See online version for legend and references.1192 Cell 143, December 23, 2010 ©2010 Elsevier Inc. DOI 10.1016/j.cell.2010.12.004

Snap

Shot:

Ext

rinsi

c A

popto

sis

Pat

hw

ays

Ing

e Ve

rbru

gg

e, R

icky

W. J

ohn

sto

ne, a

nd M

ark

J. S

myt

hR

esea

rch

Div

isio

n, T

he P

eter

Mac

Cal

lum

Can

cer

Cen

tre,

St.

And

rew

s P

lace

, Eas

t M

elb

our

ne 3

002,

Vic

tori

a, A

ustr

alia

Su

rviv

al

DEDDED C

om

ple

x II

cIA

Pan

tag

on

ists

?

?

Cas

pas

e 8/

10

Ap

op

tosi

sC

asp

ase

-in

de

pe

nd

en

t d

ea

th (

ne

cro

sis)

DED DEDDD DEDDED

DED

DED

DED

DED

DD

DED

DD

DED

DED

DED

DED

DED

DD

DD

DD

DD

DD

DD

DD

DED

DED

DD DEDDED

DED

DED

DD

DEDDED

DED

DD

DD

DD

DD

DD

DED

DDDDDDDD

DD DEDDED

DED

DED

DD

DEDDED

DED

DD

DDDDDD

DD

DD

DED

cIA

P1/

2cI

AP

1/2

DDDDDDDD

DD

XR

IPK

1

FAD

D

ME

MB

RA

NE

CY

TO

PL

AS

M

Pro

casp

ase

8/10

cFL

IP

CD

95L

CD

95

Co

mp

lex

II a

nd

cIA

Ps

in d

ea

th r

ec

ep

tor

sig

na

lin

g

DED

DED

DED

DED

DED

DED

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DEDDED

DED

DEDDED

DED

DD

DD

Bcl

-2

Ap

af-1

Pro

casp

ase

9 Cyt

och

rom

e c

Sm

ac/D

IAB

LO

Bak

Bax

O-g

lyco

syla

tio

nO

-gly

cosy

lati

on

Dyn

amin

Dyn

amin

Mit

och

on

dri

on

Mit

och

on

dri

on

Bid

Cas

pas

e 8/

10

Pro

casp

ase

8/10

Pro

casp

ase

8/10

Dea

thd

om

ain

Dea

thef

fect

or

do

mai

n

DIS

CD

ISC

ME

MB

RA

NE

CY

TO

PL

AS

M

Lip

id r

aft

(SP

OT

S)

cFL

IP

cFL

IP

CD

95L

TR

AIL

Pro

ap

op

toti

c s

ign

alin

g p

ath

wa

ys in

clu

din

g t

he

rap

uti

c in

terv

en

tio

n s

tra

teg

ies

De

ath

re

ce

pto

r a

go

nis

tsrh

TR

AIL

(AM

G95

1)a-

TR

AIL

-R1/

R2

ago

nist

ic m

Ab

s(M

apa-

/Lex

atum

umab

, A

po

mab

, A

MG

655,

PR

O95

780)

rhC

D95

L (A

PO

010)

An

tic

an

ce

r th

era

pe

uti

cs

do

wn

reg

ula

tin

g c

FL

IPIo

nizi

ng r

adia

tio

n, E

top

osi

de,

Vo

rino

stat

, P

ano

bin

ost

at,

Co

mp

oun

d C

, et

c.

Bc

l-2

an

tag

on

ists

AB

T26

3, A

T10

1,

Ob

ato

clax

, O

blim

erse

m

Sm

ac

mim

eti

cs

TL3

2711

, A

EG

4082

6, L

BW

242,

C

om

po

und

3,

8, 1

1, C

XIA

P a

nti

sen

seA

EG

3515

6

tBid

Ap

op

toso

me

Typ

e I

Typ

e I

I

XIA

P

XIA

P

Bcl

-2

Su

rviv

al/

Mig

rati

on

PI3

KS

rc f

amily

kin

ases

Cas

pas

e 8

ME

KK

1(M

AP

K)

MK

K4,

7

IKK?

AK

TN

F-κ

B(c

-Rel

)N

F-κ

B(R

elA

)A

P-1

p38

JNK

Src

ER

K

Oth

er?

Dea

thIn

flam

mat

ion

Pro

lifer

atio

n/

Su

rviv

alS

urv

ival

Dea

thTu

mo

r p

rom

oti

on

Tum

or

pro

mo

tio

n(in

vasi

ven

ess

Met

s fo

rmat

ion

)

CD

95L

CD

95

CD

95

TR

AIL

TR

AIL

-R1/

R2

TR

AIL

-R

1/R

2

An

tia

po

pto

tic

pa

thw

ays

ac

tiva

ted

by

de

ath

re

ce

pto

rs

Cas

pas

e 10

TRA

DD

TR

AF

-2

Pro

casp

ase

3/6/

7

Cas

pas

e 3/

7

Ap

op

tosi

s

Cas

pas

e 6

Cas

pas

e 3

Cas

pas

e 9

DED DED Dea

thef

fect

or

do

mai

n

Lip

id r

aft

(SP

OT

S)

PA

DED DED

DED DED

DIS

C

PAPA

DIS

C

DED DED

DED DED

DED DED

O-g

lyco

syla

tio

nO

-gly

cosy

lati

on

O-g

lyco

syla

tio

nO

-gly

cosy

lati

on

O-g

lyco

syla

tio

nO

-gly

cosy

lati

on

O-g

lyco

syla

tio

nO

-gly

cosy

lati

on

O-g

lyco

syla

tio

nO

-gly

cosy

lati

on

O-g

lyco

syla

tio

nO

-gly

cosy

lati

on

O-g

lyco

syla

tio

nO

-gly

cosy

lati

on

O-g

lyco

syla

tio

nO

-gly

cosy

lati

on

GL

DED DEDDED DED DED

DED DED

DED DED DEDDED DED

DED DED

DED DED

DED

DDDDDD

DDDDDD

PA

Bcl

-XL

Bcl

-XL

FAD

D

RIP

K1

DD

DD

ME

MB

RA

NE

CY

TO

PL

AS

M

DDDDDD

DDDDDD

DD

DED

SnapShot: Extrinsic Apoptosis PathwaysInge Verbrugge, Ricky W. Johnstone, and Mark J. SmythResearch Division, The Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne 3002, Victoria, Australia

Apoptosis plays an important role in cell and tissue homeostasis and in growth control. Two separate yet interlinked signaling pathways lead to apoptotic execution: (1) The intrinsic pathway that is activated by diverse stress signals (e.g., DNA-damaging chemotherapeutics, UV radiation, and growth factor withdrawal), and (2) the extrinsic pathway that is activated by proapoptotic receptor signals at the cell surface and is important for biological processes such as lymphocyte homeostasis. The two pathways converge at the level of effector Caspases (e.g., Caspases 3, 6, and 7), which are cysteine proteases that execute apoptosis by cleaving essential cellular proteins.

The intrinsic apoptosis pathway is regulated by proapoptotic (e.g., Bid, Bak, and Bax) and antiapoptotic (e.g., Bcl-2 and Bcl-XL) members of the Bcl-2 family, which are char-acterized by at least one conserved Bcl-2 homology (BH) domain. Activation of the intrinsic pathway ultimately leads to mitochondrial permeabilization and subsequent effector Caspase activation. The extrinsic apoptosis pathway is triggered through activation of death receptors of the tumor necrosis factor (TNF) family, including the TNF receptor 1 (TNF-R1) itself, CD95 (APO-1, Fas), TNF-related apoptosis-inducing ligand (TRAIL) receptors (TRAIL-R1 and TRAIL-R2, also known as DR4 and DR5), DR3 and DR6. The extrinsic pathway can link to the intrinsic mitochondrial pathway via cleavage of Bid. In this SnapShot, we describe the apoptotic pathways activated by the CD95 and TRAIL death recep-tors, as they are considered bona fide death receptors and induce apoptosis using similar pathways.

Cell Death Pathways Induced by Death ReceptorsSignaling through both CD95 and TRAIL-R1/-R2 leads to formation of a death-inducing signaling complex (DISC), which activates inducer Caspases 8 and 10. CD95 and TRAIL-R1/-R2 exist as preformed homotrimers on the cell membrane and contain an intracellular death domain (DD). Binding of ligand or agonistic antibodies results in death receptor oligomerization followed by recruitment Fas-associated protein with death domain (FADD) through homotypic interactions between the death domains of the receptors and those of FADD. Subsequently, FADD recruits Procaspases 8 and 10 and cellular FADD-like interleukin-1 β converting enzyme (FLICE)-like inhibitory protein (cFLIP) through homotypic interactions between their death effector domains (DEDs). Together with the death receptors, these proteins form the DISC.

Formation of the DISC and further clustering of death receptors promotes the homodimerization and activation of inducer Caspases 8 and 10, which are then processed into their mature forms. cFLIP is an important regulator of inducer Caspase activation. It closely resembles Procaspases 8 and 10 and competes for FADD binding, but cFLIP lacks the Procaspase 8/10 catalytic site, thereby preventing Procaspase 8/10 homodimerization and activation.

In cells that process only small amounts of Caspase 8 at the DISC, an amplification of the apoptotic signal through the mitochondria is often required. This is known as the “Type II” pathway and is engaged by cells such as hepatocytes. In contrast, cells that process large amounts of Caspase 8 at the DISC can activate effector Caspases directly and do not require mitochondrial amplification for death to occur. This is known as the “Type I” pathway and is utilized by cells such as lymphocytes.

In the Type II pathway, Caspase 8 cleaves the BH3-only protein Bid. Truncated Bid (tBid) activates Bak and Bax at the mitochondria, resulting in permeabilization of the mitochondrial outer membrane and release of proteins such as cytochrome c, Smac (second mitochondria-derived activator of Caspases)/DIABLO (direct inhibitor of apoptosis protein [IAP]-binding protein with low pI), and HtrA2 (high-temperature requirement protein A2)/Omi. Cytochrome c binds to the adaptor molecule APAF-1 (apoptotic protease-activating factor 1), which interacts with Procaspase 9 to form the apoptosome. At the apoptosome, Caspase 9 is activated, processed, and released, leading to the activation of effector Caspases 3, 6, and 7, and eventually disintegration of the cell.

X-linked inhibitor of apoptosis protein (XIAP), a ubiquitously expressed cytoplasmic protein, inhibits apoptosis by binding directly to Caspase 3, 7, and 9, thereby masking their active sites. Smac/DIABLO and HtrA2/Omi are released following mitochondrial permeabilization and antagonize the activity of XIAP by competing for XIAP binding with Caspases, thus promoting Caspase activation and cell death.

In the case of the CD95 pathway, a cytosolic “complex II” consisting of FADD, Procaspase 8, and cFLIP molecules conveys the apoptotic signal. In an alternative pathway, the receptor-interacting serine/threonine kinase 1 (RIPK1, also known as RIP-1) associates with the DISC through its death domain and activates a cell death pathway that is inde-pendent of Caspases. This alternative pathway is inhibited by cellular inhibitor of apoptosis proteins (cIAPs), which prevent RIPK1 accumulation in the DISC. In addition, cFLIPL in the cytosolic RIPK1 complex inhibits RIPK1-mediated death. cIAPs may also inhibit TRAIL-induced death.

The Importance of Receptor and Ligand Modification and OrganizationReceptor oligomerization is critical for apoptosis mediated by death receptors, which can depend on the formulation (native or crosslinked ligands or antibodies) and presenta-tion of death receptor agonists. In mice, membrane-bound CD95 ligand (CD95L) induces only apoptosis, whereas antiapoptotic CD95 signaling pathways are activated in mice that express only soluble CD95L, promoting autoimmunity and tumorigenesis.

Posttranslational modifications of death receptors are also required for optimal apoptosis signaling. S-palmitoylation of TRAIL-R1 (at cysteines 261–263) and CD95 (at Cys 199) or O-glycosylation of TRAIL receptors by UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14) facilitates receptor oligomerization. This leads to the localization of death receptors into specialized membrane microdomains called “lipid rafts.” Lipid rafts stabilize receptor oligomers, forming so-called “signaling protein oligomerization transduction structures” (SPOTS), which are required for inducer Caspase activation. The CD95 complex subsequently requires internalization in Type I cells for apoptosis to proceed. The “rafts” that have formed require dynamin and clathrin to be internalized, which then form specialized membrane structures termed “CD95 receptosomes.” Formation of the receptosomes also requires ezrin-mediated actin filament association. In contrast, TRAIL-R1/-R2 appears to signal for apoptosis induction from the cell surface. Although TRAIL-R1/-R2 complexes can be internalized, interfering with receptor internalization still allows apoptosis to occur.

Alternative Signaling Pathways Induced by Death ReceptorsUnder certain circumstances, death receptors may activate prosurvival and proliferation pathways involving nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), phosphoinositide 3-kinase (PI3K), and Akt. Numerous factors can determine whether death receptors promote apoptosis or survival. These include the strength or duration of receptor stimulation and the presence or activity of proteins that regulate intracellular signaling. However, the precise molecular events that mediate activation of survival pathways remain unknown.

CD95 activation has been linked to tumorigenesis and tumor invasiveness through activation of a number of signaling proteins, such as Src kinases and NF-κB and through activation of JNK-signaling pathways. Therefore, CD95 does not always act as a dedicated death receptor but instead mediates diverse functions in different tissues and under different conditions.

Clinical Application of the Extrinsic Apoptosis Pathway in CancerAs tumors frequently acquire mutations that make them resistant to apoptosis induction via the intrinsic pathway, targeting the extrinsic apoptosis pathways is attractive in cancer therapy. Because TRAIL induces apoptosis in a large proportion of long-term established tumor cell lines, but for as yet unknown reasons is generally not toxic to normal tissue, therapeutic targeting of the extrinsic apoptosis pathways has focused primarily on reagents that stimulate TRAIL-R1 and -R2. TRAIL death receptor agonists in clinical trials include recombinant human (rh) TRAIL AMG951 (phase I), α-TRAIL-R1 monoclonal antibody (mAb) Mapatumamab (phase II), α-TRAIL-R2 mAbs Lexatumamab (phase I), Apomab (phase I), and AMG655 (phase I). A CD95 agonist in a phase I clinical trial is APO010, a dimer of CD95L trimers.

In addition, cells can be greatly sensitized to apoptosis signaling mediated by death receptors via the inhibition of prosurvival Bcl-2 family proteins and downregulation of c-FLIP or IAPs. Many anticancer agents that are in use clinically, including but certainly not limited to ionizing radiation, chemotherapeutic drugs (e.g., etoposide), histone deacetylase (HDAC) inhibitors, and small molecule kinase inhibitors, have been shown to downregulate c-FLIP protein levels.

Finally, numerous agents that affect cell death mediated by death receptors are currently in clinical trials. They include IAP antagonists, such as XIAP antisense AEG35156/GEM640 (phase II) and Smac mimetics that target XIAP, cIAP1, and cIAP2. These mimetics include TL32711 (phase I), LBW242 (phase I), AEG40826/HGS1029 (phase I), com-pound 3 (phase I), compound 11 (phase I), compound C (phase I), and compound 8 (phase I). Bcl-2 antagonists, such as ABT 263 (structural homolog to ABT737, phase II), AT-101 (phase II), Obatoclax (GX15-070, phase II), and Oblimersem (antisense DNA-targeting Bcl-2, phase III), are also in clinical trials for a wide range of tumor types.

1192.e1 Cell 143, December 23, 2010 ©2010 Elsevier Inc. DOI 10.1016/j.cell.2010.12.004

SnapShot: Extrinsic Apoptosis PathwaysInge Verbrugge, Ricky W. Johnstone, and Mark J. SmythResearch Division, The Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne 3002, Victoria, AustraliaAbbreviationsAPAF-1, apoptotic protease-activating factor 1; BH, Bcl-2 homology; cFLIP, cellular FLICE (FADD-like interleukin 1 β)-like inhibitory protein; (c)IAP, (cellular) inhibitor of apoptosis protein; DD, death domain; DED, death effector domain; DIABLO, direct IAP-binding protein with low pI; DISC, death-inducing signaling complex; DR, death receptor; FADD, Fas-associated protein with death domain; GALNT14, UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 14; HtrA2, high-temperature require-ment protein A2; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; PI3K, phosphoinositide 3-kinase; rh, recombinant human; RIPK1, receptor interacting ser-ine/threonine kinase 1; Smac, second mitochondria-derived activator of caspases; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R, TRAIL receptor; XIAP, X-linked inhibitor of apoptosis protein.

ACKNOWLEDGMENTS

I.V. is funded by the Dutch Cancer Society (NKI2009-4446).

R.J. and M.J.S. are funded by the National Health and Medical Research Council of Australia (NHMRC), the Susan G. Komen Breast Cancer Foundation, the Prostate Cancer Founda-tion of Australia, Cancer Council Victoria, the Victorian Cancer Agency, and the Victorian Breast Cancer Research Consortium.

REfERENCES

Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S., Sameshima, M., Hase, A., Seto, Y., and Nagata, S. (1991). The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66, 233–243.

Kischkel, F.C., Lawrence, D.A., Chuntharapai, A., Schow, P., Kim, K.J., and Ashkenazi, A. (2000). Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 12, 611–620.

Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998). Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in reponse to activation of cell surface death receptors. Cell 94, 481–490.

Medema, J.P., Scaffidi, C., Kischkel, F.C., Shevchenko, A., Mann, M., Krammer, P.H., and Peter, M.E. (1997). FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J. 16, 2794–2804.

O’ Reilly, L.A., Tai, L., Lee, L., Kruse, E.A., Grabow, S., Fairlie, W.D., Haynes, N.M., Tarlinton, D.M., Zhang, J.G., Belz, G.T., et al. (2009). Membrane-bound Fas ligand only is essential for Fas-induced apoptosis. Nature 461, 659–663.

Pan, G., O’Rourke, K., Chinnaiyan, A.M., Gentz, R., Ebner, R., Ni, J., and Dixit, V.M. (1997). The receptor for the cytotoxic ligand TRAIL. Science 276, 111–113.

Pitti, R.M., Marsters, S.A., Ruppert, S., Donahue, C.J., Moore, A., and Ashkenazi, A. (1996). Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J. Biol. Chem. 271, 12687–12690.

Tait, S.W., and Green, D.R. (2010). Mitochondria and cell death: outer membrane permeabilization and beyond. Nat. Rev. Mol. Cell Biol. 11, 621–632.

Trauth, B.C., Klas, C., Peters, A.M., Matzku, S., Möller, P., Falk, W., Debatin, K.M., and Krammer, P.H. (1989). Monoclonal antibody-mediated tumor regression by induction of apop-tosis. Science 245, 301–305.

Walczak, H., Degli-Esposti, M.A., Johnson, R.S., Smolak, P.J., Waugh, J.Y., Boiani, N., Timour, M.S., Gerhart, M.J., Schooley, K.A., Smith, C.A., et al. (1997). TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J. 16, 5386–5397.

1192.e2 Cell 143, December 23, 2010 ©2010 Elsevier Inc. DOI 10.1016/j.cell.2010.12.004