Apoptosis and cancer. Cristina Muñoz Pinedo Bellvitge (IDIBELL)

Apoptosis and cancer Cristina Muñoz Pinedo cmunoz@idibell.cat Institut d Investigació Biomèdica de Bellvitge (IDIBELL)

Hanahan and Weinberg, Cell 2000

Apoptosis in cancer Tumors overexpress antiapoptotic proteins and display reduced levels or mutations of proapoptotic p p proteins. Damaged cells need to be eliminated to maintain genomic integrity. Growth factor deprivation should induce apoptosis. Matrix detachment should induce apoptosis. Immune response against tumors. Oncogene activation triggers apoptosis p

Apoptosis as a consequence of therapy Chemotherapy (antimetabolites, inhibitors of topoisomerases, alkylating agents ) Radioterapy Hormonal therapy (tamoxifen, aromatase inhibitors) antibodies (Trastuzumab- anti ErbB2)

Mutations ti involved in tumorogenesis, metastasis or resistance to therapy p53 frequently mutated. Bcl-2, Bcl-xL, Mcl-1 frequently overexpressed Mutations or downregulation of Bax, Bak etc. Reduced levels of caspases Amplifications in IAPs.

Apoptosis Programmed cell death? Current definition: caspase-dependent death. Morphological and biochemical criteria: - membrane blebbing; cell detachment and shrinkage; chromatin condensation. - other caspase-dependent features: - DNA ladder - subg1 DNA content - phosphatidyl-serine p exposure: Annexin-V binding

Pathways of apoptosis in vertebrates Stimulus extrinsic stimuli intrinsic stimuli Death receptor stress signal Adapter molecule DISC: FADD apoptosome: Apaf-1 Mitochondria Initiator caspase Caspase- 8/10 Caspase-9 cytochrome c Effector caspase caspase-3/7 = Apoptosis

Apoptosis phases Phagocytosis Execution Decision Apoptosome / caspase activation Cell disintegration / DNA degradation Stimulus Regulation of Bcl-2 family members Mitochondrial permeabilization Activation of death receptors De-activation of survival pathways Cell stress / damage *Goldstein et al. (2000) Nat Cell Bio. 2: 156-162

green = cytochrome c-gfp blue = Annexin-Alexa647 purple = Propidium Iodide 4 min/frame. 15h Hela ActD

Red = TMRE (mitochondrial potential) Blue = annexin Green = yopro 1 (DNA) Muñoz-Pinedo, Lab on a Chip 2005

Apoptosis versus necrosis Glucose removal thapsigargin

Other forms of death truncated apoptosis: caspase-independent cell death (CICD) lack of key molecules; caspase inhibition autophagy? unclassified: Toll-like receptors cornification some forms of neuronal cell death perforin necrosis ischemia, heat shock, irradiation etc.

Caspases

Caspases are activated in cascades Initiator caspases (caspase-8, caspase-9) Cleave executioner caspases (caspase-3,-7) caspase-3 3heterotetramer t t

Caspases caspase-3 3heterotetramer t t Lavrik et al, J. Clin. Invest. 115 (2005)

Caspase-8 is activated by homo- dimerization

Riedl and Salvesen, Nat Rev MCB 2007

Slee et al, JCB 1999

Evolution of caspases: para- and metacaspases Human caspase-8 metacaspase 9 of A. thaliana Vercammen JCB 2007

Caspase Knockout phenotype Role in apoptosis Caspase-1 Mice develop normally Role in 'pyroptosis Caspase-2 I i i / i Mice have excess oocytes Premature aging Caspase-3 Perinatal lethality Executioner caspase Brain hyperplasia Caspase-6 Mice develop normally Executioner caspase Caspase-7 Perinatal lethality Executioner caspase Caspase-8 Caspase-9 Embryonic lethality. Impaired heart development and decreased pool of hematopoietic precursors. In humans, familial mutation leads to immunodeficiency Perinatal lethality Excess brain tissue Initiator/executioner caspase Initiator of apoptosis after DNA damage and during mitotic catastrophe Initiator caspase of the death receptor (extrinsic) apoptotic pathway Proteolytically activates downstream caspases and Bid Initiator caspase of the mitochondrial (intrinsic) apoptotic pathway Caspase-10 In humans, a familial mutation is linked with Putative initiator caspase of the extrinsic Caspase-11 type II autoimmune lymphoproliferative syndrome Mice develop normally Lymphocytes have a defect in actin depolymerization pathway Proteolytically activates downstream caspases and Bid Involved in neuronal cell death induced by MPTP and other pathological stimuli Caspase-12 Mice develop normally Initiator caspase in ER stress-dependent apoptosis Caspase-14 Mice skin exhibit reduced hydration levels and enhanced sensitivity to UVB Unknown Adapted from Galuzzi et al 2008

Caspases are frequently mutated or downregulated in tumors - caspase-1 downregulated - caspase-9 mutations (truncation) in colon cancer - Polymorphism in caspase-8 8(CASP8) gene, D302H, associated with high risk of breast cancer - mutations or inactivation of caspase-8 and caspase-10 in tumors - Mutations in caspase-3 - dominant mutations in caspase-7 in several carcinomas

Pathways of apoptosis

Extrinsic pathway Intrinsic or mitochondrial pathway Induced by extracellular ligands: FasL, TNFα, TRAIL Induced by signals that activate Bcl-2-like proteins: Relevance: - hormones - elimination of infected cells - lack of growth factors - homeostasis of immune - hipoxia, hypoglicemia, lack system, immune privilege of nutrients in general - role of TRAIL in innate - chemotherapeutic drugs immunity - antitumor drugs - inflammatory signals (non - moderate heat-shock apoptotic ti function) - DNA damage - etc.

Logue and Martin, Biochem Soc Trans 2008

The extrinsic or death receptor pathway

Ligands and death receptors trigger the extrinsic pathway Wallach et al. Arthritis Res 2002

Role in cancer Fas, TNF, granzymes involved in CTL-mediated killing TNF homolog required for keeping epithelial tumors in check in Drosophila Caspase-8 behaves as a tumor suppressor Role in metastasis of caspase-8 and caspase-10 TRAIL

The extrinsic or death receptor pathway Stimulus extrinsic stimuli Death receptor Adapter molecule Initiator caspase DISC: FADD Caspase- 8/10 Effector caspase caspase-3/7 = Apoptosis

Inactivating mutations or downregulation of TRAIL receptors, caspase-8 and caspase-10 in many tumors Methylation of Fas and TRAIL-R1 FADD downregulated in thyroid and tongue carcinoma, mantle cell lymphoma FLIP expression increased in many tumors: colon and hepatocellular carcinoma, pancreatic cancer, melanoma Mutations in FADD, caspase-8 and caspase-10 higher in metastasis

Fas (CD95) DISC DISC (death-inducing signaling g complex)

TNF signalling

TRAIL signaling TRAIL-R3 TRAIL-R1 TRAIL-R2 TRAIL-R4 Osteoprotegerin DD DDDD DDDD DD FADD Caspase-8 APOPTOSIS

Wang, Oncogene 2008

TRAIL prevents tumorogenesis TRAIL mediates NK-induced cytotoxicity against tumor cells. Activated T cells exert e TRAIL-mediated apoptosis More tumors in TRAIL-deficient mice, spontaneusly and after carcinogen treatment.

TRAIL for therapy Tumor cells are more sensitive than nontransformed cells to TRAIL. rtrail, anti-trail receptor Abs efficient in xenografts In clinical trials: anti-trailr1 TRAILR1, anti-trailr2 O i ff t iti i t ll t Ongoing efforts on sensitizing tumor cells to TRAIL

Execution phase Stimulus extrinsic stimuli Death receptor Adapter molecule Initiator caspase DISC: FADD Caspase- 8/10 Effector caspase caspase-3/7 = Apoptosis

Caspases dismantle the cell by cleaving hundreds of proteins Taylor et al, Nature Reviews Molecular Cell Biology 9, 231-241 (March 2008)

Mitochondria are inactivated during execution phase: ATP levels fall - Caspases are responsible for membrane potential drop - Cells keep respiring if caspases are inhibited. - Caspases are responsible for generation of ROS

Caspases inactivate t respiratory chain Caspases OM IMS IM I Q e - II Q e - e - III C e - IV 2H + + ½ O 2 H 2 O ADP V H + + Ψm - ATP V D A C A N T

Caspases inactivate Complex I to inhibit mitochondrial membrane potential WT Uncleavable mutant t red=mitochondrial potential green= cytochrome c-gfp Hela ActD 4 min/frame

Prevention of caspase-dependent p mitochondrial dysfunction delays cell death WT uncleavable mutant green = cytochrome c-gfp blue = Annexin-Alexa647 purple = Propidium Iodide 4 min/frame

Apoptotic cells are removed by macrophages Taylor et al, Nature Reviews Molecular Cell Biology 9, 231-241 (March 2008)

Measuring apoptosis Caspase activity and cleavage (by western-blot or enzymatic assays) Chromatin condensation (by microscopy) In tissues: TUNNEL, caspase cleavage. Annexin-V (some unspecificity)

Measuring apoptosis Caspase activity and cleavage (by western-blot or enzymatic assays) Chromatin condensation (by microscopy) In tissues: TUNNEL, caspase cleavage. Annexin-V (some unspecificity) Krysko et al, Methods 2008

Measuring apoptosis % cells with sub-g1 DNA content (flow citometer) DNA ladder

The mitochondrial pathway

Stimulus extrinsic stimuli intrinsic stimuli Death receptor stress signal Adapter molecule DISC: FADD apoptosome: Apaf-1 Mitochondria Initiator caspase Caspase- 8/10 Caspase-9 cytochrome c Effector caspase caspase-3/7 = Apoptosis

Relevance for tumorogenesis and tumor maintenance - Oncogene activation - DNA damage - growth factor removal - lack of oxygen - lack of nutrients - lack of substrate adhesion - chemotherapy - radiotherapy

Deprivation of survival factors induces apoptosis through the mitochondrial pathway IL-3 BH3 Adapter molecule Initiator caspase Effector caspase apoptosome: Apaf-1 Caspase-9 Apoptosis Mitochondria cytochrome c

Stimulus Chemotherapy induces apoptosis through the mitochondrial pathway stress signal Adapter molecule Initiator caspase Effector caspase apoptosome: Apaf-1 Caspase-9 Apoptosis Mitochondria cytochrome c

Bcl-2 family proteins

Phenotypes of BCL-null mice Pro-survival family members Adapted from Youle and Strasser, NRMCB 2008 BCL-2 Abnormal death of renal epithelial progenitors, melanocyte progenitors and mature B and T cells. Fatal polycystic kidney disease (100% mortality by 6 weeks), premature greying and lymphopoenia. BCL-XL BCL-W A1A Abnormal death of fetal erythroid progenitors and neuronal cells. 100% die around embryonic day 14 Abnormal death of developing sperm cells. Causes male sterility. Abnormally accelerated death of granulocytes and mast cells in culture. MCL1 Failure in implantation. Conditional knockout causes premature death of immature and mature B and T lymphoid cells, as well as haemopoietic stem cells. Pro-apoptotic BAX/BAK family members BAX BAK Mild lymphoid hyperplasia, male sterility due to sperm-cell differentiation defect. No obvious defects detected so far.

Bcl-2 proteins regulate mitochondrial permeabilization and cytochrome c release Hela staurosporine + ZVAD

Bax translocates during apoptosis GFP-Bax Cyt. c - ReAsH merge HeLa Staurosporine + ZVAD 2 min / frame

Multidomain pro- and anti-apoptotic Bcl-2 homologs are structurally similar Bax Bcl-W Bcl-xL

Bax and Bak are required for permeabilization of the outer mitochondrial i membrane Bax/Bak Bcl-2 tbid mitochondria matrix Cyt c BH3-only tbid Bax Bak Cytochrome c Caspase-9 Apoptosis

Basáñez and Hardwick, 2008

BH3-only proteins are selective for anti- apoptotic Bcl-2 proteins Youle and Strasser, NRMCB 2008

Bcl-2 proteins in cancer -Bcl-2 discovered as an oncogene - Antiapoptotic Bcl-2 family members behave as oncogenes - Proapoptotic Bcl-2 family members behave as - Proapoptotic Bcl-2 family members behave as tumor suppressors

Antiapoptotic Bcl-2 allow tumorogenesis by c-myc Hematopoietic progenitor cells are transduced with retroviral vectors, ex vivo, followed by reintroduction into lethally irradiated syngeneic recipients Beverly et al, Oncogene, 2009

Antiapoptotic Bcl-2 proteins are overexpressed in a range of human tumors Beverly et al, Oncogene, 2009

Antiapoptotic Bcl-2 proteins are overexpressed in tumors Bcl-2 Bcl-xL follicular lymphoma lung adenocarcinoma, ovarian tumors, murine hematopoietic malignancies Mcl-1 multiple myeloma and other hematopoietic malignancies Bfl-1 diffuse large-cell lymphoma, breast tumors Bcl-w lung tumors

Tumor cells frequently inactivate BH3-onlys or downregulate Bax and/or Bak Anthony G. Letai Nature Reviews Cancer 8, 121-132 (February 2008)

Bcl2-like proapoptotic proteins are mutated or downregulated in human tumors Proapoptotic multidomain Bax oligodendroglioma, breast cancer, colon and gastric adenocarcinomas, hematopoietic ti malignancies, i Bak nonsmall cell lung carcinoma BH3-only Puma melanoma Bad squamous cell carcinomas, colon cancers Bim Bid renal cell carcinoma nonsmall cell lung carcinoma

Pro-apoptotic Puma and Noxa contribute to mycinduced lymphomagenesis Some BH3-only are upregulated in tumors: Noxa, BNIP3 Burton et al, Oncogene 2009

Bcl-2 family members modulate response to chemotherapy - In vitro, most BH3 proteins have been shown to modulate responses to drugs. -increased expression of antiapoptotic BCL2 in cell lines resistant to chemotherapy (increasing copy numbers or upregulation of mrna or protein). Frenzel et al, Apoptosis 2008

Addiction to anti-apoptotic Bcl-2 proteins Brunelle and Letai, JCS 2008

Mitochondrial permeabilization during apoptosis Stimulus intrinsic i i stimuli stress signal Adapter molecule Initiator caspase apoptosome: Apaf-1 Mitochondria cytochrome c Effector caspase Apoptosis Caspase-9

MOMP (mitochondrial outer membrane permeabilization) When mitos are permeabilized, many proteins from the intermembrane space are released: - Cytochrome c - Smac / DIABLO - Adenylate kinase 2 - Omi / HtrA2 - Endonuclease G - AIF Mitos are depolarized

Bax and Bak are required for permeabilization of the outer mitochondrial i membrane Bax/Bak Bcl-2 tbid mitochondria matrix Cyt c BH3-only tbid Bax Bak Cytochrome c Caspase-9 Apoptosis

The MOMP pore Bax and/or Bak are required for formation of the pore. It only targets the outer membrane. Different proteins up to at least 120 kda are released simultaneously from the pore.

Cytochrome c release takes 5 min within a Cytochrome c release takes 5 min within a single cell

cytochrome c-gfp and cyt.c-tetracysteinetetracysteine are released in 5 min GFP-cytocrome c cyt. c - tetracysteine REASH Hela - ActD+ZVAD 30ºC 1 min 30 /frame 2 h total

Smac/DIABLO, a second mitocondrial pro- apoptotic protein Stimulus intrinsic stimuli stress signal Adapter molecule Initiator caspase Effector caspase apoptosome: Apaf-1 Caspase-9 Apoptosis Mitochondria cytochrome c

Smac is released prior to mitochondrial depolarization, and simultaneously to cyt.c Smac-TC Smac- TC+TMRE Duration of release= 5.4 min Lag= 3.1 min Hela, Staurosporine + ZVAD

Smac is mutated t in tumors Renal cell carcinomas express less Smac than normal tissue, The expression is lower in advanced stages, Expression correlates with survival, Retransfection sensitizes RCC to TNF, TRAIL and cisplatin. Can cells survive after mitochondrial Can cells survive after mitochondrial permeabilization?

Omi/HtrA2, a serine-protease released during apoptosis. Omi and cyt.c are released simultaneously Cyt. C-GFP Omi-4cys Hela stably transfected with cyt.c- GFP; transiently with Omi-4CYS. Staurosporine + ZVAD, 3 min/picture Muñoz-Pinedo et al, PNAS 2006

AIF, apoptosis-inducing inducing factor? AIF is released slowly after cyt.c Time (min) 360 532 536 540 552 756 cyt.c- GFP merge AIF-TC

Riedl and Salvesen, Nat Rev MCB 2007

Apaf-1 promoter methylated in several leukemias Downregulated in malignant melanoma, ovarian cancer Can cells survive after cyt.c release?

IAPs ( Inhibitor of Apoptosis Protein ) IAPs ( Inhibitor of Apoptosis Protein ) part of the BIR-containing protein family

IAPs may act at different levels Stimulus extrinsic stimuli intrinsic stimuli Death receptor stress signal Adapter molecule DISC: FADD apoptosome: Apaf-1 Mitochondria Initiator caspase Caspase- 8/10 Caspase-9 cytochrome c Effector caspase caspase-3/7 = Apoptosis

Gene Disease Comments Birc2 (ciap1) Birc3 (ciap2) Birc4 (XIAP) Multiple myeloma (MM) Various carcinomas MALT lymphoma Multiple myeloma Various carcinomas X-linked lymphoproliferativ e disorder (XLP) ciap1 and ciap2 are deleted in some MM cancers, as part of a larger trend of activating mutations ti for the alternative ti NF-B pathway (Annunziata et al., 2007; Keats et al., 2007). Hence, the negative regulatory role of ciap1 or ciap2 as E3 ubiquitin ligases for NIK is removed, allowing for NIK stabilization and activation of the alternative NF-B pathway. ciap1 and ciap2 genes, at 11q22, are commonly amplified in many cancers, with visibly increased expression of ciap1. This phenomenon is also conserved in some murine tumors that also lead to co-amplification of YAP. Both ciap1 and YAP have been shown to cooperate in transformation (Zender et al., 2006). ciap1 can also cooperate with Myc to transform. t f This results from ciap1's ability to induce the proteosomal degradation of the Myc antagonist Mad1 (Xu et al., 2007). ciap2 is translocated in t(11;18)(q21;q21)-bearing lymphomas (Dierlamm et al., 1999). The chromosomal translocation fuses the three BIR domains of ciap2 with the paracaspase domain of MALT1, while preserving the ciap2, NF-B-responsive, promoter. See above ciap1 and ciap2 genes, at 11q22, are commonly amplified in many cancers, with visibly increased expression of ciap1. While ciap2 expression is often overlooked. This DNA amplification is also conserved in some murine tumors, which also leads to co-amplification of YAP (Zender et al., 2006). ciap2 may also cooperate in transformation in these cases. Apoptosis of lymphocytes from XIAP-deficient patients is enhanced in response to various stimuli including CD3, Fas and TRAIL. In addition, XIAP-deficient XLP patients (XLP-2), like SAP-deficient XLP patients (XLP-1), have low numbers of NK T cells. XLP patients can exhibit a fatal response to EBV, the viral cause of infectious mononucleosis (presumably due to a failure(s) in NK and lymphocyte homeostasis and signaling) (Rigaud et al., 2006). Adapted from LaCasse et al, Oncogene 2008

IAPs and their inhibitors are altered in cancer - XAF1 is an IAP inhibitor underexpressed in many cancer cell lines (LOH), and a prognostic marker in bladder cancers. - Smac/DIABLO inhibits XIAP, ciap1, ciap2, survivin, livin and BRUCE.

IAP small molecule antagonists in clinical development Target Compound Class Stage Company XIAP AEG35156/GEM640 Antisense Phase 2 Aegera; Idera Xantags (1396-11, 1396-12), TWX006, TWX024 Caspase-3 de-repressor Preclinical Embelin and derivatives Natural product Preclinical ciap1, ciap2, AEG40826/HGS1029, LBW242, compound 3, XIAP, livin compound 11 Joyant, Pfizer, Abbott Smac mimetic Phase 1 Aegera (Human Genome Sciences, USA), Novartis, Joyant Pfizer Abbott Compound C, compound 8, Smac mimetic Phase 1 Genentech, SF, CA BV6 GT-T, compound A Smac mimetic Preclinical Tetralogic, Malvern, PA, USA (Amgen, CA) SM-164, SM-122 Smac mimetic Preclinical Ascenta, Malvern, PA, USA ciap1 Bestatin methyl ester ciap1 degrad. promoter Preclinical Nippon Kayaku Co., Japan Bestatin actinonin hybrid, HAB-5(30b) ciap1 degrad. promoter Preclinical Nippon Kayaku Co. ciap2 Ro106-9920 IKK ubiquitination Preclinical Roche, Palo Alto, CA, USA inhibitor Survivin LY2181308/ISIS23722 Antisense Phase 2 Eli Lilly, Indianapolis, IN, USA (Isis, CA, USA) YM155 Transcriptional repressor Phase 2 Astellas, Tokyo, Japan Terameprocol (EM-1421) Transcriptional repressor Phase 2 Erimos, Raleigh, NC, USA Adapted from LaCasse et al, Oncogene 2008