Role of the survival proteins hsp27 and survivin in a small molecule sensitization to TRAIL mediated apoptosis 1

119 Modulation of Hsp27 expression can modulate HeLa cells viability upon treatment with TRAIL, alone or in combination with LY30.... 121 Silencing of Hsp27 reduces long term cell viabi

ROLE OF THE SURVIVAL PROTEINS HSP27 AND SURVIVIN IN A SMALL MOLECULE SENSITIZATION

TO TRAIL-MEDIATED APOPTOSIS

GRÉGORY MELLIER

(B.Sc., M.Sc., Claude Bernard University – Lyon 1, Lyon, France)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF

Of course, huge thanks to all my labmates, both past and present, for making the lab such a lively place

To my friend and flatmate, Dr Andrea Lisa Holme for her encouragements during the writing of this thesis and her help during the past few years

To Dr Alan Prem Kumar and the rest of the gang for their friendship

And last but not least, I would like to thank my family for their never ending support despite the distance Dad, thank you for believing in me

To my mother

TABLE OF CONTENTS

Acknowledgments i

Table of contents iii

Summary ix

List of tables xi

List of Figures xii

List of Abbreviations xiv

Introduction 1

1 General apoptotic mechanisms 1

1.1 Apoptosis and necrosis 1

1.1.1 Necrosis 1

1.1.2 Apoptosis 1

1.1.2.1 Characteristics of apoptosis 2

1.1.2.2 Apoptosis is an evolutionary conserved mechanism 3

1.1.2.3 Physiological role of apoptosis 3

1.2 Mechanisms of apoptosis induction 4

1.2.1 Molecular players in apoptosis 6

1.2.1.1 Caspase family 6

1.2.1.2 Inhibitors of apoptosis 8

1.2.1.2.1 Survivin 11

1.2.1.3 Bcl-2 family 13

1.2.2 Mitochondrial, or intrinsic, pathway 14

1.2.3 Death receptors, or extrinsic, pathway 17

1.2.3.1 Ligands and receptors of the TNF-! superfamily 17

1.2.3.2 Signalization pathway 18

2.4 TRAIL as a therapeutic modality 24

2.5 TRAIL resistance in cancer 26

2.5.1 Resistance in the death receptor pathway 28

2.5.1.1 Resistance involving the death receptors 28

2.5.1.2 DISC components: c-FLIP and caspase-8 29

2.5.2 Resistance in the mitochondrial pathway 29

2.5.2.1 Upstream of the mitochondria: the Bcl-2 family 30

2.5.2.2 Downstream of the mitochondria: the IAP family 30

2.6 Strategies to restore cancer cell sensitivity to TRAIL 31

2.6.1 Modulation of the extrinsic pathway 31

2.6.1.1 Up-regulation of death receptors 32

2.6.1.2 Oligomerization of DRs 32

2.6.1.3 Down-regulation of c-FLIP 33

2.6.2 Modulation of the intrinsic pathway 33

2.6.2.1 Down-regulation of IAPs 34

2.6.2.2 Down-regulation of the anti-apoptotic Bcl-2 proteins 34

2.6.3 Simultaneous modulation of extrinsic and intrinsic pathways 35

2.7 Pre-clinical and clinical evaluation of TRAIL 36

2.7.1 Recombinant human TRAIL 36

2.7.2 Agonistic antibodies 37

2.7.3 Combinational therapies 37

3 Heat Shock Proteins and Hsp27 39

3.1 High molecular weight Hsps 39

3.1.1 Hsp90 40

3.1.2 Hsp70 40

3.1.3 Hsp60/10 41

3.2 Small Heat Shock Proteins 41

3.2.1 Cellular characteristics 43

3.2.1.1 Cellular localization 43

3.2.1.2 Expression/Regulation 43

3.2.1.2.1 Expression following a cellular stress 44

3.2.1.2.2 Pathological expression 45

3.2.1.2.3 Physiological expression 46

3.2.2 Biochemical characteristics 46

3.2.2.1 Structure 47

3.2.2.1.1 Primary/Secondary 47

3.2.2.1.2 Tertiary/Quaternary 49

3.2.2.2 Phosphorylation 50

3.2.2.2.1 Phosphorylation signaling 51

3.2.2.2.2 Effect of phosphorylation on the quaternary structure 51

3.2.3 Cellular functions 52

3.2.3.1 Protection against heat shock and oxidative stress 54

3.2.3.2 Cytoskeleton protection 57

3.2.3.3 Anti-apoptotic functions 57

3.2.3.4 Chaperone activity 60

3.2.3.4.1 Mode of action 60

3.2.3.4.2 Importance of the large oligomers for the chaperone activity 61 3.2.3.4.3 Dynamics of oligomers 63

3.2.3.4.4 Additional role of Hsp27 chaperone activity 63

4 LY303511 65

Material and Methods 68

Cell line 68

Reagents and chemicals 68

Antibodies 69

Plasmids and siRNAs 70

Transient transfection of plasmids 70

Transient silencing of protein expression 71

Cell viability assay 71

Colony forming assay 72

Laser Scanning Cytometry (LSC) 72

Cell cycle, PI uptake 72 Mitochondrial membrane potential measurement and mitochondrial

Soluble / Insoluble fractions 75

Mitochodrial fractionation 76

Nuclear fractionation 76

Non-radioactive Electro-Mobility Shift Assay (EMSA) 77

Chromatin Immuno-Precipitation (ChIP) 77

Reverse-Transcriptase Polymerase Chain Reaction 79

Total RNA extraction 79

RT-PCR 79

Hsp27 oligomerization analysis 80

In vivo chaperone activity assay 81

Densitometry 82

Statistical analysis 82

Aims of the study 83

Results 84

LY30 restores HeLa cells sensitivity to TRAIL-induced cell death 84

LY30 and TRAIL combined treatment decreases HeLa cells ability to form colonies 85

LY30, alone or in combination with TRAIL, induces an early mitochondrial membrane potential depolarization ("#m) and mitochondrial aggregation 88

LY30 and TRAIL treatment engages the mitochondrial apoptotic pathway 91

LY30 and TRAIL combined treatment induces, and is dependent on, caspase activation 93

LY30 and LY30+TRAIL treatment decrease Hsp27 protein level in cleared RIPA cell lysates 97

LY30-mediated decrease in Hsp27 protein level is not due to transcriptional regulation nor proteasomal degradation 99

LY30, alone or in combination with TRAIL, does not affect Hsp27 expression but instead induces a long lasting translocation of Hsp27 to a nuclei-enriched fraction 101

Hsp27 specifically translocates to the nucleus upon exposure to LY30 106

Hsp27 phosphorylation increases upon exposure to LY30 108

Hsp27 increased phosphorylation is dependent on LY30-mediated p38 protein kinase activation 111

Hsp27 increased phosphorylation is dependent on LY30-mediated Protein

Phosphatase 2A (PP2A) inhibition 113

LY30 disrupts the equilibrium between small size and large size Hsp27 oligomers 117

LY30 affects Hsp27 molecular chaperone activity in vivo 119

Modulation of Hsp27 expression can modulate HeLa cells viability upon treatment with TRAIL, alone or in combination with LY30 121

Silencing of Hsp27 reduces long term cell viability/colony formation ability of HeLa cells when combined with TRAIL treatment 124

Modulation of Survivin expression by LY30 at the transcriptional level 127

Modulation of Survivin expression by LY30 at the post-transcriptional level 129 Survivin expression silencing decreases HeLa cells viability and sensitizes them to LY30 and TRAIL, alone or in combination 131

Silencing of survivin expression has a drastic effect on medium- and long-term cell survival alone or in combination with TRAIL 133

LY30 treatment induces the expression of Egr-1, a repressor of Survivin expression 135

Egr-1 binds to the survivin gene promoter upon LY30 treatment 137

Egr-1 silencing protects HeLa cells from cell death by partially reducing LY30-mediated down-regulation of Survivin 139

Double knock-down of Hsp27 and survivin sensitizes HeLa cells to TRAIL 141

Discussion 143

LY30 enhances TRAIL-induced cell death 143

LY30 regulates Hsp27 functions 145

LY30 does not affect Hsp27 expression but its localization 145

LY30 mediates Hsp27 phosphorylation through activation of p38 MAPK and inhibition of PP2A 148 LY30-mediated Hsp27 phosphorylation affects its oligomerization and

LY30 promotes survivin proteasomal degradation and inhibits its transcription

155

Importance of survivin protection in HeLa cells 156

Possible consequences of survivin down-regulation based on the litterature156 Mechanisms of LY30-mediated transcriptional regulation of survivin 158

LY30 induces Egr-1 159

Egr-1 represses survivin expression 161

Hsp27 and survivin as key targets of LY30 163

Potential use of LY30 and TRAIL combination in chemotherapy 164

Conclusion 166

References 169

Appendix A 206

Supplemental figures 206

Appendix B 208

Publications 208

Conferences 208

SUMMARY

In the last decade, TRAIL has been highlighted as a tumor selective molecule capable of engaging, depending upon the cell type, both the intrinsic and extrinsic apoptotic pathway, making it a promising therapeutic candidate However, the observation that tumor cells were resistant or could acquire resistance to TRAIL treatment sparked the search for molecules capable of enhancing or restoring sensitivity to TRAIL One such molecule, LY303511, an inactive analogue of the PI3K inhibitor LY294002, has previously been shown by our group to sensitize different type of tumor cells to TRAIL-induced apoptosis This sensitization was linked to ROS production, MAPK activation, up-regulation of death receptors expression and clustering, and overall enhancement of DISC formation Based on the premises that both quercetin and LY29, from which LY30 is derived, could affect the small heat shock protein Hsp27 and the IAP survivin, we set out to investigate the possibility for LY30 to have the same effect

Firstly, the model of LY30 sensitization to TRAIL was assessed in our cell system We show that pre-incubation of HeLa cells with LY30 significantly amplifies TRAIL signaling as evidenced by decrease cell viability and reduction in the cancer cells colony formation ability This increase in TRAIL sensitivity involved mitochondrial membrane permeabilization resulting in the release of cytochrome c

and the dissociation of Hsp27 oligomers is responsible for a marked inhibition of Hsp27 chaperone activity Furthermore, these effects are combined with a slow and sustain nuclear sequestration Thirdly, this study demonstrates that LY30 induces the down-regulation of survivin both at the transcriptional and post-translational level The transcriptional regulation was shown to be due to the induction of Egr-1, a repressor of survivin Lastly, this study provides evidence of the importance of both survival proteins in the resistance to TRAIL as well as in LY30-mediated sensitization

These findings present a novel mechanism of action of LY30 in sensitization

to TRAIL-mediated apoptosis and confirm its potential for the treatment of tumor cells

LIST OF TABLES

Table 1: The ten human small heat shock proteins ……… 49

LIST OF FIGURES

Figure 1: Apoptotic signaling 5

Figure 2: Structure of the caspase family members and their associated function 7

Figure 3: IAPs family members 10

Figure 4: Major Bcl-2 family members 15

Figure 5: TRAIL receptors 23

Figure 6: Mechanisms involded in TRAIL resistance 27

Figure 7: Structural properties of human Hsp27 48

Figure 8: Regulation of Hsp27 oligomerization by its phosphorylation 53

Figure 9: Role of Hsp27 in glutathione metabolism 56

Figure 10: Anti-apoptotic functions of Hsp27 59

Figure 11: Chaperone-like activity of Hsp27 62

Figure 12: Structures of quercetin, LY294002 and LY303511 67

Figure 13: Effect of LY30 treatment on TRAIL-mediated apoptosis 86

Figure 14: Effect of LY30 and TRAIL treatment on colony forming ability of HeLa cells 87

Figure 15: Effect of LY30 and TRAIL treatment on mitochondrial membrane polarization 89

Figure 16: Effect of upon LY30 and TRAIL treatment on mitochondrial aggregation 90

Figure 17: Effect of LY30 and TRAIL treatment on cytochrome c and Smac release from the mitochondria 92

Figure 18: Effect of LY30 and TRAIL treatment on caspase activation 95

Figure 19: Effect of caspases inhibitors on cell viability upon LY30 and TRAIL treatment 96

Figure 20: Effect of LY30 and TRAIL on Hsp27 and other Hsps expression 98

Figure 21: Effect of LY30 treatment on Hsp27 transcriptional regulation and proteasomal degradation 100

Figure 22: Effect of LY30 and TRAIL treatment on Hsp27 cellular localization 104

Figure 23: Effect of heat-shock on Hsp27 cellular localization 105

Figure 24: Nuclear translocation of Hsp27 upon LY30 and TRAIL treatment 107

Figure 25: Effect of LY30 treatment on Hsp27 phosphorylation status 110

Figure 26: Effect of LY30 treatment on p38 MAPK activity 112

Figure 27: Effect of LY30 treatment on PP2A activity 115

Figure 28: Effects of p38 MAPK and PP2A inhibitors on cell viability 116

Figure 29: Effect of LY30 treatment on Hsp27 oligomers size distribution 118

Figure 30: Effect of LY30 treatment on in vivo chaperone activity 120

Figure 31: Effect of Hsp27 expression on LY30 sensitization to TRAIL-mediated apoptosis 123

Figure 32: Effect of Hsp27 silencing and TRAIL treatment on medium- and long-term cell survival 125

Figure 33: Effect of LY30 treatment on Survivin expression 128

Figure 34: Effect of LY30 and TRAIL treatment on Survivin degradation 130

Figure 35: Effect of Survivin silencing on HeLa cells viability alone or combined with LY30 and TRAIL treatment 132

Figure 36: Effect of Survivin silencing and TRAIL treatment on medium- and long-term cell survival 134

Figure 37: Effect of LY30 treatment on Egr-1 expression 136

Figure 38: Egr-1 binding to the survivin promoter upon LY30 treatment 138

Figure 39: Effect of Egr-1 silencing on Survivin expression and cell viability upon LY30 and TRAIL treatment 140

Figure 40: Cumulative effect of survivin and Hsp27 silencing on TRAIL sensitivity 142

Figure 41: Mechanisms of LY30 sensitization to TRAIL 168

LIST OF ABBREVIATIONS

5-FU: fluorouracil

6PG: 6-phosphoglucono-!-lactone

AIF: apoptosis inducing factor

ANT: adenine nucleotide transporter

AP-1: activating protein-1

APAF: apoptosis activating factor-1

Asp: aspartate

ATP: adenosine triphosphate

Bad: Bcl-2 antagonist of cell death

Bak: Bcl-2 homologous antagonist killer

Bax: Bcl-2-associated X protein

Bcl-2: B cells lymphoma 2

Bcl-XL: B-cell lymphoma-extra large

BH domain: Bcl-2 homology domains

Bid: BH3 interacting domain death agonist

BIR: baculoviral IAP repeat

BIRC: baculoviral IAP repeat-containing protein

c-FLIP: cellular FLICE-like inhibitory protein

CAD: caspase-activated DNAse

cAMP: cyclic adenosine aono-phosphate

CARD: caspase recruitment domain

Caspase: cysteine aspartate-specific proteases

CED: cell death protein

ChIP: chromatin immuno-precipitation

CHOP: C/EBP Homologous Protein

cIAP1/2: cellular IAP 1/2

CiCCP: carbonyl cyanide m-chlorophenylhydrazone

DEM: diethyl maleate

DISC: death-inducing signaling complex

DNA: deoxyribonucleic acid

DNAse: deoxyribonuclease

DR4: death receptor 4

DR5: death receptor 5

DTT: dithiothreitol

Egr-1: early growth response protein 1

eIFG4: eukaryotic initiation factor G4

EMSA: electromobility shift assay

ERK: extracellular signal-regulated kinase

FADD: Fas-associated protein with death domain

FasL: Fas ligand

GSSG: oxidized glutathione

HDAC: histone deacetylase

HSE: heat shock element

Hsf-1: heat shock factor 1

Hsp: heat shock protein

IAP: inhibitor of apoptosis

ICAD: inhibitor of caspase-activated DNAse

IKK: inhibitor of "B kinase

ILP2: IAP-like protein-2

INCEP: inner centromere protein

JNK: c-Jun N-terminal kinase

LSC: laser scanning cytometry

LY29: LY294002: 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one

LY30: LY303511: 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one

MAPK: mitogen-activated protein kinase

Mcl1: myeloid cell leukemia sequence 1 (Bcl2-related)

mRNA: messenger RNA

mTOR: mammalian target of rapamycin

NADPH: nicotinamide adenine dinucleotide phosphate

NES: nuclear export signal

NF-$B: nuclear factor $B

NIAP: neuronal apoptosis-inhibitory protein

OMM: outer mitochondrial membrane

OMP: outer membrane protein

OPG: osteoprotegerin

p53: protein 53

PAK: p-21 activated kinase

PARP: poly-(ADP-ribose) polymerase

PI: propidium iodide

PI3K: phosphoinositide 3-kinase

PKC: protein kinase C

PLAD: pre-ligand assembly domain

PP2A: protein phosphatase 2A

PTPC: permeability transition pore complex

Puma: p53 upregulated modulator of apoptosis

RING: really interesting new gene

RIP: receptor interacting protein

RNA: ribonucleic acid

ROCK1: rho-associated kinase 1

ROS: reactive oxygen species

Ser: serine

sHsp: small heat shock protein

siRNA: small interfering RNA

TMRE: tetramethylrhodamine, ethyl ester, perchlorate

TNF: tumor necrosis factor

VDAC: voltage-dependent anion-selective channel protein 1

XAF1: XIAP-associated factor 1

XIAP: X-linked IAP

"#m: mitochondrial transmembrane potential

INTRODUCTION

1 GENERAL APOPTOTIC MECHANISMS

1.1 Apoptosis and necrosis

to either undergo necrosis (low concentration of ATP) or apoptosis (high concentration of ATP) [2] Lastly, the DNA of necrotic cells is randomly degraded by endonucleases activated by serine proteases [3, 4]

1.1.2 Apoptosis

1.1.2.1 Characteristics of apoptosis

In 1972, Kerr et al coined the term “apoptosis”, in reference to the Greek term

meaning “fall of the leaves or petals”, while describing a cell death process different from necrosis [5]

Apoptosis is an active programmed cell death process defined by the ability of

a cell to undergo cell death in a controlled manner which involves a specific set of molecular players [6, 7] The cell undergoes a series of biochemical modifications successively resulting in: 1) the reduction of the cell volume, 2) chromatin condensation and inter-nucleosomal fragmentation [7-9], 3) membrane blebbing and formation of apoptotic bodies that are subsequently phagocyted by macrophages or neighboring epithelial cells During this process, the cell maintains the integrity of the plasma membrane, hence preventing an inflammatory reaction [8] However, plasma membrane undergoes an early modification consisting in the inversion of membrane phospholipids, including extracellular exposure of phospatidylserines [10]

The successive apoptotic events can be subdivided in three phases: a reversible initiation step followed by a regulatable amplification step and an irreversible execution step During the initiation stage, there is integration of the apoptotic signals by specialized protein complexes: either the Death-Inducing Signaling Complex (DISC) when apoptotic signals are transduced by the death

receptors, or the apoptosome in the presence of a nuclear stimulus (e.g DNA damage)

or a cytoplasmic stimulus (e.g oxidative stress) These complexes activate initiator

caspases that, in turn, activate executory caspases - hence the term of caspase cascade Activation of the latter triggers the executive phase of apoptosis

1.1.2.2 Apoptosis is an evolutionary conserved mechanism

The mechanisms involved in apoptosis are extremely well conserved

throughout evolution, from prokaryotes to mammals Studies on Caenorhabditis elegans allowed the characterization of the molecular components of the apoptotic machinery For a correct development in C elegans, 131 cells out the 1090 cells

undergo apoptosis Mutational studies led to the identification of the genes involved

The apoptotic pathway uncovered in C elegans consists of ced-3 and ced-4, which are required for the induction of apoptosis, and a third gene, ced-9, coding for a

protein that is able to block the apoptotic process [11, 12] These genes have a strong homology with mammalian genes Indeed, mammalian homologues of CED-3, CED-

4 and CED-9 are members of the caspase family, Apoptosis Activating Factor-1 (APAF-1) and B Cells Lymphoma 2 (Bcl-2) family, respectively [13-16]

1.1.2.3 Physiological role of apoptosis

Apoptosis is involved in numerous physiological processes During embryogenesis, the formation of inter-digit spaces occurs via apoptosis, allowing the proper separation of fingers [17] Apoptosis also takes part in tissue homeostasis by eliminating senescent or degenerative cells In addition, it plays an important role in the immune system by eliminating lymphocytes that synthesize inactive or autoimmune antibodies [18]

1.2 Mechanisms of apoptosis induction

Following numerous studies over the last two decades, two main apoptotic modes of action have been identified, mostly based on the caspase activation sequence (Figure 1) On one hand, an extrinsic apoptotic pathway, in response to extracellular signals such as death ligands, that involves DISC formation and where the initiator caspase-8 plays a major role On the other hand, an intrinsic pathway, in response to signals affecting the mitochondria, that involves the formation of the apoptosome where the initiator caspase-9 plays the central role

Figure 1: Apoptotic signaling

Engagement of the death receptor apoptotic pathway (extrinsic pathway) by TRAIL The mitochondrial pathway (intrinsic pathway) can also be engaged through cleavage

of Bid The intrinsic pathway is either part of an amplification loop in type I cells or the primary apoptotic pathway in type II cells

These two pathways are not mutually exclusive and are often inter-connected 24]

[22-1.2.1 Molecular players in apoptosis

Despite the important diversity in apoptotic signals, all cells undergoing apoptosis display similar morphological and biochemical modifications, suggesting the existence of a common executioner phase

1.2.1.1 Caspase family

Caspases (cysteine aspartate-specific proteases) are a family of intracellular proteins involved in the initiation and execution of apoptosis Caspases are highly

homologous to C elegans cell death gene CED-3 The mammalian family contains

many members, most participating in apoptosis, whilst the remaining family members are involved in cytokine processing and inflammation [25-27] (Figure 2) Caspases share some common properties: they all have a conservative penta-peptide active site containing a cysteine residue; they are all cysteine proteases that recognize a tetra-peptide site containing an aspartate residue [28]; they are all synthesized in an inactive form (zymogen or proenzyme)

Figure 2: Structure of the caspase family members and their associated function

The caspase family can be subdivided into initiators, which are able to auto-activate and initiate the proteolytic processing of other caspases, and effectors, which are activated by other caspase molecules The effector caspases cleave the vast majority

of substrates during apoptosis

Pro-caspase activation requires two cleavages at specific Asp-X bonds that promote the assembly of two small and two large sub-units and also release the prodomain The tetramer is the active form of the caspase [29] In addition to their sub-units, pro-caspases contain a prodomain of varying length in their N-terminal part [30, 31] Caspases with a long pro-domain, called upstream or initiator caspases, contain other domains such as Death Effector Domain (DED) for caspase-8 and -10 or Caspase Recruitment Domain (CARD) for caspase-2 and -9 These domains allow their recruitment by adaptor proteins into the DISC or the apoptosome Recruitment

of the initiator pro-caspases to these complexes results in their oligomerization and autocatalytic activation [32] Pro-caspase-3, -6 and -7, called downstream or executioner caspases, have a shorter pro-domain that prevent their recruitment, oligomerization and autoactivation Executioner caspases are necessarily activated by other active caspases Their activation results in the cleavage of numerous key cellular substrates [33-35] such as poly-(ADP-ribose) polymerase (PARP) [36] or ICAD, the Inhibitor of caspase-activated DNAse PARP is a nuclear protein implicated in DNA repair and is one of the earliest proteins cleaved by caspases Cleavage of ICAD allows the release and translocation of CAD into the nucleus, which results in the internucleosomal fragmentation of DNA, a hallmark of apoptosis [37, 38] Caspases also mediate nuclear shrinkage by cleaving lamins [39] and membrane blebbing by targeting proteins such as Rho-associated kinase 1 (ROCK1), p-21 activated kinase (PAK) and gelsolin [40-42]

1.2.1.2 Inhibitors of apoptosis

The IAP (Inhibitor of Apoptosis) gene was first identified in baculovirus where it was shown to enhance virus replication by protecting infected cells from

death Since then, IAP homologues have been identified in a wide range of organisms [43] The IAP proteins are thought to provide the final level of regulation of apoptosis

by inhibiting caspase-3, -7 and -9 [44-46] The most potent inhibitor of apoptosis, and also the one drawing the most interest, is XIAP (X-linked IAP) [47, 48]

IAPs are characterized by a domain of about 70 amino acids called BIR (baculoviral IAP repeat) [49, 50] The other defining motif found in IAPs is the C-terminal RING Zinc finger [51] In addition, some human IAPs contain a CARD domain between the BIR and RING domains [52, 53]

To date, there are eight IAP members in humans: NIAP (BIRC1), c-IAP1 (BIRC2), c-IAP2 (BIRC3), XIAP (BIRC4), survivin (BIRC5), BRUCE (BIRC6), Livin (BIRC7) and ILP2 (BIRC8) [43] (Figure 3)

Figure 3: IAPs family members

Proteins of the inhibitor of apoptosis (IAP) family include XIAP (X-linked IAP), c-IAP1, c-IAP2, ILP2 (IAP-like protein-2), ML-IAP (melanoma IAP)/Livin, NAIP (neuronal apoptosis-inhibitory protein) and survivin, and are also known as MIHA/ILP1, MIHB/HIAP2, MIHC/HIAP1, Ts-IAP, KIAP, BIRC1 and TIAP, respectively The eight mammalian IAPs are characterized by different composition of functional domains: BIR, RING and CARD

IAPs are believed to exert their function via direct caspase inhibition Several IAPs have been shown to bind and inhibit specific caspases For instance, XIAP, c-IAP-1, c-IAP-2 and survivin directly bind and inhibit caspase-3, -7 and -9 but not caspase-1, -6, -8, or-10 activity NIAP, however, does not seem to bind to caspases Caspases exist as inactive pro-caspases that require proteolytic activation; in this regard the IAPs have also been shown to inhibit the activation of procaspase-9 (Deveraux, Roy

et al 1998) Interestingly, c-IAP1, c-IAP2 and XIAP have been reported to retain their anti-apoptotic ability in the absence of the RING domain In c-IAP1 and c-IAP2, which also possess a CARD domain, a N-terminal fragment with only the BIR domain is sufficient to inhibit apoptosis, suggesting that the CARD domain is not necessary for their anti-apoptotic activity [53-55] It has also been reported that in addition to targeting caspases, there are evidences of non-caspase interactions [46, 50, 51]

1.2.1.2.1 Survivin

Survivin was first described in 1997 and named after its anti-apoptotic function [56] However, survivin was later shown to be a bifunctional protein as it also plays a crucial role during mitosis [57] Hence, survivin stands at the crossroad between cell cycle and death

It is the smallest member of the IAP family and is composed of a single BIR domain without the RING domain common to the other IAPs, it also has a nuclear

out mice embryos do not survive more than five days following fertilization [58] It is important to note that four splice variants of survivin have been observed but that all are expressed at much lower level and a clear role for all of them has yet to be defined In most cancer cells however, survivin expression is aberrant and independent of the cell cycle [59] Most studies demonstrate a role at the transcriptional level to explain the deregulation of survivin expression It is explained

by the fact that transcription factors regulating survivin expression such as p53, pRb and Sp1 [59-61] are themselves de-regulated in most cancer cells Besides transcriptional regulation, survivin expression is also subject to regulation by the ubiquitin-proteasome degradation system [62, 63] Interestingly, survivin proteasomal degradation is enhanced by a complex formed by XIAP and its inhibitor XIAP-associated factor 1 (XAF1) [64]

In its mitosis regulation role, survivin is associated to INCENP (Inner Centromere Protein), Borealin and Aurora B in a complex called the Chromosomal Passenger Complex (CPC) This complex ensures a correct mitosis by regulating the structure between chromosomes and mitotic spindle, controlling the correct segregation of sister chromatids and ensuring a correct cytokinesis [65] Survivin is also responsible for the correct localization of the CPC In addition, invalidation of survivin expression by siRNA results in major mitotic defects such as increased ploidy

For a long time, the presumption was that survivin protected cells from apoptosis solely by directly inhibiting caspases as shown for other IAPs However, a number of studies points to a more complex antiapoptotic role Indeed, survivin can either indirectly inhibit caspases or inhibit apoptosis independently of its caspase inhibitory function Firstly, survivin has been shown to favor XIAP antiapoptotic role

by binding to it and preventing its ubiquitination by UbcH5 thereby protecting it from proteasomal degradation [66] Secondly, survivin can bind to Smac/DIABLO (Second Mitochondria-derived Activator of Caspase/Direct Inhibitor of Apoptosis-Binding protein with LOw pI), a known inhibitor of XIAP, and prevent its interaction with XIAP Lastly, survivin can block apoptosis independently of caspase inhibition by preventing the release of AIF (Apoptosis Inducing Factor) into the cytosol [67]

1.2.1.3 Bcl-2 family

Apoptosis is also controlled by a highly conserved family of proteins: Bcl-2 proteins The gene coding for Bcl-2 was the first to be identified as a proto-oncogene in B lymphocytes [11] This family is composed of more than 20 members sharing a low homology when their full sequences are considered However, they display a very strong homology between particular regions, called Bcl-2 Homology domains (BH), essential for protein-protein interaction [68] (Figure 4) All members contain at least one of the four different BH domains (BH1 to BH4) Bcl-2 family is divided in two groups: the pro-apoptotic and the anti-apoptotic members [69] The latter group includes Bcl-2, Bcl-XL (B-cell lymphoma-extra large), Bcl-w and Mcl-1 (myeloid cell leukemia sequence 1 (Bcl2-related)) They contain all 4 BH domains (with the exception of Mcl-1, lacking a BH4 domain) as well as a hydrophobic C-terminal transmembrane domain targeting them to the nucleus envelope, the endoplasmic reticulum or the outer mitochondrial membrane (OMM) Bcl-2 is located in the outer

proteins and the BH3-only proteins [75] Bax (Bcl-2-associated X protein) and its relatives, such as Bak (Bcl-2 homologous antagonist Killer) and Bok (Bcl-2-related ovarian Killer), contain the domains BH1 to BH3 Bax is found in the cytoplasm under physiological conditions and is known to translocate to and oligomerize in the OMM upon exposure to an apoptotic signal Bak, however, is an integral OMM protein that also oligomerizes during apoptosis Bax/Bak assembly in large structures

is thought to contribute to the Mitochondrial Outer Membrane Permeabilization (MOMP) either by forming channels allowing cytochrome c release [76, 77] and/or

by interacting with components of the mitochondrial permeability transition pore complex (PTPC), such as VDAC (Voltage-dependent anion-selective channel protein 1), inducing an opening of the pores

The second anti-apoptotic family includes Bim, Bid (BH3 Interacting domain death agonist), Bad (Bcl-2 Antagonist of cell Death), Noxa and Puma (p53 Upregulated Modulator of Apoptosis) All members only contain the BH-3 domain Their main mode of action is to bind to, and neutralize, their pro-survival relatives [78, 79] or to bind to, and activate, the other pro-apoptotic Bcl-2 members [80-83]

1.2.2 Mitochondrial, or intrinsic, pathway

For a long time, the mitochondria as been considered solely as the organelle hosting the Krebs cycle responsible for ATP synthesis Adding to this role

of the powerhouse of the cell, the mitochondria has been shown to be a crucial component of apoptosis Extra- or intracellular stimuli such as hypoxia, reactive oxygen species,

Figure 4: Major Bcl-2 family members

Bcl-2-family proteins have a crucial role in the regulation of apoptosis through

gamma or UV radiations, growth factor withdrawal and cytotoxic molecules can induce a perturbation of the mitochondrial membrane ultimately leading to DNA fragmentation and apoptosis, dependently or independently of caspases activation [84, 85] MOMP is frequently associated with the dissipation of the inner mitochondrial membrane potential ("%m) [86, 87] and leads to the release into the cytosol of pro-apoptotic proteins normally localized in the inter-membrane mitochondrial space, such as cytochrome c, AIF [88] and Smac/DIABLO [89, 90] After release in the cytosol, cytochrome c interacts with APAF-1 and pro-caspase-9, hence forming, in the presence of ATP, the apoptosome Apoptosome formation then leads to the autocatalytic processing and activation of initiator caspase-9 [91-93] responsible for the activation of the executioner caspase cascade (incl caspase-3, -6 and -7) and the subsequent controlled demise of the cell

The mechanism by which MOMP occurs involves the formation of pores in the mitochondrial membrane The formation of such pores can be attributed to members of the Bcl-2 family [6, 94-96] and/or to the PTPC [97] PTPC is formed at the contact sites between the outer and inner mitochondria membranes and have been suggested to play an important role in cytochrome c release from the mitochondria The major components of the PTPC include the outer membrane protein (OMP), the adenine nucleotide transporter (ANT) on the inner mitochondria membrane as well as VDAC [98, 99] Various stimuli such as changes in the mitochondrial membrane

potential levels, reactive oxygen species [100], and changes in ions concentration (e.g

Ca2+ and Mg2+) [101, 102] are able to induce a change in the conformation of the PTPC, switching it from its usual closed conformation to an open conformation, thereby allowing a massive entry of water and solutes Interestingly, the Bcl-2 family can also regulate PTPC opening [103-107] The ensuing swelling of the mitochondria

leads to the physical rupture of the outer mitochondria membrane and the release of pro-apoptogenic proteins Ultimately, the mitochondrial functions essential for cell survival are lost

1.2.3 Death receptors, or extrinsic, pathway

1.2.3.1 Ligands and receptors of the TNF-! superfamily

Apoptosis can also be induced by extra-cellular signals (membrane-bound or soluble proteins) coming from neighboring or distant cells These signals are cytokines belonging to the TNF-! superfamily They are expressed as type II

transmembrane proteins (i.e extracellular C-terminal part and cytosolic N-terminal

part) Proteolysis of their C-terminal part by metalloproteases allows the release of the cytokines in their soluble forms These ligands have a 20-30% homology in a 150 amino acid domain, known as the TNF homology domain (THD), which is responsible for the binding to their respective receptors

TNF-! superfamily ligands interact with members of the TNF-receptor superfamily They form a group of about 29 proteins including TNF-R1 (p55), TNF-RII (p75), CD40, p75 NGFR, TRAMP, Fas, DR4 and DR5 These receptors are

expressed as type I transmembrane proteins (i.e cytosolic C-terminal part and

extracellular N-terminal part) with two exceptions: DcR-1, which is bound to the

disulfide bounds between cysteines of CRDs of the extracellular domain of a receptor promotes the stable formation of a functional trimeric or multimeric TNF superfamily receptor The CRDs are also responsible for the binding of the receptor to its cognate ligand [110]

A small group of receptors, including Fas (CD95/APO-1), TNF-R1, DR4 and DR5, is able to induce apoptosis These receptors, known as Death Receptors, have in common the presence of a conserved intracellular region called Death Domain (DD), which is a proteic sequence of about 80 amino acids allowing the transduction of the apoptotic signal initiated by the binding of the ligand to the receptor [110-112]

1.2.3.2 Signalization pathway

Binding of specific ligands to death receptors is the first step of the extrinsic apoptotic pathway Transmission of extracellular death signal is triggered upon the ligation of trimerized death ligands (FasL, TNF or TRAIL) to death receptors (Fas, TNF-R1 or DR4 and DR5) [113, 114] which leads to a conformational change in their death domains that represents, along with the subsequent trimerization, oligomerization and clustering of the receptors, the functional activation of the receptors However, Fas and TNF-R1 have been reported to be present at the plasma membrane in a trimerized form prior to the binding of their ligand This is made possible by the presence of a particular domain in their N-terminal part, Pre-Ligand Assembly Domain (PLAD) This implies that ligand binding does not result in trimerization of these receptors but, instead, could induce a change in the receptor conformation that would allow the interaction with adaptator proteins Activation of the death receptors allows for the recruitment of FADD (Fas-Associated protein with

Death Domain) via homophilic interaction between the respective death domains [115] Alternatively, ligation of TNF-! to TNF-R1 leads to the recruitment of the TNF-R1 Associated Death Domain protein (TRADD) [116] TRADD can then recruit FADD Acting as an adaptor protein, FADD also allows for the recruitment of pro-caspases-8/-10 via interaction of their respective death effector domains [117] The resulting multi-protein complex is referred to as the DISC [118] and results in the autocatalytic activation of the initiator caspases [119] in accordance with the induced proximity model Activated caspase-8/-10 in turn targets the effector caspase-3 for proteolytic cleavage, which, once activated, cleaves other caspases as well as numerous regulatory and structural proteins [120, 121] resulting in the appearance of the hallmarks of apoptosis Depending on the type of cells, some might require the involvement of mitochondria to amplify the death signal for efficient apoptosis [122] Composition of the DISC is, however, not definite and varies between death receptors and/or cell type For example TRADD is able to bind to a range of intermediate proteins allowing for diverse signaling with distinctive biological outcomes For example, TRADD can recruit TRAF-2 (TNF-# receptor associated factor-2) [123] and the serine-threonine kinase RIP (Receptor interacting protein) instead of FADD, and initiate the activation of NF-$B (Nuclear Factor $B) and JNK (c-Jun N-terminal Kinase) signaling pathways [124]

1.2.3.3 Activation of the mitochondrial pathway by the death receptors