The role of autophagy in apoptotic response of lung cancer cells under starvation. Master Thesis. For the attainment of the academic degree

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The role of autophagy in apoptotic response of lung cancer cells under starvation

Master Thesis

For the attainment of the academic degree

Master of Science

From the University of Applied Sciences FH Campus Wien

Submitted by:

Georg Baumgartner

Personal identity code:

1410544009

Supervisor:

Vitaliy Kaminskyy, PhD Karolinska Institutet

Institute of Environmental Medicine | Division of Toxicology Nobels väg 13

171 77 Stockholm Sweden

Submitted on:

22.12.2016

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Abstract English

KEYWORDS: autophagy, apoptosis, caspase-8, starvation, NSCLC

BACKGROUND: The ‘self-eating’ mechanism macroautophagy (hereafter simply ‘autophagy’) is an essential and highly conserved process involved in maintenance of cellular homeostasis and providing adaption to various intra- and extracellular stresses. In contrast, apoptosis constitutes the intracellular ‘suicide program’ which allows a controlled destruction of a cell. Both pathways have been shown to interact with each other and cytoprotective functions of autophagy have been demonstrated to limit efficacy of several cancer therapeutics targeting the apoptotic pathway.

OBJECTIVES: Several studies have investigated the crosstalk between the two pathways but it is still not fully understood how apoptosis is activated following nutrient depletion. In order to explore the role of autophagy in lung cancer cells following amino acid and growth factors withdrawal and the effects on initiation of apoptotic cell death, the present study was performed using the non-small cell lung carcinoma (NSCLC) cell line U1810.

RESULTS: Autophagy-inhibition by targeted deletion of ATG13 resulted in increased levels of apoptotic cell death following nutrient withdrawal, which was particularly dependent on caspase-8. It was demonstrated that starvation caused inhibition of protein translation and led to a significant decrease of cellular FLICE-inhibitory protein (cFLIP) levels. Furthermore, knockdown of cFLIPs revealed its important function in caspase-8 inhibition. Moreover, it was shown that mitochondrial functions play a role in the induction of apoptotic response, since processing of caspase-8 could be blocked by inhibition of mitochondrial respiration but not glycolysis. Similar to starvation, UVC radiation was demonstrated to cause caspase-8 dependent apoptosis in ATG7 knockout cells.

Additionally, the small redox protein thioredoxin was shown to be involved in the apoptotic response and caspase-8 activation following UVC radiation.

CONCLUSION: The presented data indicate that amino acid and growth factor withdrawal as well as UVC radiation can induce activation of caspase-8, which is facilitated by depletion of autophagy.

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Kurzfassung Deutsch

SCHLAGWÖRTER: Autophagie, Apoptose, Caspase-8, Nährstoffentzug, NSCLC

HINTERGRUND:Der als Makroautophagie bezeichnete Mechanismus (nachfolgend nur

‚Autophagie‘ genannt) ist ein essenzieller und hochkonservierter Prozess, der bei der Aufrechterhaltung der zellulären Homöostase sowie bei der Anpassung an verschiede- ne intra- und extrazelluläre Stressfaktoren beteiligt ist. Im Gegensatz dazu stellt die Apoptose das intrazelluläre Suizidprogramm dar und ermöglicht einen kontrollierten Zelltod. Beide Prozesse interagieren miteinander und beeinflussen sich maßgeblich: so können beispielsweise zytoprotektive Funktionen von Autophagie die Wirksamkeit von Krebstherapeutika, welche apoptotischen Zelltod in Tumorzellen auslösen, beträchtlich senken.

FRAGESTELLUNG: Obwohl beide Prozesse sowie deren gegenseitige Regulation cha- rakterisiert wurden, ist noch nicht bekannt, wie Apoptose als Reaktion auf Nährstoff- entzug aktiviert wird. Um die Rolle von Autophagie in Lungenkrebszellen nach dem Entzug von Aminosäuren und Wachstumsfaktoren sowie die Auswirkungen auf die Initiation des apoptotischen Zelltodes zu untersuchen, wurde die vorliegende Arbeit unter Verwendung der nichtkleinzelligen Bronchialkarzinom-Zelllinie U1810 durchge- führt.

ERGEBNISSE: Die Inhibition von Autophagie durch ein Knockout von ATG13 führte nach Reduzierung der Nährstoffe zu einer Erhöhung des Zelltods, welcher spezifisch ab- hängig von Caspase-8 war. Weiters wurde gezeigt, dass durch den Nährstoffentzug die Proteintranslation gehemmt wird. Dies führte zu einer signifikanten Reduktion des zellulären FLICE-inhibitory Proteins (cFLIP), welchem generell eine maßgebliche Rolle bei der Inhibierung von Caspase-8 zukommt. Da die Aktivierung von Caspase-8 nach Inhibition der oxidativen Phosphorylierung nicht jedoch der Glykolyse blockiert werden konnte, spielt auch die Funktionalität von Mitochondrien bei der Initiation des apoptotischen Prozesses eine entscheidende Rolle. Überdies aktivierte auch UVC-Strahlung Caspase-8 in ATG7-Knockout-Zellen und führte dadurch zu apoptotischem Zelltod.

Außerdem wurde gezeigt, dass das kleine Redoxprotein Thioredoxin bei der Aktivie- rung von Caspase-8 nach UVC-Strahlung involviert ist.

SCHLUSSFOLGERUNGEN:Die hier gezeigten Daten zeigen, dass ein Entzug von Amino- säuren und Wachstumsfaktoren sowie UVC-Strahlung die Aktivierung von Caspase-8 verursachen, was durch die Inhibition von Autophagie erleichtert wird.

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T ABLE OF CONTENTS

INTRODUCTION ... 9

1.1 Cancer ...9

1.1.1 Lung cancer ... 10

1.1.2 Programmed cell death pathways in cancer ... 10

1.2 Autophagy ... 10

1.2.1 The autophagic machinery and molecular mechanism ... 11

1.2.2 Activation and inhibition of autophagy ... 14

1.2.3 Autophagic cell death ... 15

1.2.4 The role of autophagy in cancer ... 16

1.3 Apoptosis ... 16

1.3.1 Phases of apoptotic signalling ... 18

1.3.2 The molecular process of mammalian apoptosis ... 18

1.3.3 Type-I and type-II cells ... 22

1.3.4 Proteins regulating apoptosis ... 22

1.3.5 The role of apoptosis in tumorigenesis ... 23

1.4 The crosstalk between autophagy and apoptosis ... 24

1.4.1 Inhibition of apoptosis by autophagy ... 24

1.4.2 Autophagy as a trigger of apoptosis ... 25

1.4.3 Inhibition and activation of autophagy by apoptosis ... 26

1.4.4 The role of the autophagy-apoptosis crosstalk in cancer ... 26

1.5 The present study ... 27

1.5.1 Goal of research ... 27

1.5.2 Methodology and experimental design ... 28

MATERIAL & METHODS ... 31

2.1 Cell culture ... 31

2.1.1 Construction of ATG7 and ATG13 knockout cells ... 31

2.1.2 Knockdown of caspase-8 by shRNA ... 32

2.1.3 Small interfering RNA (siRNA) silencing ... 32

2.1.4 Cell starvation and treatments ... 33

2.1.5 Measurement of protein biosynthesis ... 34

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2.2 Cell extract preparation ... 34

2.2.1 Whole cell lysate ... 34

2.2.2 Fractionation (nuclear and membrane/cytosolic fraction) ... 34

2.2.3 Measurement of protein concentration ... 35

2.3 Co-immunoprecipitation of caspase-8 ... 35

2.4 Immunoblotting ... 36

2.5 Immunocytochemistry... 37

2.6 Flow cytometry analysis ... 37

2.6.1 Annexin V / propidium iodide staining ... 37

2.6.2 TMRE staining ... 37

2.7 Determination of intracellular ATP-levels ... 38

2.8 Statistical analysis ... 38

RESULTS ... 39

3.1 Amino acid and serum depletion promotes caspase-8-dependent apoptosis in autophagy-deficient U1810 cells ... 39

3.1.1 Knockout of autophagy protein ATG13 inhibits autophagy during serum and amino acid deprivation ... 39

3.1.2 Cell death in response to starvation is mediated by apoptosis... 41

3.1.3 Apoptotic response following starvation is facilitated by depletion of autophagy and particularly dependent on caspase-8 ... 42

3.1.4 Active caspase-8 is present in nuclear fraction following starvation ... 44

3.2 Levels of protein translation are reduced during starvation and correlate with caspase-8 activation ... 45

3.2.1 Translation of caspase-8 inhibitory protein cFLIP is inhibited during starvation and degraded by the ubiquitin-proteasome system ... 46

3.2.2 Removal of cFLIP promotes caspase-8 activation in autophagy-deficient cells ... 47

3.3 The apoptotic response following starvation is energy dependent ... 48

3.3.1 Activation of caspase-8 can be suppressed by inhibition of mitochondrial respiration ... 49

3.3.2 ATP levels increase following nutrient depletion ... 50

3.3.3 Apoptotic cell death is converted to necrosis following ATP-depletion ... 52

3.4 Identification of caspase-8 activation platform under conditions of starvation ... 52

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3.5 Cell death response following UVC radiation is dependent on caspase-8 and

facilitated by autophagy-depletion ... 54

3.5.1 Autophagy-deficiency promotes cell death following UVC radiation ... 55

3.5.2 Cell death following UVC radiation is mainly mediated by caspase-8 and facilitated by autophagy inhibition ... 56

3.5.3 Apoptotic response following UVC radiation is facilitated by autophagy inhibition ... 58

3.5.4 The thioredoxin-system is possibly involved in caspase-8 activation following UVC radiation 58 DISCUSSION ... 60

4.1 The role of caspase-8 activation following starvation ... 60

4.2 The role of translation in caspase-8 activation ... 62

4.3 Inhibitory protein cFLIP and its effects on caspase-8 inhibition ... 63

4.4 Implication of energy metabolism in caspase-8 processing ... 65

4.5 Identification of caspase-8 activation platform ... 66

4.6 The connection between caspase-8 and thioredoxin ... 68

4.7 Conclusions ... 69

4.8 Open questions & future perspectives ... 70

4.9 Concluding remarks ... 72

REFERENCES ... 74

ACKNOWLEDGMENTS ... 82

STATUTORY DECLARATION ... 84

APPENDIX ... 85

8.1 List of figures ... 85

8.2 List of tables ... 86

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List of abbreviations

2DG 2-Deoxy-D-Glucose

AA Antimycin A

ADP Adenosine diphosphate

AIF Apoptosis inducing factor

Ambra1 Autophagy and Beclin 1 regulator 1

AMP Adenosine monophosphate

AMPK Adenosine monophosphate-activated protein kinase APAF1 Apoptotic protease activating factor 1

ATG Autophagy-related protein

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-cell lymphoma 2 BCL-XL BCL extra-large

BH3 BCL-2 homology 3

BID BH3 interacting-domain death agonist CARD Caspase activation and recruitment domains Caspase Cysteine-aspartic protease

CD95 Cluster of differentiation 95

cFLIP-L/S/T cellular FLICE/caspase-8 -like inhibitory protein long/short/total

cl. cleaved

dATP Deoxyadenosine triphosphate

DED Death effector domain

DEDD Death effector domain-containing protein

DEPTOR DEP domain-containing mTOR-interacting protein DIABLO/SMAC Second mitochondria-derived activator of caspases DISC Death-inducing signalling complex

EGFR Epidermal growth factor receptor

ELAVL1/HuR Embryonic lethal, abnormal vision)-like protein 1

EndoG Endonuclease G

ER Endoplasmic reticulum

FACS Fluorescence-activated cell sorting

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FADD Fas-associated protein with death domain FIP200 200 kDa FAK family kinase-interacting protein FLICE FADD -like interleukin-1β-converting enzyme GAPDH Glyceraldehyde 3-phosphate dehydrogenase HADHB Hydroxyacyl-CoA dehydrogenase subunit beta IAP Inhibitory apoptosis proteins

IP Immunoprecipitation

JNK JUN N-terminal protein kinase

LC3/ATG8 Microtubule-associated proteins 1A/1B light chain 3A LMP Lysosomal membrane permeabilization

MCL1 Myeloid leukaemia cell differentiation mLST8 Mammalian lethal with SEC13 protein 8 MMP (Δψm) Mitochondrial membrane potential

MOMP Mitochondrial outer membrane permeabilization mTOR Mechanistic target of rapamycin

mTORC1 mTOR complex 1

NAC N-acetyl-L-cystein

NO Nitric oxide

NSCLC Non-small cell lung cancer

OM Oligomycin A

p62/SQSTM1 62-kDa protein (Sequestosome-1) PARP Poly (ADP-ribose) polymerase

PE Phosphatidylethanolamine

PRAS40 40 kDa Pro-rich AKT substrate

PUMA p53 upregulated modulator of apoptosis Rab GTPase Ras-related in brain monomer G proteins RAPTOR Regulatory-associated protein of mTOR RIP Receptor-interacting protein kinase

ROS Reactive oxygen species

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis shRNA small hairpin RNA

siRNA small interfering RNA

SMAC/DIABLO Second mitochondria-derived activator of caspases SNARE Soluble N-ethylmaleimide-sensitive fusion attachment pro-

tein receptor

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TMRE Tetramethylrhodamine ethyl ester TNFR1 Tumor necrosis factor-alpha receptor 1

TNFSF10 Tumor necrosis factor (ligand) superfamily member 10 TNF-α Tumor necrosis factor-alpha

TRADD Tumour necrosis factor receptor type 1-associated death domain protein

TRAIL Tumour necrosis factor related apoptosis inducing ligand

UBL Ubiquitin-like

ULK1 Uncoordinated 51-like kinase-1 VMP1 Vacuole membrane protein 1 VPS34 Class III PI 3-kinase

XIAP X-linked inhibitor of apoptosis

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1

I NTRODUCTION

1.1 Cancer

Cancer is one of the leading causes of deaths worldwide, accounting for 8.2 million deaths and 14 million new cases in 2012 (Stewart and Wild, 2014). In general, the disease is characterized by the uncontrolled growth of cells with the potential to spread and invade other parts of the body, which might eventually result in death. Cancer can be caused by both external factors such as tobacco smoking, unhealthy diet or infec- tious organisms, as well as internal factors like inherited mutations, immune conditions or abnormal hormone production (Torre et al., 2015). However, the major contribution towards cancer risk is by around 90-95% due to environmental factors rather than inherited genetics accounting with approximately 5-10% (Anand et al., 2008).

In an attempt to reduce the complexity of cancer to a small number of characteristics which contribute to tumour growth and metastasis, Hanahan and Weinberg proposed the six hallmarks of cancer: self-sufficiency in growth signals, insensitivity to anti- growth signals, evading apoptosis, limitless replicative potential, sustained angiogene- sis, as well as tissue invasion and metastasis (Hanahan and Weinberg, 2000). In an updated version, the list was expanded by four new hallmarks: deregulated metabo- lism, evading the immune system, genome instability, and inflammation (Hanahan and Weinberg, 2011). All those hallmarks are thought to govern and contribute to the transformation of a normal cell to a malignant tumour cell.

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1.1.1 Lung cancer

Both in men and women, lung cancer is the leading cause of death by cancer worldwide despite the fact that most overall diagnosed cancers are breast and prostate cancer, respectively. An estimated number of around 1.8 million new lung cancer cases occurred in 2012, representing approximately 13% of total diagnosed cancers. Moreo- ver, 1.6 million people died of lung cancer of which the vast majority was caused by long-term smoking (Stewart and Wild, 2014; Torre et al., 2015).

The heterogeneous disease can be classified according to its histopathology and consists in general of two main subtypes: small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), the latter one accounting for around 85% of all lung cancers.

Although NSCLC subtypes often start from different types of lung cells, they are grouped together since the treatment and therapeutic outlook are in most cases quite similar (Novaes et al., 2008).

1.1.2 Programmed cell death pathways in cancer

In general, the term programmed cell death refers to at least three morphologically distinct processes named autophagy, apoptosis and programmed necrosis (necroptosis).

All three mechanisms are capable to decide jointly the fate of a (cancer) cell, but, however, they strongly differ in their contribution: apoptosis and necroptosis are only involved in the execution of cell death, whereas the autophagic process can provide both pro-survival and pro-death functions. Targeting the apoptotic pathway has been shown to be a promising therapeutic strategy to selectively kill tumour cells, including NSCLC.

However, cytoprotective mechanisms such as autophagy often limit the therapeutic success of such therapies. Therefore, deciphering those processes and their interplay might support the discovery of new anti-cancer targets and thus the development of new therapeutic strategies (Ouyang et al., 2012).

1.2 Autophagy

Autophagy, literally meaning ‘self-eating’, constitutes an intracellular degradation system delivering cytoplasmic components such as proteins or cellular organelles for degradation to lysosomes. In the past decades, lot of efforts were put into deciphering the molecular mechanisms behind autophagy since the process can be connected with a wide range of pathophysiological conditions in humans such as cancer, diabetes or neurodegenerative diseases (Feng et al., 2014). Overall, there are at least three dis-

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tinctive types of autophagy, which differ in their mechanism for selecting and guiding the cytoplasmic content to the lysosomes: macroautophagy, microautophagy, and chaperone-mediated autophagy (Klionsky, 2005; Massey et al., 2006).

The process of microautophagy mediates the direct sequestration and engulfment of small cytoplasmic portions by lysosomes, enabled by invaginations of the lysosomal membrane. However, the knowledge about the process in mammalian cells is still very limited (Mijaljica et al., 2011).

In contrast, chaperone-mediated autophagy constitutes a selective degradation system of individual proteins. The process consists of the recognition of specific proteins by heat shock cognate protein of 70kDa (Hsc70), unfolding of the selected protein before its degradation, and the following translocation into the lysosome (Kaushik and Cuervo, 2012; Massey et al., 2006).

Macroautophagy is thought to be the most prevalent form of autophagy and – com- pared with the two other classes – by far the best studied one. After initiation of the process, a double-membraned compartment is formed (termed phagophore or isolation membrane), sequestering cytoplasmic constituents. Following, the construct matures to the so-called autophagosome, which then fuses with a lysosome. Eventually, the cargo is degraded and resulting macromolecules are released back into the cytosol for further use (Feng et al., 2014).

Studies over the years have revealed the importance of macroautophagy for the degradation of not only macromolecules such as proteins or lipids, but also invasive mi- crobes (Gutierrez et al., 2004; Ogawa et al., 2005) or cellular organelles such as mitochondria. As an example, mitophagy – the specific elimination of mitochondria – has been identified in both yeast and mammals (Rodriguez-Enriquez et al., 2004; Youle and Narendra, 2011). For the sake of simplicity and due to the focus of this thesis, macroautophagy will be referred to simply as ‘autophagy’ hereafter.

1.2.1 The autophagic machinery and molecular mechanism

The whole autophagic process is tightly controlled by the activity of several regulatory components. To date, over 30 autophagy-related (ATG) proteins have been discovered and characterized, most exhibiting high homology between yeast and mammalian ge- nomes. In brief, the highly conserved steps during the autophagic process are the (1)

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mation and vesicle elongation, and, eventually, (3) the fusion with lysosomes and degradation of the cargo (Mizushima et al., 2011), as can be seen schematically in Figure 1.

Source: Marino, G., Niso-Santano, M., Baehrecke, E.H., and Kroemer, G. (2014). Self-consumption: the interplay of autophagy and apop- tosis. Nat Rev Mol Cell Biol 15, 81-94 [reprinted by permission from Nature Publishing Group, © 2014]

Figure 1: The molecular process of mammalian autophagy.

(a) The initiation and progression of the autophagic process needs a temporarily coordinated activation of several proteins and molecular components. Those include amongst others the ULK1 complex, which is functionally coupled to mammalian target of rapamycin complex 1 (mTORC1), a negative regulator complex of autophagy. Active mTORC1 hyper-phosphorylates ATG13 at multiple serine residues thus preventing ULK1 binding and complex formation. The inactivation of mTORC1 due to starvation or rapamycin treatment results in the partial dephosphorylation of the ULK1 complex components and phosphorylation of FIP200 leading to ULK1 complex assembly thus promoting isolation membrane (or phagophore) formation.

(b) The Beclin1-VPS34-complex, which can be inhibited by anti-apoptotic BCL-2 family members (such as BCL-2, MCL1 or BCL-XL), promotes the nucleation process of the isolation membrane.

(c) The transmembrane proteins VMP1 and ATG9 are probably responsible for lipid recruitment to the isolation membrane.

(d) Two ubiquitin-like (UBL) protein conjugation systems, namely LC3 UBL and ATG12 UBL, respective- ly, catalyse the conjugation of phosphatidylethanolamine (PE) to LC3 as well as of ATG12 to ATG5, leading to elongation of the autophagosome membrane.

(e) Following, several proteins out of the Rab GTPase and SNARE protein families promote the docking and fusion of lysosomes with autophagosomes.

(f) Finally, lysosomal acid hydrolases degrade the engulfed cargo and generate a macromolecule nutri- ent pool which is then recycled back to the cytosol

(Kamada et al., 2010; Marino et al., 2014; Yang and Klionsky, 2010).

AMPK, AMP-activated protein kinase; ATG, autophagy-related protein; BCL-2, B-cell lymphoma 2, BCL-XL, BCL extra-large; BH3, BCL-2 homology 3; DEPTOR, DEP domain-containing mTOR-interacting protein; LC3, microtubule-associated proteins 1 light chain 3B; MCL1, myeloid cell leukaemia sequence 1; mLST8, mammalian lethal with SEC13 protein; mTOR, mammalian target of rapamycin; PRAS40, 40 kDa Pro-rich AKT substrate; RAPTOR, regulatory-associated protein of mTOR; SNARE, soluble N-ethylmaleimide-sensitive-factor attachment receptor; ULK1, Unc-51-like kinase 1; VMP1, vacuole membrane protein 1; VPS34, Class III PI 3-kinase

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(1) Initiation of autophagosome formation and vesicle nucleation: The initiation and progression of the process needs a temporarily coordinated activation of several proteins and molecular components. The conserved checkpoint protein kinase mammalian target of rapamycin (mTOR), which is a major effector of cell growth and proliferation, regulates amongst others the biosynthesis of proteins and further links the cellular nutrient status to the activation of downstream events such as autophagy (Hay and Sonenberg, 2004).

The mTOR complex 1 comprising beside mTOR also mLST8, DEPTOR, RAPTOR and PRAS40, is functionally coupled to the ULK 1 complex. The ULK1 complex consists of Unc-51-like kinase 1 (ULK1), which is a serine-threonine protein kinase, FIP200, ATG13 and ATG101, and initiates autophagosome formation. Overall, the whole autophagic process is initiated by the dephosphorylation of ULK1 following stimuli such as nutrient depletion or rapamycin treatment which causes the dissociation of the ULK1 complex from mTORC1 (Chang and Neufeld, 2009; Hosokawa et al., 2009; Jung et al., 2009).

After the initiation, the Beclin-1-VPS34 complex consisting of Beclin-1, VPS34, VPS15 and ATG14L is recruited to the site of autophagosome formation and responsible for building up the initial autophagosome structure, called phagophore or isolation membrane. The phosphorylation of Ambra1 by ULK1 leads to the relocalization to the endoplasmic reticulum enabling membrane formation and positioning of the core complex (Di Bartolomeo et al., 2010). Furthermore, activated ULK1 phosphorylates Beclin-1 thus enhancing the activity of the Beclin-1-VPS34 complex (Russell et al., 2013). Con- secutively, the phosphatidylinositol 3-phosphate (PI(3)P) produced by VPS34 recruits the effector protein DFCP1, promoting the nucleation of the double-membrane vesicle (Axe et al., 2008).

(2) Vesicle elongation: The following vesicle elongation of the autophagosome is primarily mediated by the ATG proteins which can be separated into two ubiquitin-like (UBL) protein conjugation systems: the microtubule-associated protein light chain 3 (LC3/ ATG8) UBL and the ATG12 UBL. Both systems share the autophagy related protein ATG7 as E1-like ligase: The ATG12 UBL system executes the conjugation of ATG12 to ATG5, involving ATG7 and ATG10, an E2-like protein. Following, the ATG12-ATG5 conjugate binds ATG16 forming an E3-like enzyme complex that directs

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Heretofore, ATG4 generates LC3 from pro-LC3 by cleaving off its carboxyl terminus, whilst ATG7 and the E2-like enzyme ATG3 mediate the conjugation of LC3 to the membrane lipid phosphatidylethanolamine (PE) thus leading to its lipidation (Geng and Klionsky, 2008). This consecutive conjugation of LC3 to PE is essential for the formation and elongation of the autophagosome, and it is assumed that the amount of LC3 correlates with the final vesicle size (Xie et al., 2008).

(3) Autophagosome-lysosome fusion and degradation of the autophagic cargo: After completion of autophagosome formation, several sets of protein families such as Ras- related in brain monomer G proteins (Rab GTPases), SNARE (soluble N- ethylmaleimide-sensitive fusion attachment protein receptors)-like proteins and others mediate the docking and fusion process between the autophagosome and lysosomes, forming the so-called autophagolysosomes (or autolysosomes) (Ao et al., 2014;

Moreau et al., 2013). Finally, lysosomal acid hydrolases degrade the engulfed cargo and generate a macromolecule nutrient pool which is then recycled back to the cytosol.

The whole autophagic process can be terminated by reactivation of mTOR, for example by nutrients generated during the process, and requires the degradation of autoph- agolysosomal products. This feedback mechanism inhibits a too exaggerated activation of autophagy during starvation periods. Moreover, the restored mTOR activity generates proto-lysosomal tubules and vesicles extruding from autophagolysosomes, finally maturing into functional lysosomes thus restoring the full capacity of lysosomes in a cell (Yu et al., 2010).

1.2.2 Activation and inhibition of autophagy

As already mentioned, mTOR forms a complex with several other proteins building up mTORC1 and acts as a master regulator of autophagy by inhibition of the ULK1 complex. The mTOR complex senses both nutrients and growth factors and, exemplarily, the presence of amino acids has been shown to be essential for mTORC1 activity (Jewell et al., 2013).

1.2.2.1 Basal levels of autophagy

In general, autophagy is active on a basal level in mostly all cells and is more and more considered as a critical housekeeping pathway. The process serves not only as a nutrient supply during starvation periods, but is also responsible for the removal and recy- cling of aggregated and dysfunctional proteins or cellular organelles thus maintaining a

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proper cellular environment and protecting the cell from cytotoxic effects (Mizushima et al., 2008).

1.2.2.2 Activation of autophagy as a starvation response

Autophagy can be stimulated by various physiological stimuli, but the most studied one is nutrient deprivation. Autophagy constitutes a cellular key response upon nutrient withdrawal, for instance it has been shown that autophagy-deficient yeast mutants are not capable to survive nitrogen starvation (Tsukada and Ohsumi, 1993). Furthermore, the level of autophagy is immediately upregulated in several tissues of mice following birth due to interruption of the placental nutrient supply. Mice defective for autophagosome formation die within one day after delivery, indicating the importance of the autophagic degradation of cellular components for maintenance of energy and amino acid metabolism (Kuma et al., 2004). However, it is important to note that the induction of autophagy by nutrient starvation is strongly dependent on the tissue since some tissues show high levels of active autophagy even without nutrient deprivation (Mizushima et al., 2004).

1.2.2.3 Other modes of activation

mTOR is capable to integrate also other signals such as hypoxia, energy levels, growth factors and other stresses into an autophagic response thus maintaining a metabolic homeostasis. For example, hypoxia regulates mTORC1 by the activation of AMPK and following mTORC1 phosphorylation due to ADP and AMP accumulation. Beside the increase during hypoxia, the intracellular ATP:AMP ratio is in general an important indi- cator of cellular energy levels and is capable to activate AMPK. Low levels of oxygen further upregulate hypoxia-responsive genes that are capable of suppressing mTORC1 by several mechanisms (Laplante and Sabatini, 2012).

Beside the regulation by mTORC1, the protein kinase AKT and the epidermal growth factor receptor (EGFR) are capable of regulating and modulating autophagy by direct phosphorylation of Beclin-1 (Wang et al., 2012; Wei et al., 2013).

1.2.3 Autophagic cell death

Excessive autophagy has been observed in connection with various forms of cell death. In the beginning, the term ‘autophagic cell death’ described a cell death accom- panied by autophagy but since autophagy is turned on by cellular stresses and dying cells often show autophagic features, the idea of an own cell death modality has

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agosomes and the dependency of cell death on some autophagic proteins supports this idea. Nevertheless, it has been debated whether the cells die by autophagy itself or the process tries to prevent the cellular ruin and autophagic cellular features are thus just a ‘by-product’. Therefore, the term was redefined as a mode of cell death that is suppressed by the inhibition of autophagy. Nevertheless, this type of cell death remains poorly defined and is still controversially debated (Denton et al., 2012; Liu and Levine, 2015).

1.2.4 The role of autophagy in cancer

In general, the autophagic process has an important but very context-dependent role in many diseases, amongst others in cancer. Since autophagy is essential for maintaining a proper cellular homeostasis by both detoxification and providing adaption to various kinds of stresses, it also limits DNA damage and thus the initiation of tumorigenesis.

Contrarily, the process can assist and promote cancer cell survival and growth by providing adaption to environmental stresses such as nutrient deprivation or hypoxia, and promote tumour progression in this manner. Furthermore, the process can be ex- ploited to deal with the toxicity of certain anticancer drugs thus limiting their function and efficiency. With that said, it is clear that the autophagic process has an important albeit complex role in cancer development, progression and treatment. However, since the process is connected with many cancer promoting networks and pathways (such as p53, mTOR or RAS), and suppression of autophagy often sensitizes cancer cells to radiation or chemotherapeutics, the process represents an interesting target for the development of new clinical autophagy modulators to increase efficacy of existing (and new) cancer therapeutics (Liu and Ryan, 2012; Wu et al., 2012).

1.3 Apoptosis

The term apoptosis, which represents the main form of programmed cell death, has been first used in 1972 describing a morphologically distinct mode of cell death (Kerr et al., 1972). The apoptotic process constitutes a structured ‘suicide program’ of single cells which can either be triggered by exogenous or endogenous stimuli such as genotoxic chemicals, DNA damage and ultraviolet radiation, respectively. In contrast to other forms of cell death such as necrosis, the process is carried out actively by a genet- ically pre-determined program. The whole mechanism is strictly controlled and ensures the elimination and removal of relevant cells without harming surrounding tissue and invoking inflammation (Bohm and Schild, 2003).

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Source: Marino, G., Niso-Santano, M., Baehrecke, E.H., and Kroemer, G. (2014). Self-consumption: the interplay of autophagy and apop- tosis. Nat Rev Mol Cell Biol 15, 81-94 [reprinted by permission from Nature Publishing Group, © 2014]

Figure 2: Molecular mechanism behind the extrinsic and intrinsic pathways of apoptosis.

(a) The activation of death receptors such as FasR/CD95, TNF receptor 1 or TRAILR by binding of their corresponding ligands leads to recruitment and subsequent activation of caspase-8 by the Fas- associated protein with death domain (FADD) and tumour necrosis factor receptor type 1 (TNFR1)- associated death domain (TRADD) forming the so-called death-inducing signalling complex (DISC). The active form of caspase-8 can then forward signalling in two different ways: both direct proteolytic cleavage and activation of downstream executioner caspases (such as caspase-3, -6 or -7) and/or by co- activation of the intrinsic pathway.

(b) Both external stress such as radiation or cytokine deprivation as well as intrinsic signals such as ac- tivated initiator caspases such as caspase-8 or DNA damage can activate the intrinsic pathway, which is primarily marked by mitochondrial outer membrane permeabilization (MOMP). This step is mainly mediated by the activation of BH3-only proteins and as a result BAX and BAK oligomerize, cause the disruption of the outer mitochondrial membrane and leads to release of pro-apoptotic regulators such as cytochrome c, SMAC/DIABLO or apoptosis inducing factor (AIF). This step triggers the thereafter ATP- dependent formation of the apoptosome consisting of apoptotic protease-activating factor 1 (APAF1), cytochrome c and recruited caspase-9. After its activation, the proteolytical activity of caspase-9 leads to processing of effector caspase-3 and execution of apoptosis.

(Bratton and Salvesen, 2010; Marino et al., 2014; Riedl and Shi, 2004; Scaffidi et al., 1998)

AIF, apoptosis inducing factor; APAF1, Apoptotic protease activating factor 1; BAD, BCL-2-antagonist of cell death; BCL-2, B-cell lymphoma 2; BID, BH3 interacting-domain death agonist; CD95, cluster of differentiation 95; DISC, death inducing signalling complex; EndoG, Endonuclease G; FADD, Fas-associated protein with death domain; JNK, c-Jun N-terminal kinase; LMP, lysosomal membrane permeabilization; MOMP, mitochondrial outer membrane permiabilization; PUMA, p53 upregulated modulator of apoptosis; RIP, receptor-interacting protein kinase; ROS, reactive oxygen species; TNF(R1), tumor necrosis factor alpha (receptor 1); TRADD, tumor necrosis factor receptor type 1-associated death domain protein

The morphological characteristics of the apoptotic process are cell shrinkage, chroma- tin condensation, nuclear fragmentation as well as plasma membrane blebbing. Follow-

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apoptotic bodies, which are then engulfed by neighbouring cells or macrophages. This controlled degradation of cells avoids not only damage to neighbouring cells but further prevents an immune response.

However, the usual classification of cell death modalities by morphology is increasingly replaced by the characterization of underlying biochemical pathways (Marino et al., 2014).

1.3.1 Phases of apoptotic signalling

The apoptotic process can be structured into three phases, namely initiation, effector and degradation phase. The initiation of the apoptotic signalling cascade results from stimuli such as DNA damage, inflammation or oncogenic stimulation causing the increased production of pro-apoptotic factors and can be performed by two different pathways: the extrinsic and intrinsic pathway. As the names imply, the intrinsic pathway is triggered by intracellular signals while the extrinsic pathway is elicited by exogenous stimuli. In the thereafter following effector or execution phase, mitochondrial membranes are disrupted (mitochondrial outer membrane permeabilization; MOMP) leading to the release of mitochondrial proteins, cytochrome c and other proteases into the cytosol. The released proteins serve mainly as signalling and degradation molecules while the release of cytochrome c has major implications in the energy status of the cell. The following morphological changes mark the outcome of the effector phase and, eventually, the apoptotic process peaks in the condensation (pyknosis) and fragmentation (karyorrhexis) of nucleosomal DNA and the formation of apoptotic bodies (Kerr et al., 1972).

1.3.2 The molecular process of mammalian apoptosis

Depending on the stimulus, the initial steps of apoptosis can be executed by either the intrinsic or extrinsic pathway as summarized in Figure 2. Although they differ with regard to the initiation, both pathways converge in the execution phase leading to the activation of effector caspases (mainly caspase-3, -6 and -7).

1.3.2.1 The role of caspases in the apoptotic process

Caspases (for cysteinyl aspartate-specific proteases) are an evolutionary conserved family of enzymes that has been shown to be the main regulators and executioners of apoptosis (Thornberry and Lazebnik, 1998). In general, all of these enzymes contain a critical cysteine in the active site that attacks and cleaves their target protein following a C-terminal aspartic acid residue (Alnemri et al., 1996). By now, 14 different caspases

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have been identified and in general all of them share a number of common structural features as can be seen in Figure 3. They are all expressed as inactive zymogens that can be categorized into three major groups according to their pro-domain: pro- inflammatory caspases (caspase-1, -4, -5, -11, -12, -13 and -14), apoptotic initiator (caspase-2, -8, -9 and -10) and effector/executioner caspases (caspase-3, -6, -7) (Earnshaw et al., 1999; Nicholson, 1999; Olsson and Zhivotovsky, 2011; Shi, 2002).

Source: Riedl, S.J., and Shi, Y. (2004). Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol 5, 897-907.

Figure 3: Structure of mammalian apoptotic effector and initiator caspases.

The structure of effector caspases (red) and initiator caspases (purple) are shown, including their catalytic cysteine residue as a red line. In general, caspases are expressed as inactive, immature pro-forms (or zymogens) with three main domains: a pro-domain, a large (~p20) and small (~p10) catalytic subunit. The pro-domains (such as DED or CARD) allow the recruitment and dimerization of the procaspase, which can be assisted by adaptor proteins such as FADD. The activation and processing of caspases involves a two-step proteolytic cleavage: the first activating cleavage between the large (p20) and small subunit (p10) is highlighted by black arrows, whereas grey arrows mark the second cleavage site. This proteolytic cleavage of the zymogen at specific aspartic acid residues results in the formation of a caspase tetramer, containing two large and two small subunits (not shown).

(Riedl and Shi, 2004; Sprick et al., 2002; Zhao et al., 2010)

DED, Death effector domain; CARD, Caspase activation and recruitment domain; FADD, Fas-Associated protein with death domain

Caspases are mainly present in the cytosol, but might be also found in other cellular compartments such as nucleus, Golgi apparatus or the endoplasmic reticulum (Paroni et al., 2002; Qin et al., 2001; Samali et al., 1998; Zhivotovsky et al., 1999). Although sharing common properties, their mode of activation is different: the activation of initia-

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(auto-) proteolytic cleavage between the domains, and, finally, the formation of a heterodimer between the small and the large subunit. In contrast, effector caspases lack the protein-protein interaction motifs and are already present as inactive homodi- mers within a cell. After initiator caspase-mediated cleavage, they are responsible for the cellular destruction process by cleavage of specific substrates causing amongst others the inactivation of apoptotic inhibitory proteins, disassembly of cellular structures and loss/gain of function of proteins due to site-specific cleavage (Au et al., 1999;

Kothakota et al., 1997; Rhéaume et al., 1997).

1.3.2.2 The death-receptor mediated extrinsic pathway of apoptosis

The extrinsic pathway is induced by binding of exogenous death-inducing signalling molecules such as tumour necrosis factor α (TNF-α), Fas/CD95 ligand or TNF ligand superfamily member 10 (TNFSF10) to their cognate receptors including TNF-α receptor 1, Fas/CD95 or tumour necrosis factor related apoptosis inducing ligand (TRAIL) receptors 1 and 2 (TRAILR1/2), respectively (Schutze et al., 2008; Wajant, 2002). This binding leads to the formation of the so-called death-inducing signalling complex (DISC) at the intracellular domain of the receptor, which consists of adaptor proteins such as tumour necrosis factor receptor type 1-associated death domain protein (TRADD) and Fas-associated death domain (FADD), respectively, and serves as an activation platform for the initiator caspases (caspase-8 and caspase-10). Following their activation by autocatalytic cleavage, the caspases dissociate from the receptor, move to the cytoplasm and activate effector caspases (mainly caspase-3, -6 and -7), which then start to cleave each other thus amplifying the proteolytic cascade (Rathmell and Thompson, 1999). Beside the initiation of the apoptotic response, activated caspase-8 can further amplify the apoptotic signal and trigger the activation of the intrinsic pathway which is mediated by the caspase-8 dependent cleavage of B-cell lymphoma 2 (BCL-2)-homology domain 3 (BH3)-domain-containing protein BID (BH3-interacting- domain death agonist) (Fischer et al., 2003).

In general, caspase-8 is predominantly present as a monomeric cytoplasmic protein which dimerizes following its recruitment to the DISC where it is cleaved auto- catalytically. Beside its recruitment, post-translational modifications such as polyubiquitination at the DISC have been shown to be involved in its processing and activation (Jin et al., 2009). Although caspase-10 shows a very homologous structure to caspase-8, it cannot functionally substitute caspase-8 completely. Its recruitment to the DISC happens similarly in a FADD-dependent manner but its function at the DISC

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is still not completely understood (Sprick et al., 2002). Nevertheless, overexpression of several caspase-10 isoforms revealed that resistance to TRAIL treatment caused by absence of caspase-8 could be reverted by caspase-10 overexpression underlining their similarity in respect to their function (Mühlethaler-Mottet et al., 2011).

1.3.2.3 The mitochondria-mediated intrinsic pathway of apoptosis

The intrinsic mitochondrial pathway is triggered by diverse cellular stresses such as DNA damage, irradiation or cytokine deprivation, leading to the activation of BH3-only proteins such as BID, BAD (BCL-2 antagonist of cell death), PUMA (p53 upregulated modulator of apoptosis) or others as shown in Figure 2. These activated proteins neu- tralize anti-apoptotic BCL-2 proteins (BCL-2, BCL-extra-large (BCL-XL) or myeloid leukaemia cell differentiation 1 (MCL1)), liberating BCL-2 associated X protein (BAX) and BH antagonist or killer (BAK) (Youle and Strasser, 2008). Those two proteins lead to the subsequent disruption and permeabilization of the mitochondrial outer membrane (MOMP) causing the release of second mitochondrial activator caspases (SMAC/DIABLO) as well as cytochrome c into the cytosol, which marks a pivotal step in the apoptotic process and is often considered as ‘point-of-no-return’ (Du et al., 2000;

Liu et al., 1996).

Following its release, cytochrome c binds apoptotic protease activating factor 1 (APAF1), which promotes in the presence of dATP apoptosome complex formation.

This complex serves as heptameric activation platform for caspase-9, leading to the recruitment of procaspase-9 by its caspase activation and recruitment domain (CARD).

Similar to caspase-8 and -10, the cleaved activated form of caspase-9 propagates the apoptotic signal by activating effector procaspases-3, -6 and -7 eventually leading to apoptotic cell death (Bratton and Salvesen, 2010).

1.3.2.4 Caspase-independent cell death

Beside activation of caspases, apoptosis can also proceed by a caspase-independent pathway, which is mainly mediated by mitochondrial proteins such as AIF or endonuclease G (EndoG) as a result of MOMP. This caspase-independent cell death might also be a result from stimuli causing permeabilization of lysosomal membranes (LMP).

However, this caspase-independent mode of apoptosis is still poorly defined and understood (Kroemer and Martin, 2005).

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1.3.3 Type-I and type-II cells

Since the intrinsic and extrinsic apoptotic pathways ‘share’ some of their proteins, one can easily imagine that there is also a crosstalk between the two pathways. In fact, in so-called ‘type-I cells’ activation of caspase-8 is mediated by death receptors and sufficient to induce processing of effector caspases such as caspase-3, -6 or -7 which happens independently of mitochondria. In contrast, ‘type-II cells’ do strongly rely on the mitochondrial pathway since activation of caspase-8 alone is not sufficient to propagate the apoptotic stimulus. In general, the amount of active caspase-8 generated at the DISC seems to determine whether apoptosis is dependent on participation of mitochondria or not (Scaffidi et al., 1998). In type-II cells, active caspase-8 cleaves the pro- apoptotic BCL-2 family member BID which’s truncated form tBID localizes to mitochondria and forwards the apoptotic signal to the mitochondrial membrane. This leads to loss of mitochondrial membrane potential, release of cytochromce c and other mito- chondrial proteins and, moreover, propagates the activation of caspase-9 thus leading to activation of effector caspases (Li et al., 1998).

1.3.4 Proteins regulating apoptosis

1.3.4.1 BCL-2 family proteins

The B-cell lymphoma 2 (BCL-2) family is an evolutionary conserved protein family regulating apoptosis. It consists of the protein BCL-2 and its homologues and proteins of that family can exhibit either pro-apoptotic (including e.g. BAX and BAD) or anti- apoptotic (such as BCL-2 or BCL-XL) functions. In general, those proteins are main regulators of mitochondrial events taking place following activation of the intrinsic pathway such as release of cytochrome c (Kluck et al., 1997). So far, more than 30 different proteins related to this family have been discovered, most of them sharing BCL-2 homology domains (BH1-4) and a transmembrane domain. Overall, the family can be categorized into three subgroups according to their function in apoptosis: two pro-apoptotic subfamilies (BH3-only proteins such as BID and PUMA and death effec- tors like BAX and BAK) and one anti-apoptotic subfamily (including BCL-2 and BCL- XL) (Czabotar et al., 2014).

1.3.4.2 Cellular FLICE-like inhibitory protein (cFLIP)

In addition to DISC adaptor proteins and initiator caspases-8 and -10, also caspase inhibitory proteins such as cellular FLICE-like inhibitory protein (cFLIP) are recruited to the DISC following initiation of receptor-mediated apoptosis. cFLIP, mainly consisting of the two splice variants cFLIP-long (cFLIP-L) and cFLIP-short (cFLIP-S), is one of the

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main regulators of caspase-8. In regard to its structure it shares similar features with caspase-8 such as a death effector domain (DED) but it lacks the catalytic cysteine residue (Safa, 2013). Beside its inhibitory function during receptor mediated apoptosis, it is further involved in inhibition of the death receptor independent apoptotic platform named ripoptosome (consisting of the core components RIP1, FADD and caspase-8) which is mainly activated following genotoxic stress (Tenev et al., 2011). However, the role of cFLIP in cell death is still not completely understood.

1.3.5 The role of apoptosis in tumorigenesis

The development of cancer is a result of several genetic changes which promote the transformation of a normal healthy cell to a transformed malignant cell. Evading programmed cell death, especially apoptosis, has been proposed to be one of the key characteristics and hallmarks in tumour development and progression (Hanahan and Weinberg, 2011). Since the apoptotic process is based on a pre-defined genetic program like any other cellular developmental or metabolic process, its functionality can be disrupted for example by genetic mutations. The following deregulation of the apoptotic pathway represented by reduction or resistance to apoptotic stimuli has been shown to be implicated in several pathological conditions, including cancer, autoimmune diseases or neurodegenerative disorders (Eguchi, 2001; Mattson, 2000; Thompson, 1995). In general, a disturbance of the apoptotic process can be caused by several factors such as a up/downregulation or mutations in pro- and anti-apoptotic proteins (such as BCL-2 family proteins, p53 or IAPs), a reduction in caspase activity (e.g. due to downregulation or lacking expression) or an impaired death receptor signalling (caused by impair- ment of receptor function or expression) (Wong, 2011).

Exemplarily, p53 – also known as the ‘guardian of the genome’ – was the first tumour suppressor gene which was linked to apoptosis. Mutations in p53 occur in most of hu- man tumours and although p53 was first discovered as cell-cycle arrest protein, it has been further shown that it is also capable to induce cell death (Yonish-Rouach et al., 1991). The promotion of apoptosis by p53 happens by both transcription-dependent and -independent mechanisms to ensure an efficiently performed cell death program which can be activated following hypoxia, DNA damage or other cellular stresses.

Therefore, disruptions of its function can lead to cellular immortalization and inappro- priate cell survival which is caused amongst others by inhibition of apoptosis and thus promotes cancer development (Vousden and Lu, 2002).

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1.4 The crosstalk between autophagy and apoptosis

As already mentioned, autophagy is in general a cytoprotective mechanism removing protein aggregates and damaged cellular organelles thus providing a survival ad- vantage to cells undergoing stress. Nevertheless, it has also been shown that autophagy can be directly linked to death processes. Regardless the controversial discussion of autophagic cell death, autophagy and apoptosis have been reported to both cooper- ate and counteract directly and indirectly, and their relationship seems to be strongly context-dependent (Amaravadi and Thompson, 2007).

1.4.1 Inhibition of apoptosis by autophagy

Autophagy can specifically inhibit apoptosis by reducing levels of pro-apoptotic cytosolic proteins. Ubiquitinated proteins are recognized by a series of autophagy receptors such as sequestome-1 (SQSTM1/p62) which is followed by their autophagic degradation. Exemplarily, it was shown that active caspase-8 can be degraded by TRAIL- mediated autophagy in BAX-deficient colon carcinoma cells (Hou et al., 2010). In a similar way, knockout of ATG7 increased caspase-8 activity in hepatocytes following TNF-induced apoptosis most probably due to failed removal of caspase-8 (Amir et al., 2013). Moreover, the autophagic protein SQSTM1 is degraded by autophagy which – if overexpressed – stimulates ROS production and leads to cell death (Mathew et al., 2009).

Another mechanisms by which autophagy inhibits the activation of the apoptotic process is by removal of whole mitochondria (mitophagy). Since survival and death signals converge at least to some extent at mitochondria, mitophagy might also participate in the decision whether intrinsic apoptosis is initiated or not (Youle and Narendra, 2011).

Several factors such as lipids, proteins and metabolites, affect the integrity and thus functionality of mitochondrial membranes. The disruption of the mitochondrial outer membrane (MOMP) causes the release of several pro-apoptotic signal molecules (such as apoptosis-inducing factor (AIF), cytochrome c or SMAC/DIABLO) and a drop of the mitochondrial membrane potential (MMP; Δψm) – which results in an energetic catas- trophe and marks a ‘point-of-no-return’ in the progression of apoptosis. Especially damaged mitochondria are prone to activate apoptosis and, therefore, their removal by mitophagy can increase the threshold of apoptosis induction (Galluzzi et al., 2012).

It was further shown that autophagy is involved in cell-cycle arrest regulation. The cy- clin-dependent kinase (CDK) inhibitor p27/Kip1 has been shown to link the induction of

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autophagy with inhibition of apoptosis following nutrient withdrawal. Resulting decline of ATP:AMP ratio stimulates the activity of AMP-dependent kinase (AMPK) due to accumulation of AMP. The AMPK-dependent phosphorylation of p27/Kip1 leads to its stabilization and activates autophagy to cope with the metabolic stress (Liang et al., 2007).

Altogether, these findings provide an important link for autophagy-dependent inhibition of apoptosis and show the specific role of cytoprotective autophagy in a cell.

1.4.2 Autophagy as a trigger of apoptosis

In contrast to the previously presented role of autophagy in inhibition of apoptosis, several examples have been reported in which autophagy activates or at least facilitates activation of apoptosis. Previously published data suggests that autophagosome formation rather than the complete autophagic process provides a platform for caspase-8 activation (Young et al., 2012). However, activation of caspase-8 does normally not occur when autophagy is induced. Knockout of ATG7, a protein acting early in autophagy, was shown to facilitate caspase-8 activation following stimulation by TNF in hepatocytes in vivo (Amir et al., 2013). However, it is still elusive which proteins determine whether autophagosomes stimulate caspase-8 activation.

Another way how autophagy might stimulate apoptosis is by depletion of endogenous apoptotic inhibitors. In organisms such as Drosophila melanogaster the interplay be- tween inhibitory apoptosis proteins (IAP) and caspases regulate the apoptotic process and specific degradation of IAPs result in activation of the apoptotic process (Nezis et al., 2010).

Moreover, ATG proteins might also contribute to cell death signalling independently of their participation in the autophagic process. Exemplarily, ATG12 is considered to participate in caspase activation through the mitochondrial pathway since its depletion reduces activation of caspases following various apoptotic stimuli. ATG12 has been shown to interact with BCL-2 family members thus representing an important point of contact between the autophagy and apoptosis (Rubinstein et al., 2011). Furthermore, ATG7 but not ATG5 facilitates activation of apoptosis following lysosomal photo- damage probably due to lysosomal membrane permeabilization which similarly to MOMP is capable to trigger apoptosis (Kessel et al., 2012).

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1.4.3 Inhibition and activation of autophagy by apoptosis

The induction of autophagy shows a cell’s capability and demand to adapt to stress following stress stimuli such as chemotherapeutics, ionizing radiation or lack of essential nutrients. In these cases, autophagy is normally activated to a certain extent to cope with the stress before the stress exceeds a certain limit and cell death pathways are activated (Kroemer et al., 2010). If apoptosis is suppressed, induction of autophagy is often intensified for example by removal of pro-apoptotic proteins such as BAX or BAK (Shimizu et al., 2004).

When the severity or duration of stress reaches a certain limit, mechanisms allowing adaption to the stress such as autophagy are overpowered and cell death programmes are activated. For example, caspases are capable to digest essential autophagic proteins leading to inactivation of the autophagic programme probably to interrupt and stop its cytoprotective function and to precipitate the ruin of the cell. Targets include amongst others ATG3 and Beclin-1, which causes a loss of their autophagy-stimulatory function and further enhances the apoptotic process by promoting release of mitochondrial pro-apoptotic factors (Oral et al., 2012; Wirawan et al., 2010). Moreover, frag- ments of autophagic proteins resulting from caspase cleavage are assumed to acquire pro-apoptotic functions. For instance, cleaved Beclin-1 localizes to mitochondria and permeabilizes the membrane which causes the release of cytochrome c and other pro- apoptotic factors (Wirawan et al., 2010).

Notably, also anti-apoptotic proteins were shown to influence autophagic process. The caspase-8 inhibitory protein cFLIP has been shown to suppress autophagy by preventing processing of LC3 by an inhibitory action on ATG3. Thus it does not only provide anti-apoptotic functions but also influences autophagy by its anti-autophagic interfer- ence (Lee et al., 2009).

1.4.4 The role of the autophagy-apoptosis crosstalk in cancer

Cell death in vivo involves a very complex and context-dependent interaction between all programmed cell death pathways, which occur either independently of or simultane- ously with each other. Many processes promote tumour development but might also sensitise cancer cells to death stimuli such as chemotherapeutic drugs. One outcome of tumour development is that cells often gain abilities to evade and escape cell death mechanisms by inactivation of the corresponding pathways or upregulation of protec-

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tive pathways. This situation is further complicated since for example autophagy can exhibit both pro- and anti-apoptotic functions.

Exemplarily, TRAIL is a cytokine and promising candidate for treatments of different cancer types. Following binding to TRAIL receptors (TRAILR) it causes caspase-8 dependent apoptosis primarily in tumour cells. Research in the past year has revealed that autophagy is capable to affect and delay death-receptor induced MOMP and thus apoptosis by regulation of PUMA levels. In this manner, autophagy inhibition sensitizes cancer cells to TRAIL treatment by upregulation of PUMA (Thorburn et al., 2014).

Moreover, it was shown that inhibition autophagy enhances heat-induced apoptosis through ER stress pathways and further contributes to chemo-resistance in hypoxic conditions (Lee et al., 2015; Xie et al., 2016).

Therefore, targeting autophagy might be a promising strategy to bypass and circum- vent resistance and enhance efficacy of cancer therapies for cancer patients.

1.5 The present study

Autophagy is deregulated in various diseases. Furthermore, accumulating evidence suggests that in tumours autophagy is activated in areas most distal from blood supply thus providing tumour cells with energy and promoting their survival. Many drugs used in cancer therapy kill tumour cells via the apoptotic pathway and upregulation of autophagy can cause and promote resistance to those apoptotic stimuli. Autophagy seems to play either a direct or an indirect role in inhibition of apoptosis especially following nutrient depletion although the precise mechanism of activation is still not understood.

1.5.1 Goal of research

The main aim of this thesis was to investigate the effects of autophagy on cellular apoptotic response as well as to reveal the general crosstalk between these two pathways. The specific goal was to elucidate the mechanism of induction of the apoptotic process following autophagy-depletion under conditions of starvation (amino acid and serum depletion). It was shown before that glucose deprivation activates caspase-8 (Caro-Maldonado et al., 2010) hence caspase-8 was thought to be responsible for initiation of apoptotic response following depletion of other nutrients as well. Therefore, a particular focus was set on caspase-8 and the identification of pathways and proteins in

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Moreover, it was tried to further define apoptotic response following other types of stresses.

The main goals of this thesis were:

1. To explore activation of apoptotic response following different stress conditions such as nutrient deprivation or UVC radiation in autophagy-deficient cells 2. To characterize the role of caspase-8, its localization and mode of activation af-

ter induction of apoptosis, particularly following amino acid and growth factor depletion

3. To reveal the possible involvement and effects of altered cellular processes (such as energy metabolism, protein synthesis and degradation) on apoptosis in cells with inhibited autophagy

4. To determine and characterize proteins involved in caspase-8 activation or inhibition

1.5.2 Methodology and experimental design

This part provides a short introduction, overview and justification of the methods used in this thesis. The detailed descriptions and protocols can be found in material and methods.

Induction and evaluation of autophagy

It was shown previously that the depletion of amino acids and growth factors is an appropriate way to induce autophagy in cells (Klionsky et al., 2012). Therefore, starvation conditions using serum and amino acid free HBSS medium containing only glucose were used to induce the autophagic process for different amounts of time, and cellular responses in respect to cell death were assessed by appropriate methods as described below. In general, inhibition of autophagy was achieved by creation of stable knockout cell lines lacking essential autophagic proteins by CRISPR/Cas9 system. The efficiency was assessed by detection of autophagy-specific proteins such as ATG7 or ATG13, accumulation of SQSTM1/p62 due to lacking degradation as well as co-localization of autophagosomes with lysosomes.

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Assessment of apoptotic cell death

Characteristic features of apoptosis are morphological changes such as cell membrane blebbing, cell shrinkage, and nuclear condensation, and were in general assessed by microscopy. Another characteristic feature of the apoptotic process is the translocation of the phospholipid-membrane component phosphatidylserine from the inner to the outer side of the plasma membrane. This exposure can be detected with the Ca²⁺- dependent phospholipid-binging protein Annexin V, which exhibits a high affinity for phosphatidylserine. A commercial kit was used with recombinant Annexin V (conjugat- ed to fluorescein) and red fluorescent propidium iodide, which allows distinguishing living and apoptotic cells from necrotic cells due to ruptured cell membrane and nucleic acid binding. Beside the exposure of phosphatidylserine, cells undergoing apoptosis exhibit a drop in mitochondrial membrane potential (MMP, ΔΨm) due to MOMP. The cationic fluorochrome dye tetramethylrhodamine ethyl ester (TMRE) accumulates in mitochondria exhibiting normal function. The drop in membrane potential resulting from apoptotic response causes a release of TMRE which results in a decrease in fluorescence intensity and a shift in the fluorescence emission spectrum. For both methods, cells were stained with corresponding dyes and changes were assessed and analysed by flow cytometry.

Determination of caspase activation and progression of apoptotic response

Caspases require undergoing proteolytic cleavage in order to be activated. This activation process of caspases can be assessed by using specific antibodies in Western blot analysis. In this study, antibodies for detection of full-length procaspases and their cleaved (active) forms (e.g. procaspase-8 and cl. caspase-8) were used. Thus, detection of specific cleavage products was used as a readout for the activation of caspases and progression of the apoptotic process. Another characteristic feature of apoptosis is cleavage of the DNA damage repair protein poly (ADP-ribose) polymerase (PARP) by activated caspases (Lazebnik et al., 1994). Accordingly, a specific antibody against cleaved PARP was used to detect caspase activity in immunoblotting.

Identification of proteins interacting with caspase-8

Immunoprecipitation uses specific antibodies to precipitate and isolate a protein of in- terest from a lysate containing various proteins. Co-immunoprecipitation is the precipi- tation of a protein together with its interacting proteins that is used to determine possible protein-protein interactions in vitro. For the identification of the caspase-8 activation