Apoptosis. Sebas&ano Ba+aglia, PhD. Dept. Pharmacology and Therapeu&cs Roswell Park Cancer Ins&tute

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1 Apoptosis Sebas&ano Ba+aglia, PhD Dept. Pharmacology and Therapeu&cs Roswell Park Cancer Ins&tute

2 Reference books The Biology of Cancer (Robert Weinberg) + other online references

3 What is Apoptosis Role of Apoptosis Mechanisms of Apoptosis Apoptosis related human diseases

4 WHAT IS APOPTOSIS

5 WHAT IS APOPTOSIS Programmed cell death mechanism (type I cell death, type II is autophagy) First observed in 1842 Further described in 1965/72 as apoptosis (from greek apo=without/from ptosis=falling) as organized cell death event

6 Image from: h1p:// - h1p://en.wikipedia.org/wiki/apoptosis

7 APOPTOSIS vs NECROSIS Inflamma&on response Release of cell content in the surrounding environment Can follow physical trauma Image from: h1p://medicinembbs.blogspot.com/2011/03/programmed- cell- death- apoptosis.html

APOPTOSIS vs AUTOPHAGY Degrada@on of cell s components through lysosomes Crea@on of vescicles that fuse

8 APOPTOSIS vs AUTOPHAGY of cell s components through lysosomes Crea@on of vescicles that fuse with lysosomes Can degrade en@re organelles: Macroautophagy Microautophagy Chaperone mediated autophagy

9 ROLE OF APOPTOSIS Number of cells is &ghtly regulated Fine control over ra&o prolifera&ng/dying cells When a cell is not needed anymore, it commits a suicide Image from: h1p://science.kukuchew.com/2008/10/30/brain- cell- apoptosis

tadpole tail s cells undergo apoptosis, therefore, tail is lost

10 ROLE OF APOPTOSIS - DEVELOPMENT Apopto&c cells are removed aber ~12 months and shape digits in the mouse paw Hi, I am a Tadpole The tadpole tail s cells undergo apoptosis, therefore, tail is lost during development Images from: Molecular Biology of the Cell. 4th edi@on.

11 ROLE OF APOPTOSIS GERM LINE During spermatogenesis, degenera&on and death of germ cells can be a+ributed to intrinsic or acquired disturbance of cell viability Apoptosis in the testes leads to the op&mal ra&o between germ cells/sertoli cells Mature spermatozoa can undergo apoptosis, oben observed as DNA fragmenta&on Apoptosis in the germ line (R John Aitken et al) Reproduc&on 2010

12 ROLE OF APOPTOSIS GERM LINE 99% of germ cells generated during ovarian development are lost through apoptosis and never reach ovula&on Apoptosis of the endometrium during menstrual cycle Follicular Atresia (dele&on of the follicles that are not selected for ovula&on) is mainly regulated by apoptosis

13 ROLE OF APOPTOSIS MAMMARY Regression of the cells in the mammary gland INVOLUTION: About 90% of the epithelial cells accumulated to produce milk, will die apoptosis aber weaning of the offspring

14 Apoptosis in Neural Development and Disease Annual Review of Neuroscience Vol. 23: (Volume date March 2000) ROLE OF APOPTOSIS NEURONAL Neurons, glia and neural progenitor cells undergo apoptosis during development About half of the popula&on is lost Op&miza&on of synap&c connec&on, pa+ern forma&on and removal of unnecessary neurons The more connec&on a neuron has, the higher are its chances to survive (kinda like in mankind..)

15 MECHANISMS OF APOPTOSIS So how does this happen?

16 But there are common mechanisms

17 APOPTOSIS - MECHANISM - Apopto&c cell die neatly, without damaging its neighbors. - shrinks and condenses - cytoskeleton collapses - nuclear envelope disassembles - nuclear DNA breaks up into fragments. - Cell surface is altered, allowing neighboring cell or macrophages to phagocyte the dying cell before leakage of its contents occurs Molecular Biology of the Cell. 4th edi@on. Alberts B, Johnson A, Lewis J, et al.

18 MECHANISM MEMBRANE INTEGRITY DAPI binds strongly and selec&vely to the minor groove of adenine- thymine regions of DNA. Bound DAPI has a fluorescence intensity approximately 20 fold higher than that of unbound DAPI, directly propor&onal to the amount of DNA present. As the apopto&c cell membrane is compromised, more DAPI enters the cell and stains a stronger blue color. Tumor Biology Volume 31, Number 3,

19 MECHANISM DNA FRAGMENTATION

20 MECHANISM DNA FRAGMENTATION DNA fragmenta&on is mediated by a heterodimeric factor of 40 and 45 kda in humans [DNA fragmenta&on factor (DFF) 40 and 45] DFF45 is cleaved by caspases in three smaller fragments and dissociates from DFF40 DFF40 homodymerize in a larger complex with Dnase ac&vity

APOPTOSIS ANNEXIN V/PROPIDIUM DISTINGUISH BETWEEN NECROSIS AND APOPTOSIS Morphological changes of the plasma membrane that exposes phospholipid phosphaidylserine (PS) on the outer leaflet of the

21 APOPTOSIS ANNEXIN V/PROPIDIUM DISTINGUISH BETWEEN NECROSIS AND APOPTOSIS Morphological changes of the plasma membrane that exposes phospholipid phosphaidylserine (PS) on the outer leaflet of the membrane Annexin V is a kda Ca2+ dependent phospholipid- binding protein that has a high affinity for PS Propidium iodide can only enter into necroic cells because of the damaged membrane Image from: hup:// ProductCode=ANNEX100F&SearchType=Simple&SearchString=Annex100F#imageTag1

22 MECHANISM - CASPASES Caspases are a family of proteases that have a cysteine at their ac&ve site and cleave proteins at specific aspar&c acid Apoptosis- based therapies for hematological malignancies Petronelli, A., Riccioni, R., Pasquini, L., Petrucci, E., Testa, U. Drugs Fut 2005, 30(7): 707

23 MECHANISM - CASPASES Caspases are synthe&zed as zymogens (= inac&ve enzymes), or pro- caspases including: Prodomain Small + Large subunit Ini&ator caspases have a longer prodomain that allows them to interact to other proteins through: CARD domain (- 2, - 9) DED domain (- 8, - 10)

24 INTRINSIC PATHWAY Ini&ated from within the cell: DNA damage Defec&ve cell cycle Hypoxia Other cell stress Relies on the balance between pro/an&- apopto&c signals BCL- 2 superfamily

INTRINSIC PATHWAY Image from: h1p://www.biooncology.

25 INTRINSIC PATHWAY Image from: h1p:// pathways/intrinsic/index.html

26 BCL- 2 SUPERFAMILY PRO ANTI - > 2 subgroups based on the # of BH domains - > BH3 only (Bid, Bad, Puma, NOXA ) ac&vate those with several BHs (Bax, Bak) - > BCL- 2, BCL- X L - > prevent permeabiliza&on of mitochondrial membrane

27 CYTOCHROME C Physiologically transfers electrons during oxida&ve process Sits between inner/ outer mitochondrial membrane Image from: hup:// science/metabolomics/enzyme- explorer/learning- center/cytochrome- c.html

28 INTRINSIC PATHWAY - > Mitochondrial membrane permeabiliza&on - > release of Cytochrome c and SMAC/DIABLO - > SMAC/DIABLO bind to the IAPs (Inhibitor of Apoptosis Proteins) releasing repression on the Caspases enzymes - > Cyt- c + Apaf- 1 + Casp- 9 complex to form APOPTOSOME - > Ac&va&on of effector caspases Image from: h1p:// educa@on/ apoptosis/pathways/intrinsic/index.html

29 EXTRINSIC PATHWAY Begins outside of the cell Ac&va&on of pro- apopto&c receptors (TNF- R family) with ligands such as: Apo2L/TRAIL (binds DR4-5) CD95L/FasL (binds CD95/Fas) Intracellular binding of FADD (Fas Associated Death Domain) protein Binding and ac&va&on of Casp- 8

Receptor binding EXTRINSIC PATHWAY Recruitment of FADD (Fas- Associated Death Domain) + Casp- 8/- 10 Forma&on of the DISC (Death- Inducing Signal Complex) Ac&va&on of

30 Receptor binding EXTRINSIC PATHWAY Recruitment of FADD (Fas- Associated Death Domain) + Casp- 8/- 10 Forma&on of the DISC (Death- Inducing Signal Complex) Ac&va&on of Casp- 3/- 6/- 7 Convergence into the INTRINSIC PATHWAY Huang X et al. J. Biol. Chem. 2007;282: by American Society for Biochemistry and Molecular Biology

31 EXTRINSIC PATHWAY TYPE I/II TYPE I No need for mitochondrial amplifica&on of the signal Compensated with increased expression of Casp- 8 TYPE II Minimal producion of Casp- 8 Requires mitochondrial amplificaion of CD95 signal - > Bid (Bcl2 family) cleaved by Casp- 8 - > translocates to mitochondria - > release of mitochondial factors

be used for later apopto&c events Cleaved PARP rou4nely used to asses apoptosis in

32 APOPTOSIS PARP Poly(ADP- Ribose) Polymerase: - > enzyme involved in DNA repair mechanism - > when cleaved by Casp- 3 is inac&vated: - > saves NAD and ATP that will be used for later apopto&c events Cleaved PARP rou4nely used to asses apoptosis in the lab Oncogene (1998) 16, 387 ± 398 Image from: h1p://www2.brc.riken.jp/dna/pathway.html?img=7

33 AND HOW ABOUT p53? p53 can trig apoptosis in a transcrip&onal dependent or independent manner p53 can promote transcrip&on of membrane receptors such as Fas, DR5 and PERP BCL2 family members such as Bax, Noxa, Puma BH3 only proteins, favoring Cyt- c release APAF- 1 through E2F1 p53 can localize in the mitochondria favoring pre- Cyt- c release and Casp- 3 ac&va&on Apoptosis - the p53 network - October 15, 2003 J Cell Sci 116,

35 Apoptosis - the p53 network - October 15, 2003 J Cell Sci 116,

36 MUTANT p53 IN CANCER

37 p53 INDEPENDENT APOPTOSIS ANOIKIS death of cells that lose anchorage to extracellular matrix BLUE Human mammary epithelial cells RED Integrins. Bind to the ECM proteins GREEN Casp- 3 staining = apopto&c cells

38 APOPTOSIS AND DISEASE

39 APOPTOSIS AND DISEASE Cancer: con&nuous growth of aberrant cells (p53, Bcl- 2, Myc muta&ons) Restenosis: local overgrowth of vascular smooth muscle in cells in the walls of arteries Autoimmune diseases: failure to eliminate autoreac&ve lymphocytes Viral response: certain viruses (adenovirus, poxvirus) can inhibit apoptosis in infected cells Extract from: Apoptosis and disease R. Ramírez Chamond, J. Carracedo Añón, C. Moreno Aguilar y F. Guerra Pasadas Allergy Service and Research Unit, Reina So@a University Hospital, Córdoba, Spain

40 APOPTOSIS AND DISEASE Neurodegenera&ve: Alzheimer Parkinson Re&ni&s pigmentosa Amyotrophic Lateral Sclerosis Haematologic diseases Myocardial infarc&on, ischaemic renal damage or cerebrovascular accidents (cells surrounding insult undergo apoptosis) AIDS: IHV infec&on depletes CD4+ lymphocytes pool Extract from: Apoptosis and disease R. Ramírez Chamond, J. Carracedo Añón, C. Moreno Aguilar y F. Guerra Pasadas Allergy Service and Research Unit, Reina So@a University Hospital, Córdoba, Spain

41 Extract from: Apoptosis and disease R. Ramírez Chamond, J. Carracedo Añón, C. Moreno Aguilar y F. Guerra Pasadas Allergy Service and Research Unit, Reina So@a University Hospital, Córdoba, Spain

42 APOPTOSIS AND CANCER Tumor cells evade apoptosis by: Increased an&- apopto&c signals (Bcl- 2, Bcl- x L ) Increased survival signals (Igf1/2) Downregula&on pro- apopto&c factors (Bax, Bid, Puma) Blocking the extrinsic pathway Loss of p53 ability to induce PUMA/NOXA leads to survival of abnormal cells

APOPTOSIS AND CANCER Hypermethyla@on of APAF- 1 - > reduced apoptosome forma@on Hyperac@va@on of PI3K - > AKT/PKB pathway: Increased PIP

43 APOPTOSIS AND CANCER of APAF- 1 - > reduced apoptosome forma@on Hyperac@va@on of PI3K - > AKT/PKB pathway: Increased PIP 3 through inac@va@on of PTEN Ac@va@on of AKT/PKB Inhibi@on of pro- apopto@c proteins (Bad, Casp- 9, IkB) Ac@va@on of Mdm2 à p53 inac@va@on

44 APOPTOSIS, MYC AND CANCER c- MYC is a transcrip&on factor with dual role: Proto- oncogene Can ac&vate apoptosis c- MYC complexes with Max to ac&vate downstream effectors (CPP32 protease) Most probably linked to Casp- 3 PARP pathway CPP32 in c- Myc- induced apoptosis A Kangas et al. Oncogene, 1998

45 APOPTOSIS, MYC AND CANCER p53 dependent p53 independent ApoptoDc signaling by c- MYC B Hoffman and D A Liebermann Oncogene, 2008

46 APOPTOSIS AND CANCER THERAPY Radia&on and chemotherapy induce DNA damage à intrinsic pathway Target pro- apopto&c receptors with PARAs (Pro- Apopto&c Receptor Agonists) to ac&vate the extrinsic pathway Evades need of p53 Used in combina&on with ani- angiogenic or immuno therapy Bcl- 2 antagonists (Genasense) Bax inducer (Bortezomib) Nature Reviews Drug Discovery 7, (December 2008)

47 APOPTOSIS AND CANCER THERAPY PARAs targe&ng DR4- DR5 are well tolerated and in phase- I trial recombinant human Apo2L/TRAIL (rhapo2l/trail) mab agonis&c to DR4- DR5 (mapatumumab, Apomab) Rituximab: treatment for non- Hodgkin s lymphoma, probably through apoptosis Mdm2 inhibitors Nature Reviews Drug Discovery 7, (December 2008) Carcinogenesis (2005) 26 (2):

48 APOPTOSIS AND CANCER THERAPY Oncoly&c Adenoviruses (ONYX- 015): mediate lysis of p53- deficient cells Adenovirus- mediated p53 delivery (INGN Advexin): Allows transcrip&on of func&onal p53 following intra/peri- tumoral injec&on Cancer Res March 3, ; 1193 Nature Medicine 6, (2000) Expert Opin Biol Ther Aug;6(8):

49 APOPTOSIS AND CANCER THERAPY Cancer cells have lost cri&cal checkpoint controls (G2 - > M phase) Target this lesion with chemo won t allow the cell to repair the damage Tumor cells will enter mito&c catastrophe Damage is so big that trigs the residual MITOTIC CATASTROPHE

50 QUESTIONS?!

51 VARIOUS RESOURCES h+p:// educa&on/apoptosis/introduc&on/index.html

Apoptotic Pathways in Mammals Dr. Douglas R. Green

Apoptotic Pathways in Mammals Douglas R. Green 1 Apoptosis A form of cell death that is defined morphologically, and features a number of biochemical events Programmed cell death Cell death that occurs