytic processing and activation of caspase-8 and cas-

pase-10, which can then stimulate the effector caspases.

Commitment to apoptotic death often requires coopera-

tion of the extrinsic and intrinsic pathways: Caspase-8

mediates an amplification loop through cleavage of Bid to

activate the intrinsic pathway (Figure 1). Although ago-

nists of the death receptors TNFR1 and Fas/CD95 such

as TNFa and Fas/CD95L also can trigger apoptotic cell

death, to date their utility as cancer therapeutic agents

has been limited by systemic toxicity, likely caused by

lack of selectivity toward tumor versus normal tissues [5].

Therefore, attention has focused on developing proa-

poptotic agonists of DR4 and DR5, which have consist-

ently displayed relative selectivity for malignant over

healthy cells [3,6,7].

Apo2L/TRAIL signaling — physiological role intumor suppression?Studies in mice suggest that the Apo2L/TRAIL path-

way has a role in immune surveillance. Mice deficient in

either Apo2L/TRAIL or mDR5 (the sole mouse ortho-

log of human DR4 and 5) show increased susceptibility

to tumorigenesis and pathogen infection [3,8]. In cer-

tain models, however, tumor development does not

appear to be affected by mDR5 deficiency; for example,

there was no impact on the incidence of lymphomas

in p53-null mice or intestinal polyps in the APCmin

model [9]. Tumors develop various mechanisms to

escape anti-tumor innate and adaptive immune surveil-

lance — a process referred to as immunoselection or

immunoediting [10]. Inflammatory conditions during

tumorigenesis may induce Apo2L/TRAIL expression

by tumor-infiltrating immune cells [11], perhaps exert-

ing an early immunoselective pressure in malignant

lesions. Consistent with this possibility, chemically


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http://dx.doi.org/10.1016/j.ceb.2010.08.001

838 Cell division, growth and death

Figure 1

Proapoptotic Death Receptor 4 or 5 signaling in tumor cells. DR4 and DR5 activation by PARAs (either trimeric rhApo2L/TRAIL or agonistic DR4 or

DR5-specific antibodies) or Apo2L/TRAIL expressed by innate immune cells. FADD is recruited to DR4 or DR5 located within lipid raft containing

regions of the membrane, which promotes receptor clustering and autocatalytic processing of the apoptosis initiating proteases caspase-8 or

caspase-10 to form the active DISC. Caspase-8 can be polyubiquitylated at the DISC by a cullin-3/Rbx1-based E3 ubiquitin ligase, which facilitates

caspase-8 activation. This process is negatively regulated by the de-ubiquitinating enzyme, A20. The signaling adaptor p62 can bind to ubiquitilated

caspase-8 and translocate it to ubiquitin-rich foci, which may also enhance its activity. In many cancer cells, proapoptotic signaling involves the

mitochondrial pathway via caspase-8-mediated cleavage of Bid to t-Bid. Proapoptotic signaling through the intrinsic pathway is further regulated by

pro apoptotic and anti apoptotic members of the Bcl-2 family. Receptor tyrosine kinase (RTK) signaling and chemotherapy or radiotherapy can further

modulate the intrinsic proapoptotic pathway through targeting Bcl-2 family members. Under certain circumstances, DR4 or DR5 signaling can promote

alternative signaling pathways such as JNK, MAPK or NFkB, which may require recruitment of RIP1 and TRAF2 or TRAFs5 to form secondary signaling

complexes. Depicted in blue are inhibitors that may enhance proapoptotic signaling by PARAs by targeting mechanisms of resistance in tumor cells.

induced tumors from Apo2L/TRAIL-deficient mice

show increased sensitivity to DR5 agonists, as com-

pared with those isolated from wild-type mice [11,12].

Therefore, at least for some cancers, acquisition of

a capacity to evade Apo2L/TRAIL signaling may

represent an important requirement of tumor initiation

and progression.


PARAs — co-opting the Apo2L/TRAILpathway for cancer therapyRecombinant Apo2L/TRAIL (dulanermin) and agonistic

monoclonal antibodies targeting DR4 or DR5 (Figure 1)

may have broad potential for cancer therapy [7,13]. Proa-

poptotic anti-tumor activity has been demonstrated in

models reflecting diverse tumor types, both in vitro and in


Proapoptotic DR4 and DR5 signaling in cancer cells Yang, Wilson and Ashkenazi 839

tumor xenograft settings. However, despite the wide

expression of DR4 and DR5, various cancer cell lines

and primary tumor isolates exhibit partial responsiveness

or resistance to DR4 and DR5 agonists [14��,15–17]. This

resistance might be caused by a specific selective pressure

to evade Apo2L/TRAIL-based immune surveillance, or it

might be due to a less specific capacity to evade apoptosis

in response to various types of cellular stress. An import-

ant challenge for clinical translation is to identify tumor

types and patients that may respond robustly to PARAs,

alone or in combination with ‘sensitizing’ agents. Emer-

ging Phase I and II clinical trials have confirmed that

PARAs targeting the Apo2L/TRAIL pathway are gener-

ally safe and well-tolerated at doses associated with pre-

clinical efficacy [13]. These studies underscore a unique

opportunity to harness the proapoptotic activity of DR4

and DR5 PARAs without significant systemic toxicity. To

date, however, only modest overall anti-tumor activity has

been observed in patients with advanced malignancies.

Hence, in order to achieve meaningful clinical benefit, it

is critical to understand which specific tumor resistance

mechanisms may suppress propapoptotic signaling by

DR4 or DR5.

Role of death and decoy receptors inresistance to PARAsDR4 and DR5 mRNAs are widely detected in healthy

tissues [18]; however, protein expression appears

restricted to damaged, infected, or malignant cells

[19,20]. Cell-surface levels of DR4 and DR5 do not

generally correlate with tumor cell sensitivity to

Apo2L/TRAIL signaling [14��], although various agents

can upregulate receptor expression and sensitize resistant

tumor cells to PARAs [8,21]. This suggests that low

receptor density may be a relevant feature of tumor cell

resistance. Indeed, DR4 and DR5 are located on a region

of chromosome 8p21-22 that can undergo hemizygous

deletion in certain cancers [22]. Rare somatic mutations or

polymorphisms in the DR4 and DR5 genes also have

been identified in several tumor types, and mechanistic

studies suggest that some of these sequence changes

result in functionally inactive — or even dominant-inter-

fering — receptor proteins [22,23]. Epigenetic silencing,

particularly of DR4, has been found in up to 70% of

glioblastomas [24], 30% of ovarian carcinomas [25], and in

some melanoma cell lines [26] while DR5 did not appear

to be affected.

Accumulating evidence that post-translational modifi-

cation of DRs is important for robust proapoptotic sig-

naling corroborates the notion that receptor modulation

may contribute to resistance. In particular, O-glycosyla-

tion of DR4 and DR5 enhances ligand-dependent re-

ceptor clustering, which is necessary for efficient

proapoptotic signaling (Figure 1). High expression levels

of the O-glycosyltransferases GALNT14, GALNT3,

FUT3 and FUT6 closely correlate with Apo2L/TRAIL


sensitivity in a variety of cancer cell lines, and modulation

of these enzymes by overexpression, small-interfering

RNA (siRNA), or pharmacologic inhibition yields corre-

sponding changes in Apo2L/TRAIL responsiveness

[14��]. This observation uncovers the potential for re-

ceptor O-glycosylation status or components of the under-

lying biochemical pathway to be used as biomarkers

predictive of sensitivity. Recently, immunohistochemical

assays for GALNT14 and FUT3/6 have been developed

for implementation in clinical trials [27]. Additional modi-

fications, including palmitoylation and S-nitrosylation of

DR4, have been implicated in receptor oligomerization,

localization to lipid rafts, and stimulation of caspase-8

[28�,29�]. These modifications and their impact for pre-

dicting response to PARAs remain to be clinically eval-

uated.

The subcellular location of DRs also has been linked with

resistance to PARAs. Whereas internalization appears to

play a stimulatory role in apoptosis signal transduction by

CD95 [30], this process dampens caspase activation in

response to DR4 and DR5 ligation [4]. Colon cancer cells

selected for resistance to Apo2L/TRAIL exhibited

defects in DR4 transport to the cell surface [31].

Similar findings were reported for breast cancer, where

resistance was associated with constitutive endocytosis

and decreased cell-surface expression of DR4 and DR5

[32]. Immunohistochemical staining of patient tumor

samples from recent clinical trials underscores the notion

that DR expression may not be as ubiquitous as seen in

cancer cell lines. Although a majority (>70%) of tumors

showed some DR4 expression, lack of staining was

observed in a percentage of cells within most samples.

Even in samples where most tumor cells stained positive,

predominantly cytoplasmic receptor localization was

noted [33,34]. We caution, however, that these types of

analyses are subject to important technical pitfalls, in-

cluding poor epitope preservation and antibody sensi-

tivity. Notwithstanding, there is apparent heterogeneity

in receptor level and localization, suggesting that DR

expression may be useful for predicting responsiveness to

PARA-based therapy. This possibility is supported by the

observation that many agents that sensitize tumor cells to

PARAs — including chemotherapy, ionizing radiation,

and histone deacetylase inhibitors (HDACi) — appear

to act in part by upregulating DR expression [8,21].

The contribution of DR4 versus DR5 to apoptotic sig-

naling may not be equivalent or interchangeable in

different tumors and settings. Using receptor-selective

agonists — including Apo2L/TRAIL variants and mono-

clonal antibodies targeting either DR4 or DR5 — several

studies have demonstrated contextually preferential sig-

naling through one receptor versus the other. For

instance, DR5-selective agonists exhibited greater

potency in colon and breast cancer cell lines [35], while

DR4 was shown to be the dominant proapoptotic receptor



in chronic lymphocytic leukemia (CLL) [36]. It is also

possible that, in some cancers, both receptors contribute

to apoptotic signaling and dual-specificity PARAs such as

Apo2L/TRAIL may have a more optimal effect.

Finally, there are three additional receptors for Apo2L/

TRAIL in humans — DcR1, DcR2, and osteoprotegrin

(OPG). Known as ‘decoy’ receptors (DcRs), they bind

Apo2L/TRAIL without transmitting downstream apop-

totic signaling [37]. DcRs may compete for ligand binding

and also sequester DR5 in non-functional complexes

[38,39]. Although these observations provide a mechan-

istic basis for DR inhibition, evidence of a role for

endogenous DcRs in PARA resistance is lacking [8,14��].

Defects within the DISCThe formation of a functional DISC is a central feature of

DR signaling. Engagement of DR4 or DR5 by Apo2L/

TRAIL or other agonists induces the recruitment of

FADD and caspase-8 or caspase-10 to the plasma mem-

brane. Additional requirements, including translocation

of DISC components into membrane lipid rafts, facilitate

receptor clustering and autocatalytic processing of the

initiator caspase (Figure 1) [40]. These membrane-prox-

imal events are critical for apoptotic signaling via the

extrinsic pathway, and hence their dysregulation may

contribute to resistance.

Cellular FLICE Inhibitor Protein (c-FLIP) antagonizes

caspase-8 and caspase-10 (Figure 1) [41]. c-FLIP shares

structural and sequence homology with these caspases —

notably in the death-effector domains (DEDs) which are

required for binding to FADD — but lacks enzymatic

activity. Consequently, c-FLIP can be recruited to the

DISC, and heterodimerize with caspase-8 or caspase-10.

Long and short isoforms of c-FLIP (c-FLIPL and c-

FLIPS) may impair DISC activity in distinct ways. c-

FLIPS is a truncated protein lacking the C-terminal

caspase-like domain and likely acts as a competitive

inhibitor by preventing binding or processing of cas-

pase-8 and caspase-10 in the DISC. In contrast, c-FLIPL

possesses a caspase-like domain, so its hetero dimeriza-

tion with caspase-8 or caspase-10 may augment the lat-

ter’s enzymatic activity; however, at higher levels, it may

displace initiator caspases from FADD and inhibit apop-

tosis signaling [41]. Selective depletion of c-FLIPL in

cancer cell lines enhances proapoptotic signaling by

Apo2L/TRAIL [42], while a novel cleavage product of

c-FLIPL, p22, was recently shown to mediate nuclear

factor-kB (NF-kB) activation in non-apoptotic cells [43].

Additionally, c-FLIP, together with Receptor-Interacting

Protein (RIP1), may antagonize proapoptotic signaling by

promoting distribution of the Apo2L/TRAIL DISC into

non-lipid raft fractions [44].

The transcription factor c-Myc was shown to repress the

c-FLIP gene directly, and Apo2L/TRAIL sensitivity in


tumor cell lines correlated with c-Myc overexpression

[45��]. In APC-deficient cells, c-FLIP down regulation by

c-Myc overexpression was a key component of sensitiz-

ation to Apo2L/TRAIL [46��]. Moreover, a high-through-

put screen for modulators of Apo2L/TRAIL sensitivity

revealed both c-Myc and Wnt signaling pathways as

sensitizers of DR4/5-induced apoptosis [47], suggesting

that activation of these pathways may be associated with

increased tumor sensitivity to PARAs.

Recent work from our laboratory revealed that in response

to Apo2L/TRAIL, casapse-8 is polyubiquitylated at the

DISC by a cullin-3/Rbx1-based E3 ubiquitin ligase

(Figure 1). This modification promotes full activation

of caspase-8 by driving its association with the ubiquitin-

binding protein p62, and translocation into ubiquitin-rich

foci [48��]. The deubiquitinase A20 antagonizes this

process, consistent with its inhibitory role in NF-kB

signaling. Whether defects in caspase-8 ubiquitylation

play a role in PARA resistance has yet to be interrogated.

The mitochondrial apoptosis pathway inresistance to PARAsIn simplest terms, the mitochondrial apoptosis pathway is

regulated by the balance of pro apoptotic versus anti

apoptotic members of the Bcl-2 family. In most cancer

cell lines, an effective apoptotic response to DR signaling

requires signal amplification through the mitochondria.

As such, alterations in the expression or function of the

Bcl-2 family not only contribute to tumor development

and chemoresistance, but also represent likely barriers to

the therapeutic potential of PARAs.

The importance of the propapoptotic Bcl-2 proteins to

DR signaling was clearly demonstrated by comparing

isogenic HCT116 human colon carcinoma cell lines that

differed only in their Bax status. Although DISC for-

mation in response to Apo2L/TRAIL was similar in both

cell lines, Bax deletion caused cells complete resistance

to Apo2L/TRAIL-induced apoptosis [49]. Moreover, in

two DNA mismatch repair-deficient colon carcinoma cell

lines, resistant clones that emerged after selection with

Apo2L/TRAIL showed inactivating frameshift mutations

of Bax [49]. Interestingly, treatment of these resistant

derivatives with genotoxic drugs restored Apo2L/TRAIL

sensitivity, via upregulation of DR5 and the Bax relative,

Bak. Proteasome degradation of Bax, which can be

reversed by the proteasome inhibitor bortezomib, also

has been implicated in Apo2L/TRAIL resistance in B-

cell lymphoma [50]. These findings suggest that inacti-

vation of proapoptotic Bcl-2 proteins, particularly Bax,

may be a key resistance mechanism for DR-mediated

apoptosis.

Overexpression of anti-apoptotic members of the Bcl-2

family has been associated with resistance to Apo2L/

TRAIL. Bcl-2 protected neuroblastoma, glioblastoma,



and breast carcinoma cell lines from Apo2L/TRAIL-

induced death [51], while high levels of Bcl-XL conferred

resistance in pancreatic adenocarcinoma [52]. More

recently, a role for Mcl-1 in Apo2L/TRAIL resistance

has been highlighted. Mcl-1 downregulation has been

pinpointed as the primary basis for sensitization of tumor

cells to Apo2L/TRAIL by the multikinase inhibitor Sor-

afenib (Figure 1) [53�]. Mcl-1 may be especially import-

ant in Bax-deficient tumors, as it appears to be a major

block to Bak-mediated compensation [54]. The devel-

opment of selective Bcl-2 antagonists [55] should help

elucidate which specific proteins mediate resistance in

various tumor cells, and thus facilitate rational combi-

nation strategies based on PARAs and inhibitors of anti-

apoptotic Bcl-2 family members (Figure 1).

Downstream of the Bcl-2 family, caspase activity is

regulated by the IAPs. First identified in baculovirus,

IAPs can bind to and inhibit executioner caspase-3 and

caspase-7, as well as the initiator caspase-9. Mammalian

homologs of IAPs, including XIAP, cIAP1 and 2, have

been associated with tumor development and with resist-

ance to treatment in a wide range of human cancers [56].

Genetic targeting or RNAi-mediated depletion of XIAP

greatly sensitized tumor cells to Apo2L/TRAIL [57,58].

Smac is an endogenous inhibitor of IAPs; its release from

mitochondria augments apoptotic signaling in response to

Apo2L/TRAIL [59]. Indeed, IAP antagonists that mimic

Smac have shown impressive synergy with PARAs

[60�,61��,62��]; these effects were attributed mainly to

relief of caspase inhibition by XIAP (Figure 1)

[61��,62��].

Alternative signaling pathways–apoptosisfriend or foe?In addition to the primary DISC, engagement of DRs can

induce a secondary signaling complex, containing core

DISC components, as well as RIP1 and TRAF2 (Figure

1) [63]. This complex may activate additional signaling

cascades, including the NF-kB, extracellular signal-

regulated kinase (ERK), c-Jun N-terminal kinase

(JNK) and p38-mitogen activated protein kinase (MAPK)

pathways [8,64]. Many of these alternative signaling

events have been proposed to counter proapoptotic

activity, although there is considerable debate regarding

their physiological kinase significance and impact on

response to PARAs.

Particular emphasis has been placed on the NF-kB path-

way, which is mostly associated with pro-survival pro-

grams, especially the transcriptional activation of c-FLIP,

IAPs, and anti-apoptotic Bcl-2 genes, by canonical NF-kB

signaling [65]. Genetic or pharmacologic inhibition of the

NF-kB pathway confers greater sensitivity to Apo2L/

TRAIL in many cancer cells [8]. However, DR-mediated

NF-kB activation is unlikely to be a major basis for

resistance to PARAs. Rather, constitutive activity of


the NF-kB pathway in certain tumors may set a higher

threshold for an apoptotic response [66]. Therefore, strat-

egies aimed at combining NF-kB inhibitors with PARAs

may be important for overcoming tumor cell resistance to

apoptosis (Figure 1). Confounding this notion, however,

is the capacity of various NF-kB subunits to hetero

dimerize and thus yield distinct — even opposing —

transcriptional programs. For example, RelA complexes

can actually induce expression of DR4 and DR5 while

repressing IAPs, thereby potentiating, rather than inhi-

biting, cell death [67].

Similar considerations apply to interactions between DR

pathways and other signaling cascades. Inhibition of

PI3K, JNK, or p38-MAPK signaling has been shown to

sensitize tumor cells to PARA-mediated apoptosis, but

these kinases can also facilitate Apo2L/TRAIL-mediated

cell death [8]. Thus, alternative signaling pathways may

exert context and cell-type dependent impact on apop-

totic responsiveness to PARAs. A rational basis for com-

bination therapies will require further elucidation and

understanding of the cross-talk between DRs and other

receptor-based signaling modalities.

Last, DR activation may cooperate with the endoplasmic

reticulum (ER) stress response in tumor cells. ER stress

has been shown to sensitize cells to PARAs, possibly via

upregulation of DR5 [68]. This synergy may involve

Prostate Apoptosis Response Protein 4 (Par-4). Previous

work showed that Par-4 augments Apo2L/TRAIL-

mediated apoptosis by activating caspases and downre-

gulating c-FLIP, Bcl-2 and IAPs [69]. While this activity

was attributed to intracellular Par-4, it was recently shown

that either Apo2L/TRAIL treatment or ER stress leads to

increased secretion of a soluble Par-4 variant, which then

binds the chaperone GRP78 to induce apoptosis [70�].The cross-talk between DR signaling, Par-4, and the ER

stress pathways may provide a novel basis for predictive

biomarkers or combination strategies, but this requires

further study.

Conclusions/perspectiveThe selective killing of malignant versus normal cells by

PARAs targeting the Apo2L/TRAIL pathway provides a

unique opportunity to investigate how best to harness the

extrinsic apoptosis pathway for cancer therapy. Given

that many cancers are inherently refractory to apoptosis

activation, the successful clinical translation of PARAs

will likely require better understanding of the most

relevant determinants of sensitivity versus resistance.

Insights from preclinical studies regarding predictive

biomarkers such as DR expression and O-glycosylation,

together with rational combinations with agents targeting

other cellular signaling pathways that might intersect

with the extrinsic apoptotic pathway, may further

enhance the clinical utility of PARAs. Moreover, mol-

ecular improvements — such as those to strengthen



potency or alter receptor selectivity — may augment

PARA activity in specific cancers. Together, these

approaches should help uncover the full potential of

the cell-extrinsic apoptosis pathway as a means of con-

trolling malignant disease.

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest

�� of outstanding interest

1. Brenner D, Mak TW: Mitochondrial cell death effectors. CurrOpin Cell Biol 2009, 21:871-877.

2. Vaux DL, Silke J: IAPs, RINGs and ubiquitylation. Nat Rev MolCell Biol 2005, 6:287-297.

3. Wilson NS, Dixit V, Ashkenazi A: Death receptor signaltransducers: nodes of coordination in immune signalingnetworks. Nat Immunol 2009, 10:348-355.

4. Gonzalvez F, Ashkenazi A: New insights into apoptosissignaling by Apo2L/TRAIL. Oncogene 2010, 29:4752-4765.

5. Daniel D, Wilson NS: Tumor necrosis factor: renaissance as acancer therapeutic? Curr Cancer Drug Targets 2008, 8:124-131.

6. Ashkenazi A: Targeting death and decoy receptors of thetumour-necrosis factor superfamily. Nat Rev Cancer 2002,2:420-430.

7. Ashkenazi A: Directing cancer cells to self-destruct with pro-apoptotic receptor agonists. Nat Rev Drug Discov 2008,7:1001-1012.

8. Johnstone RW, Frew AJ, Smyth MJ: The TRAIL apoptoticpathway in cancer onset, progression and therapy. Nat RevCancer 2008, 8:782-798.

9. Yue HH, Diehl GE, Winoto A: Loss of TRAIL-R does not affectthymic or intestinal tumor development in p53 andadenomatous polyposis coli mutant mice. Cell Death Differ2005, 12:94-97.

10. Dunn GP, Koebel CM, Schreiber RD: Interferons, immunity andcancer immunoediting. Nat Rev Immunol 2006,6:836-848.

11. Cretney E, Takeda K, Yagita H, Glaccum M, Peschon JJ,Smyth MJ: Increased susceptibility to tumor initiation andmetastasis in TNF-related apoptosis-inducing ligand-deficient mice. J Immunol 2002, 168:1356-1361.

12. Takeda K, Smyth MJ, Cretney E, Hayakawa Y, Kayagaki N,Yagita H, Okumura K: Critical role for tumor necrosisfactor-related apoptosis-inducing ligand in immunesurveillance against tumor development. J Exp Med 2002,195:161-169.

13. Wiezorek J, Holland P, Graves J: Death receptor agonists as atargeted therapy for cancer. Clin Cancer Res 2010,16:1701-1708.

14.��

Wagner KW, Punnoose EA, Januario T, Lawrence DA, Pitti RM,Lancaster K, Lee D, von Goetz M, Yee SF, Totpal K et al.:Death-receptor O-glycosylation controls tumor-cellsensitivity to the proapoptotic ligand Apo2L/TRAIL. Nat Med2007, 13:1070-1077.

This study identifies O-glycosylation of DR4 and DR5 as a modificationthat frequently augments sensitivity of cancer cell lines to Apo2L/TRAIL,providing a potentially predictive diagnostic biomarker for PARA-basedtherapies.

15. Naka T, Sugamura K, Hylander BL, Widmer MB, Rustum YM,Repasky EA: Effects of tumor necrosis factor-relatedapoptosis-inducing ligand alone and in combination withchemotherapeutic agents on patients’ colon tumors grown inSCID mice. Cancer Res 2002, 62:5800-5806.


16. Cheng J, Hylander BL, Baer MR, Chen X, Repasky EA: Multiplemechanisms underlie resistance of leukemia cells to Apo2Ligand/TRAIL. Mol Cancer Ther 2006, 5:1844-1853.

17. Hylander BL, Pitoniak R, Penetrante RB, Gibbs JF, Oktay D,Cheng J, Repasky EA: The anti-tumor effect of Apo2L/TRAIL onpatient pancreatic adenocarcinomas grown as xenografts inSCID mice. J Transl Med 2005, 3:22.

18. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M,Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI et al.:Control of TRAIL-induced apoptosis by a family of signalingand decoy receptors. Science 1997, 277:818-821.

19. Ichikawa K, Liu W, Zhao L, Wang Z, Liu D, Ohtsuka T, Zhang H,Mountz JD, Koopman WJ, Kimberly RP et al.: Tumoricidal activityof a novel anti-human DR5 monoclonal antibody withouthepatocyte cytotoxicity. Nat Med 2001, 7:954-960.

20. Brincks EL, Katewa A, Kucaba TA, Griffith TS, Legge KL: CD8 Tcells utilize TRAIL to control influenza virus infection.J Immunol 2008, 181:4918-4925.

21. Pennarun B, Meijer A, de Vries EG, Kleibeuker JH, Kruyt F, deJong S: Playing the DISC: turning on TRAIL death receptor-mediated apoptosis in cancer. Biochim Biophys Acta 2010,1805:123-140.

22. Ozoren N, El-Deiry WS: Cell surface death receptor signaling innormal and cancer cells. Semin Cancer Biol 2003,13:135-147.

23. Bin L, Thorburn J, Thomas LR, Clark PE, Humphreys R,Thorburn A: Tumor-derived mutations in the TRAIL receptorDR5 inhibit TRAIL signaling through the DR4 receptor bycompeting for ligand binding. J Biol Chem 2007, 282:28189-28194.

24. Elias A, Siegelin MD, Steinmuller A, von Deimling A, Lass U,Korn B, Mueller W: Epigenetic silencing of death receptor 4mediates tumor necrosis factor-related apoptosis-inducingligand resistance in gliomas. Clin Cancer Res 2009,15:5457-5465.

25. Horak P, Pils D, Haller G, Pribill I, Roessler M, Tomek S, Horvat R,Zeillinger R, Zielinski C, Krainer M: Contribution of epigeneticsilencing of tumor necrosis factor-related apoptosis inducingligand receptor 1 (DR4) to TRAIL resistance and ovariancancer. Mol Cancer Res 2005, 3:335-343.

26. Bae SI, Cheriyath V, Jacobs BS, Reu FJ, Borden EC: Reversal ofmethylation silencing of Apo2L/TRAIL receptor 1 (DR4)expression overcomes resistance of SK-MEL-3 and SK-MEL-28 melanoma cells to interferons (IFNs) or Apo2L/TRAIL.Oncogene 2008, 27:490-498.

27. Stern HM, Padilla M, Wagner K, Amler L, Ashkenazi A:Development of immunohistochemistry assays to assessGALNT14 and FUT3/6 in clinical trials of dulanermin anddrozitumab. Clin Cancer Res 2010, 16:1587-1596.

28.�

Rossin A, Derouet M, Abdel-Sater F, Hueber AO: Palmitoylationof the TRAIL receptor DR4 confers an efficient TRAIL-inducedcell death signalling. Biochem J 2009, 419: 185–192, 182 pfollowing 192.

29.�

Tang Z, Bauer JA, Morrison B, Lindner DJ: Nitrosylcobalaminpromotes cell death via S nitrosylation of Apo2L/TRAILreceptor DR4. Mol Cell Biol 2006, 26:5588-5594.

Refs. [28�,29�] identify DR4 modifications that correlate with increasedproapoptotic signal strength. See also Ref. [14��].

30. Schutze S, Tchikov V, Schneider-Brachert W: Regulation ofTNFR1 and CD95 signalling by receptorcompartmentalization. Nat Rev Mol Cell Biol 2008.

31. Jin Z, McDonald ER 3rd, Dicker DT, El-Deiry WS: Deficient tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)death receptor transport to the cell surface in human coloncancer cells selected for resistance to TRAIL-inducedapoptosis. J Biol Chem 2004, 279:35829-35839.

32. Zhang Y, Zhang B: TRAIL resistance of breast cancer cells isassociated with constitutive endocytosis of death receptors 4and 5. Mol Cancer Res 2008, 6:1861-1871.



33. Greco FA, Bonomi P, Crawford J, Kelly K, Oh Y, Halpern W, Lo L,Gallant G, Klein J: Phase 2 study of mapatumumab, a fullyhuman agonistic monoclonal antibody which targets andactivates the TRAIL receptor-1, in patients with advanced non-small cell lung cancer. Lung Cancer 2008, 61:82-90.

34. Trarbach T, Moehler M, Heinemann V, Kohne CH, Przyborek M,Schulz C, Sneller V, Gallant G, Kanzler S: Phase II trial ofmapatumumab, a fully human agonistic monoclonal antibodythat targets and activates the tumour necrosis factorapoptosis-inducing ligand receptor-1 (TRAIL-R1), in patientswith refractory colorectal cancer. Br J Cancer 2010,102:506-512.

35. Kelley RF, Totpal K, Lindstrom SH, Mathieu M, Billeci K, Deforge L,Pai R, Hymowitz SG, Ashkenazi A: Receptor-selective mutantsof apoptosis-inducing ligand 2/tumor necrosis factor-relatedapoptosis-inducing ligand reveal a greater contribution ofdeath receptor (DR) 5 than DR4 to apoptosis signaling. J BiolChem 2005, 280:2205-2212.

36. MacFarlane M, Inoue S, Kohlhaas SL, Majid A, Harper N,Kennedy DB, Dyer MJ, Cohen GM: Chronic lymphocyticleukemic cells exhibit apoptotic signaling via TRAIL-R1. CellDeath Differ 2005, 12:773-782.

37. LeBlanc HN, Ashkenazi A: Apo2L/TRAIL and its death anddecoy receptors. Cell Death Differ 2003, 10:66-75.

38. Clancy L, Mruk K, Archer K, Woelfel M, Mongkolsapaya J,Screaton G, Lenardo MJ, Chan FK: Preligand assembly domain-mediated ligand-independent association between TRAILreceptor 4 (TR4) and TR2 regulates TRAIL-induced apoptosis.Proc Natl Acad Sci U S A 2005, 102:18099-18104.

39. Merino D, Lalaoui N, Morizot A, Schneider P, Solary E, Micheau O:Differential inhibition of TRAIL-mediated DR5-DISC formationby decoy receptors 1 and 2. Mol Cell Biol 2006, 26:7046-7055.

40. Muppidi JR, Tschopp J, Siegel RM: Life and death decisions:secondary complexes and lipid rafts in TNF receptor familysignal transduction. Immunity 2004, 21:461-465.

41. Budd RC, Yeh WC, Tschopp J: cFLIP regulation of lymphocyteactivation and development. Nat Rev Immunol 2006, 6:196-204.

42. Sharp DA, Lawrence DA, Ashkenazi A: Selective knockdown ofthe long variant of cellular FLICE inhibitory protein augmentsdeath receptor-mediated caspase-8 activation and apoptosis.J Biol Chem 2005, 280:19401-19409.

43. Golks A, Brenner D, Krammer PH, Lavrik IN: The c-FLIP-NH2terminus (p22-FLIP) induces NF-kappaB activation. J Exp Med2006, 203:1295-1305.

44. Song JH, Tse MC, Bellail A, Phuphanich S, Khuri F, Kneteman NM,Hao C: Lipid rafts and nonrafts mediate tumor necrosis factorrelated apoptosis-inducing ligand induced apoptotic andnonapoptotic signals in non small cell lung carcinoma cells.Cancer Res 2007, 67:6946-6955.

45.��

Ricci MS, Jin Z, Dews M, Yu D, Thomas-Tikhonenko A, Dicker DT,El-Deiry WS: Direct repression of FLIP expression by c-myc is amajor determinant of TRAIL sensitivity. Mol Cell Biol 2004,24:8541-8555.

46.��

Zhang L, Ren X, Alt E, Bai X, Huang S, Xu Z, Lynch PM, Moyer MP,Wen XF, Wu X: Chemoprevention of colorectal cancer bytargeting APC-deficient cells for apoptosis. Nature 2010,464:1058-1061.

Refs. [45��,46��] implicate c-myc in regulating the levels of the FLICE(caspase-8) inhibitory protein (FLIP) in tumor cells.

47. Aza-Blanc P, Cooper CL, Wagner K, Batalov S, Deveraux QL,Cooke MP: Identification of modulators of TRAIL-inducedapoptosis via RNAi-based phenotypic screening. Mol Cell2003, 12:627-637.

48.��

Jin Z, Li Y, Pitti R, Lawrence D, Pham VC, Lill JR, Ashkenazi A:Cullin3-based polyubiquitination and p62-dependentaggregation of caspase-8 mediate extrinsic apoptosissignaling. Cell 2009, 137:721-735.

This study demonstrates that ubiquitylation of caspase-8 by a Cullin 3-based E3 ligase in response to Apo2L/TRAIL promotes caspase-8 aggre-gation and activation, and thereby, augments proapoptotic signaling.


49. LeBlanc H, Lawrence D, Varfolomeev E, Totpal K, Morlan J,Schow P, Fong S, Schwall R, Sinicropi D, Ashkenazi A: Tumor-cellresistance to death receptor-induced apoptosis throughmutational inactivation of the proapoptotic Bcl-2 homologBax. Nat Med 2002, 8:274-281.

50. Liu FT, Agrawal SG, Gribben JG, Ye H, Du MQ, Newland AC, Jia L:Bortezomib blocks Bax degradation in malignant B cellsduring treatment with TRAIL. Blood 2008, 111:2797-2805.

51. Fulda S, Meyer E, Debatin KM: Inhibition of TRAIL-inducedapoptosis by Bcl-2 overexpression. Oncogene 2002,21:2283-2294.

52. Hinz S, Trauzold A, Boenicke L, Sandberg C, Beckmann S,Bayer E, Walczak H, Kalthoff H, Ungefroren H: Bcl-XL protectspancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis. Oncogene 2000,19:5477-5486.

53.�

Kim SH, Ricci MS, El-Deiry WS: Mcl-1: a gateway to TRAILsensitization. Cancer Res 2008, 68:2062-2064.

Reviews a series of papers that highlight Mcl-1 as a resistance factor thatmay control death receptor-mediated apoptosis in tumors.

54. Gillissen B, Wendt J, Richter A, Muer A, Overkamp T, Gebhardt N,Preissner R, Belka C, Dorken B, Daniel PT: Endogenous Bakinhibitors Mcl-1 and Bcl-xL: differential impact on TRAILresistance in Bax-deficient carcinoma. J Cell Biol 2010,188:851-862.

55. Fesik SW: Promoting apoptosis as a strategy for cancer drugdiscovery. Nat Rev Cancer 2005, 5:876-885.

56. LaCasse EC, Mahoney DJ, Cheung HH, Plenchette S, Baird S,Korneluk RG: IAP-targeted therapies for cancer. Oncogene2008, 27:6252-6275.

57. Cummins JM, Kohli M, Rago C, Kinzler KW, Vogelstein B, Bunz F:X-linked inhibitor of apoptosis protein (XIAP) is anonredundant modulator of tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL)-mediated apoptosis inhuman cancer cells. Cancer Res 2004, 64:3006-3008.

58. Vogler M, Walczak H, Stadel D, Haas TL, Genze F, Jovanovic M,Gschwend JE, Simmet T, Debatin KM, Fulda S: Targeting XIAPbypasses Bcl-2-mediated resistance to TRAIL and cooperateswith TRAIL to suppress pancreatic cancer growth in vitro andin vivo. Cancer Res 2008, 68:7956-7965.

59. Deng Y, Lin Y, Wu X: TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO. Genes Dev2002, 16:33-45.

60.�

Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG:A small molecule Smac mimic potentiates TRAIL- andTNFalpha-mediated cell death. Science 2004,305:1471-1474.

61.��

Vogler M, Walczak H, Stadel D, Haas TL, Genze F, Jovanovic M,Bhanot U, Hasel C, Moller P, Gschwend JE et al.: Small moleculeXIAP inhibitors enhance TRAIL-induced apoptosis andantitumor activity in preclinical models of pancreaticcarcinoma. Cancer Res 2009, 69:2425-2434.

62.��

Varfolomeev E, Alicke B, Elliott JM, Zobel K, West K, Wong H,Scheer JM, Ashkenazi A, Gould SE, Fairbrother WJ et al.: Xchromosome-linked inhibitor of apoptosis regulates celldeath induction by proapoptotic receptor agonists. J BiolChem 2009, 284:34553-34560.

Refs. [60�,61��,62��] demonstrate the potential utility of combining PARAswith inhibitors of IAP to enhance tumor cell apoptosis.

63. Varfolomeev E, Maecker H, Sharp D, Lawrence D, Renz M,Vucic D, Ashkenazi A: Molecular determinants of kinasepathway activation by Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand. J Biol Chem 2005,280:40599-40608.

64. Falschlehner C, Emmerich CH, Gerlach B, Walczak H: TRAILsignalling: decisions between life and death. Int J Biochem CellBiol 2007, 39:1462-1475.

65. Karin M, Cao Y, Greten FR, Li ZW: NF-kappaB in cancer: frominnocent bystander to major culprit. Nat Rev Cancer 2002,2:301-310.



66. Braeuer SJ, Buneker C, Mohr A, Zwacka RM: Constitutivelyactivated nuclear factor-kappaB, but not induced NF-kappaB,leads to TRAIL resistance by up-regulation of X-linkedinhibitor of apoptosis protein in human cancer cells. MolCancer Res 2006, 4:715-728.

67. Chen X, Kandasamy K, Srivastava RK: Differential roles of RelA(p65) and c-Rel subunits of nuclear factor kappa B in tumornecrosis factor-related apoptosis-inducing ligand signaling.Cancer Res 2003, 63:1059-1066.

68. Brookd AD, Jacobsten KM, Li W, Shanker A, Sayers TJ:Bortezomib sensitizes human renal cell carcinomas to TRAILapoptosis through increased activation of caspase-8 in the


death-inducing signaling complex. Cancer Res 2010,8:729-738.

69. Boehrer S, Nowak D, Puccetti E, Ruthardt M, Sattler N, Trepohl B,Schneider B, Hoelzer D, Mitrou PS, Chow KU: Prostate-apoptosis-response-gene-4 increases sensitivity toTRAIL-induced apoptosis. Leuk Res 2006,30:597-605.

70.�

Burikhanov R, Zhao Y, Goswami A, Qiu S, Schwarze SR,Rangnekar VM: The tumor suppressor Par-4 activates anextrinsic pathway for apoptosis. Cell 2009, 138:377-388.

This work links Par-4 and the ER stress pathway to Apo2L/TRAILsignaling and cell death.


Proapoptotic DR4 and DR5 signaling in cancer cells: toward clinical translation

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