Types of cell death and apoptosis resistance mechanisms. Institut for Experimental Cancer Research

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1 Types of cell death and apoptosis resistance mechanisms Prof.Dr.rer. rer. nat.anna Trauzold Institut for Experimental Cancer Research

2 Physiological cell death Embryogenesis Control of the tissue size Renewal of epithelia Selection (immune system) Elimination of tumor cells Elimination of infected/injured cells

3 (Patho)physiological cell death (too much) Neurodegenerative diseases (Morbus Parkinson) Infections (HIV, HBV, HCV) Cardiovascular diseases (cardiac infarction) Radio /Chemotherapy Cirrhosis of the liver

4 (Patho)physiological cell death (too little) Efficient induction and successful execution of cell death is an aim of many anti tumor tumor therapies Hanahan & Weinberg, 2011, Cell 144:

5 Until recently Apoptosis vs. Necrosis programmed vs. accidental active vs. passive

6 Until recently induced by extremely harsh physical conditions Passive process Clarke & Smyth, 2007, Nat. Biotech. 25: Accidental cell death Necrosis cell death with inflammation

7 Until recently eat me signal macrophage Active process Clarke & Smyth, 2007, Nat. Biotech. 25: Programmed (regulated) cell death Apoptosis p activation of caspases silent death without inflammation and immune response

8 Cell death modalities Number of articles in PubMed Nov Nov Nov Apoptosis Necrosis Autophagic cell death Anoikis Cornification Mitotic catastrophe Necroptosis Pyroptosis Entosis Parthanatos Netosis

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10 Cell Death Differ Jan;19(1):

11 No more valid equation! Programmed (regulated) cell death Apoptosis activation of caspases silent death without inflammation and immune response

12 No more valid equation! Programmed (regulated) cell death Apoptosis activation of caspases silent death without inflammation and immune response Existence of other forms of regulated cell death (necroptosis and autophagic cell death)

13 No more valid equation! Programmed (regulated) cell death Apoptosis activation of caspases silent death without inflammation and immune response Existence of other forms of regulated cell death (necroptosis and autophagic cell death) Apoptosis can occur also without activation of caspases, caspases can also be activated in other cell death modalities

14 No more valid equation! Programmed (regulated) cell death Apoptosis activation of caspases silent death without inflammation and immune response Existence of other forms of regulated cell death (necroptosis and autophagic cell death) Apoptosis can occur also without activation of caspases, caspases can also be activated in other cell death modalities Cells dying by apoptosis can induce inflammation and immune response

15 No more valid equation! Programmed (regulated) cell death Apoptosis activation of caspases silent death without inflammation and immune response Tumour cells dying after exposure to antracyclins (for example doxorubicin, mitoxantrone), oxaliplatin or ionizing radiation can induce strong anticancer immune response Priming of CD4+ and CD8+ lymphocytes, anti-tumor response Calreticulin (CRT) at the plasma membrane of dying cell eat me signal for antigen presenting dendritic cells Reviewed by Kepp et al., 2011, Cancer Metastasis Rev 30:61-69.

16 Immunogenic Cell Death (ICD) determines the long term success of anticancer therapy Suboptimal regimens (without inducing ICD) Alterations in cancer cells (preventing emission of immunogenic signals) Defects in the immune effectors (abolishing the perception of ICD by the immune system) All contribute to therapeutic failure ICD immunogenic cell death; DC, dendritic cell; CTL, cytotoxic T-cell lymphocytes; CRT, calreticulin Kroemer et al., 2013, Annu. Rev. Immunol. 31:51-72.

17 No more valid equation! Programmed (regulated) cell death Apoptosis activation of caspases silent death without inflammation and immune response Passive process Accidental cell death cell death Necrosis with inflammation not always! In some cases (inhibition of caspases) stimulation of cells with death ligands (CD95, TNFalpha, TRAIL) leads to necrosis = necroptosis regulated, active process

18 Emerging pathways of regulated necrosis Necroptosis, parthanatos, oxytosis, ferroptosis, NETosis, pyronecrosis and pyroptosis RIPK1/3: receptor-interacting protein kinase 1/3 MLKL: mixed lineage kinase domain-like Berghe et al., Nat Rev Mol Cell Biol. (2014) ;15(2):

19 Cell death modalities today Regulated cell death cell death occuring by dedicated molecular machinery, can be inhibited by targeted pharmacological and/or genetic intervention Apoptosis (extrinsic and intrinsic pathways) Autophagic cell death / cell death with autophagy Necroptosis (maybe some other forms) regulated necrosis Accidental cell death cell death triggered by extremely harsh physical conditions, cannot be inhibited by pharmacological and/or genetic manipulations Necrosis

20 Autophagy: Renovation of cells and tissues Major intracellular degradation system by which cytoplasmic materials aredelivered to anddegradeddegraded inthe lysosome The purpose of autophagy is not the simple elimination of materials, instead it serves as dynamic recycling system that produces new building blocks and energy for cellular renovation and homeostasis Essential role in: metabolic adaptation (starvation) intracellularquality control (degradation of damaged mitochondria) renovation during development (after fertilization maternal proteins and RNAs are extensively degraded while new proteins encoded by the zygotic genome aresynthesized) differentiation (lineage differentiation: adipocytes, erythrocytes, T cells) Impairment or activation of autophagy contributes to pathogenesis of diverse diseases Mizushima & Komatsu, Cell 2011, 147:

21 Autophagic cell death In mammals autophagy is usually a self- limiting process that protects cells from death and is essential for tissue homeostasis. mammalian cells die usually either by necrosis or apoptosis. presence of the autophagosomes usually only accompany cell death - cell death with autophagy real autophagic cell death There are only few known examples of cell death that can be prevented by genetic inhibition of autophagy in mammals in lower organisms, several developmental processes (salivary gland involution in the larvae of Drosophila, excitotoxic i cell death of Caenorabditis elegans neurons) Kroemer & Levine, Nat. Rev. Mol. Cell Biol Dec;9(12): Galluzzi et al., Cell Death Differ., 2015 Jan;22(1):58-73.

22 Apoptosis inducing signal inducing/executing machinery Apoptosis intrinsic extrinsic caspase- -dependent d caspase- -independent caspase- -dependent death receptors dependence receptors

23 Caspases the motors of Apoptosis Proteases Cysteine in active center Cleave proteins after Aspartate (Cysteine Aspartase) Present in the cytoplasm as inactive forms Procaspases Pro apoptotic stimulus Initiator caspases Effektor caspases Caspase cascade Caspases are activated via: 1. autocatalysis 2. transactivation by other caspases 3. proteolysis by other proteases (Granzyme B, Cathepsin G) Apoptosis

24 Caspase Family Regulatory Domain Catalytic Domain Caspase 1 Caspase 4 Caspase 5 Caspase 13 Caspase 2 Caspase 9 Caspase 8 Caspase 10 Caspase 3 Caspase 6 Caspase 7 Caspase 14 DED DED CARD CARD CARD CARD CARD CARD DED DED N N N CARD p20 p20 p20 p20 p20 p20 p20 p20 p20 p20 p20 p20 p10 p10 p10 p10 p10 p10 p10 p10 p10 p10 p10 p10 Inflammation Activation of cytokines Apoptosis Differentiation CARD, caspase recruitment domain; DED, death effector domain; N, N terminal peptide

25 Apoptotic Caspases are classified as Initiator or Effector Caspases Initiator Caspases: ii Pro caspases DED DED DED DED cleavage sites p20 p20 p10 p10 Caspase 2 Caspase 9 Caspase 8 Caspase 10 DED DED CARD CARD DED DED p20 p20 p20 p20 p10 p10 p10 p10 p20 p10 p10 p20 Active tetramer cleaves other caspases and initiates cell death signaling Effector Caspasen: Caspase 3 Caspase 6 Caspase 7 N N N p20 p20 p20 p10 p10 p10 Effector Caspases cleave cytoplasmic and nuclear substrates Cell death CARD, caspase recruitment domain; DED, death effector domain; N, N terminal peptide

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27 Apoptosis inducing signal executing machinery Apoptosis intrinsic extrinsic caspase- -dependent d caspase- -independent caspase- -dependent death receptors dependence receptors

28 Intrinsic apoptosis Chemotherapy Radiotherapy Mitochondria Caspase 9 Caspase 3, 6, 7 Apoptosis DNA damage p53 PUMA, NOXA BAX, BAK Cytochrome c APAF1 Cell-intrinsic pathway p53 DNA damage Ashkenazi A. Nat Rev Cancer 2002;2: , modified APAF1, apoptotic protease activating factor-1; BAK, BCL2 homologous antagonist/killer; BAX, BCL2-associated protein; BCL2, B-cell chronic lymphocytic leukemia/lymphoma 2; PUMA, p53-upregulated modulator of apoptosis;

29 Intrinsic apoptosis

30 Extrinsic apoptosis death ligand dependence receptor death receptor Activation of effector caspases Apoptosis

31 Dependence Receptors Dependence receptors have two faces: In the presence of ligand transduction of a positive signal In the absence of ligand induction of apoptosis All are cleaved by caspases All contain an ADD (addiction/dependance domain) After exposition of ADD by receptor cleavage ADD recruits additional caspase activating complexes Goldschneider & Mehlen, Oncogene 2010,

32 Death Receptors TNF FasL TL1A TRAIL? EDA1 NGF DR4 DR5 DcR2 DcR1 Cysteine-rich motif Death domain TL1, TNF-like cytokine; EDAR, Ectodermal displasia receptor EDA1, Ectodysplasin A1 NGF, Nerve growth factor

33 Physiological function of Death Receptors (CD95 & TRAIL Rs) NK CTL NK CTL CTL TRAIL-R1/R2 TRAIL NK TRAIL TRAIL-R1/R2 TRAIL-R1/R2 CD95 tumor cell CD95 tu mor tumor cell cell TRAIL NK CTL CD95L NK CTL CD95L NK CD95 CD95L CTL TNFRI mainly inflammation

34 Death receptor mediated apoptosis DL DR DR Procaspase 8, 10 FADD DISC Type I-Cells Caspase 8, 10 Caspase 3, 6, 7 Apoptosis Ashkenazi A. Nat Rev Cancer 2002;2: , modified DR, death receptor; DL, death ligand; FADD, Fas-associated death domain;

35 Death receptor mediated apoptosis DR DL DR Procaspase 8, 10 FADD DISC BID t-bid Mitochondria Caspase 8, 10 Cytochrome c APAF1 Caspase 9 Caspase 3, 6, 7 Apoptosis BAX, BAK Type II-Cells Ashkenazi A. Nat Rev Cancer 2002;2: , modified APAF1, apoptotic protease activating factor-1; BID, BH3-interacting domain death agonist; DR, death receptor; FADD, Fasassociated death domain;

36 Intrinsic and extrinsic apoptotic pathways are linked Cell-extrinsic pathway TRAIL-R1 Procaspase 8, 10 Ashkenazi A. Nat Rev Cancer 2002;2: , modified death receptor ligand TRAIL TRAIL-R2 DNA damage p53 FADD PUMA, NOXA BID BAX, BAK t-bid Mitochondria Caspase 8, 10 Cytochrome c Caspase 9 Caspase 3, 6, 7 Apoptosis APAF1 Chemotherapy Radiotherapy Cell-intrinsic pathway p53 DNA damage APAF1, apoptotic protease activating factor-1; BAK, BCL2 homologous antagonist/killer; BAX, BCL2-associated protein;; BID, BH3- interacting domain death agonist; DR, death receptor; FADD, Fas-associated death domain; PUMA, p53-upregulated modulator of apoptosis;

37 Each step of the apoptotic pathway is tightly controlled TRAIL-R1 Decoy-Receptors TRAIL TRAIL-R2 FLIP Procaspase 8, 10 DISC FADD BCL2, BCLX L, MCL1 BID t-bid BAX, BAK Mitochondria Caspase 8, 10 Caspase 9 Caspase 3, 6, 7 Apoptosis Cytochrome c SMAC/DIABLO APAF1 IAP Ashkenazi A. Nat Rev Cancer 2002;2: , modified APAF1, apoptotic protease activating factor-1; BID, BH3-interacting domain death agonist; DR, death receptor; FADD, Fasassociated death domain;

38 Decoy receptors diminish DISC formation OPG soluble Receptor for TRAIL DcR1 membrane anchored TRAIL R DR2 DcR2 TRAIL R R with truncated t ddd DR4 = TRAIL R1 R1 DR5 = TRAIL R2 DcR: Decoy Receptor; OPG: Osteoprotegerin

39 FLIP inhibits DISC activity extrinsic pathway DR DL DR FLIP Procaspase 8, 10 FLIP Caspase 8 homologue Mutation in active center DISC FADD BCL2, BCLX L, MCL1 BID t-bid BAX, BAK Mitochondria Caspase 8, 10 Caspase 9 Caspase 3, 6, 7 Apoptosis Cytochrome c SMAC/DIABLO APAF1 IAP Ashkenazi A. Nat Rev Cancer 2002;2: , modified APAF1, apoptotic protease activating factor-1; BID, BH3-interacting domain death agonist; FADD, Fas-associated death domain; FLIP

40 Members of the Bcl 2 Family regulate intrinsic pathway and extrinsic pathway in Type II cells extrinsic pathway DR DL DR Type II-Cells FLIP Procaspase 8, 10 DISC FADD BCL2, BCLX L, MCL1 BID t-bid BAX, BAK Mitochondria Caspase 8, 10 Caspase 9 Caspase 3, 6, 7 Apoptosis Cytochrome c SMAC/DIABLO APAF1 IAP intrinsic pathway Ashkenazi A. Nat Rev Cancer 2002;2: , modified APAF1, apoptotic protease activating factor-1; BID, BH3-interacting domain death agonist; DR, death receptor; FADD, Fasassociated death domain;

41 The Bcl 2 Family Guardians critical for cell survival anti apoptotic p functions Effectors upon activation Bax/Bak mediate MOMP activation of effector stages of apoptosis Sensors essential for initiation of apoptosis signaling BH: Bcl-2 homology domain

42 The Bcl 2 Family Bax and Bak form pores in the outer mitochondrial membrane Bcl2 and Bcl xl inhibit Bax and Bak BH3 only proteins influence these processes

43 Phosphorylation of Bid inhibits its proteolytic cleavage extrinsic pathway DR DL DR Type II-Cells CKI/II FLIP Procaspase 8, 10 DISC FADD BCL2, BCLX L, P MCL1 BID t-bid BAX, BAK Mitochondria Caspase 8, 10 Caspase 9 Caspase 3, 6, 7 Apoptosis Cytochrome c SMAC/DIABLO APAF1 IAP Ashkenazi A. Nat Rev Cancer 2002;2: , modified. APAF1, apoptotic protease activating factor-1; BID, BH3-interacting domain death agonist; DR, death receptor; FADD, Fasassociated death domain;

44 IAPs inhibit caspases extrinsic pathway DR4 Pro-apoptotic ligand DR5 CKI/II intrinsic pathway FLIP Procaspase 8, 10 DISC FADD BCL2, BCLX L, P MCL1 BID t-bid BAX, BAK Mitochondria Caspase 8, 10 Caspase 9 Caspase 3, 6, 7 Apoptosis Cytochrome c SMAC/DIABLO APAF1 IAP Ashkenazi A. Nat Rev Cancer 2002;2: , modified. APAF1, apoptotic protease activating factor-1; BID, BH3-interacting domain death agonist; DR, death receptor; FADD, Fasassociated death domain;

45 IAP Family IAP I hibit f A t i BIR B l i l IAP R t CARD CA R it t D i IAP: Inhibitors of Apoptosis; BIR: Baculoviral IAP Repeat; CARD: CAspase Recruitment Domain; RING: RING zing finger; NOD: Nucleotide-binding Oligomerization Domain; LRR: Leucine Rich Repeat

46 IAPs inhibit both, intrinsic and extrinsic apoptotic pathways XIAP inhibits both, initiator- caspase (Caspase-9) and effector-caspases (Caspase-3 und 7) IAPs are inhibited by Smac/Diablo, Omi/Htr2A, XAF1

47 Efficient induction and successful execution of cell death is the aim of many anti tumor therapies Hanahan & Weinberg, 2011, Cell 144:

48 Apoptosis in anti tumor therapy extrinsic pathway TRAIL-R1 Procaspase 8, 10 Ashkenazi A. Nat Rev Cancer 2002;2: , modified death receptor ligand TRAIL or agonistic antibodies TRAIL-R2 DNA damage p53 FADD PUMA, NOXA BID BAX, BAK t-bid Mitochondria Caspase 8, 10 Cytochrome c Caspase 9 Caspase 3, 6, 7 Apoptosis APAF1 Chemotherapy Radiotherapy p53 intrinsic pathway DNA damage APAF1, apoptotic protease activating factor-1; BAK, BCL2 homologous antagonist/killer; BAX, BCL2-associated protein;; BID, BH3- interacting domain death agonist; DR, death receptor; FADD, Fas-associated death domain; PUMA, p53-upregulated modulator of apoptosis;

49 Tumor cells develop multiple, parallel operating apoptosis inhibiting inhibiting strategies

50 Constitutive apoptosis resistance mechanisms in PDAC cells TRAIL induces death preferentially in tumor cells (clinical trials) TRAF2 FLIP TRAIL TRAIL-R1/R2R1/R2 Antibody Apoptosis BclxL XIAP c-iap1/2 Hinz et al., Oncogene 2000 Hinz et al., Oncogene 2000 Trauzold et al., Oncogene 2001 Trauzold et al., Br. J. Cancer 2003 Trauzold et al., Faseb J. 2005

51 Invasion instead of Apoptosis new Function of death receptors in PDAC (and many other) tumor cells Exogenous ous TRAIL induces metastasis s (via TRAIL-R1) Trauzold et al., Oncogene 2006 Trauzold et al., Oncogene 2001 Siegmund et al., Cell Signal 2007 Lemke et al., J Mol Med 2010 Ehrenschwender et al., Cell Death Diff 2010 Endogenous TRAIL induces metastasis (via TRAIL-R2) R2) in KRAS-mutated t cells von Karstedt et al., Cancer Cell 2015

52 Current TRAIL based anti tumor therapy TRAIL TRAIL-R1/R2 Antibody Bcl-xL IAPs FLIP + sensitizing agents + inhibitors Apoptosis Tumor cell Proliferation Inflammation Migration/Invasion Metastasis Enhancement of cell sensitivity Inhibition of non-apoptotic signaling Inhibition of TRAIL?

53 High intracellular levels of TRAIL Receptors correlate with poor prognosis Tumor cells express TRAIL receptors mainly in intracellular locations high intracellular levels of TRAIL-R2 correlate with poor prognosis TRAIL-R1 TRAIL-R2 Haselmann et al, Hauser, Egberts,..& Trauzold: Gastroenterology, 2014 Bertsch,, Trauzold; Cell Death & Disease, 2014

54 Tumor cells express TRAIL Rs mainly intracellularly nuclear TRAIL R2 has an oncogenic function Haselmann et al., Gastroenterology 2014

55 Nuclear TRAIL R2 enhances tumor growth and metastasis KD of TRAIL R2 : HMGA2 myc CXCR4 E cadherin Orthotopic PDAC xenotransplantation model in mouse Breast cancer bone metastasis t model dl Haselmann et al., & Trauzold, Gastroenterology 2014 Fritsche et al., Oncotarget 2015

56 Future TRAIL R based anti tumor therapy TRAIL TRAIL-R1/R2R1/R2 Antikörper Plasma membrane TRAIL Receptors enhancement sensitisation inhibition of non-apoptotic signaling Tumor cell Cell death or inhibition of TRAIL-R R neutralisation of endogenous TRAIL nuclear TRAIL-R2 i hibiti f th l i t inhibition of the nuclear import enhancement of the export inhibition of the nuclear functions

57 Efficient induction and successful execution of cell death is the aim of many anti tumor therapies Hanahan & Weinberg, 2011, Cell 144:

58 The paradox of cell death driven cancer Labi & Erlacher, Cell Death and Disease (2015) 6, e1675; doi: /cddis

59 Pro oncogenic effects of apoptotic cell death ipla2 calcium-independent phospholipase A 2 COX Cyclooxygenase eat me and find me signals FKN fractalkine, ATP, LTF lactotrasferrin Ichim & Tait, Nature Review Cancer (2016) 16:

60 Current strategies: Enhancing apoptosis while minimizing damage CSF1R Colony stimulating factor 1 receptor Ichim & Tait, Nature Review Cancer (2016) 16:

61 The paradox of cell death driven cancer Further research is necessary to understand the contribution of apoptosis in shaping tumors, as a prerequisite to generate a more comprehensive picture on tumorigenesis and allow more effective therapeutic intervention Labi & Erlacher, Cell Death and Disease (2015) 6, e1675; doi: /cddis