Signaling Apoptosis. Scott André Oakes, M.D. Dept. of Pathology Univ. of Calif-San Francisco. Cyt c Release BAX/BAK. Apoptosome Formation

Transcription

1 Signaling Apoptosis Cyt c Release BAX/BAK Apoptosome Formation Caspase Activation Scott André Oakes, M.D. Dept. of Pathology Univ. of Calif-San Francisco

2 Why do we need cell death? Sculpt Organs Control Cell Numbers Delete Structures Eliminate Abnormal Cells Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology Baehrecke EH et al 2002, 3: , Copyright 2002

3 There is more than one way to die.. Kumar et al Robbins and Cotran Pathologic Basis of Disease 8/E (Fig. 1-8) Copyright 2010 by Saunders, an imprint of Elsevier Inc. All rights reserved.

4 The many faces of cell death Shedding (epithelia) Suicide 1 (apoptosis) Suicide 2 (alternative programmed cell death or necroptosis) Necrosis (trauma) Abscission (plants, newts) Autophagy Terminal Differentiation Senescence

5 There are two primary apoptotic pathways.. Kumar et al Robbins and Cotran Pathologic Basis of Disease 8/E (Fig. 1-24) Copyright 2010 by Saunders, an imprint of Elsevier Inc. All rights reserved.

6 A Closer Look at the Components of the Apoptotic Machinery. Sensors: detect and respond to various types of cell injury. growth factor receptors ATM/ATR (DNA damage) Integrators: calculate injury, decide if damage is lethal, and signal effectors. Pro- and anti-apoptotic BCL-2 proteins Mitochondrial pro-death proteins (cytochrome c, SMAC/Diablo) IAPs (inhibitors of apoptosis) Effectors: execute cell demise. Caspases Engulfment machinery

7 Follicular Lymphoma and the Discovery of BCL-2 Follicular Lymphoma, Spleen Follicular Lymphoma, Lymph Node Kumar et al Robbins and Cotran Pathologic Basis of Disease 8/E (Fig & 12-11) Copyright 2010 by Saunders, an imprint of Elsevier Inc. All rights reserved.

8 BCL-2 Overexpression Protects Cells Against Death t(myc;ig) B Cell BCL-2

9 BCL-2 Family

10 Structure of Pro-survival BCL-2 Family Proteins Bound to BH3 Peptide BCL-X L bound to BIM BH3 peptide (purple) MCL-1 bound to BIM BH3 peptide (purple) Reprinted by permission from Macmillan Publishers Ltd., Nature Reviews Drug Discovery Lessene G et al 2008, 7: , Copyright 2008.

11 BCL-2 Family Proteins Large family of proteins that integrates upstream damage signals. Activated either transcriptionally or through post-translational modifications. The ratio/activity of pro- and anti- death members regulates outer mitochondrial membrane permeability.

12 Mitochondria: A Storehouse of Apoptotic Factors Cyto c: member of electron transport chain that triggers apoptosome formation and caspase-9 activation if released into cytosol. Smac/DIABLO: promotes apoptosis by binding to and relieving the caspase inhibitory effect of IAPs (Inhibitors of Apoptosis). Reprinted by permission from Macmillan Publishers Ltd., Nature Chemical Biology Orrenius S el al 2005, 1: , Copyright 2005.

13 The Apoptosome: A Platform for Caspase Activation Reprinted by permission from Macmillan Publishers Ltd., Cell Death and Differentiation Cecconi F 2008,15: , Copyright 2008.

14 Caspases (Cys Asp Proteases) Homologs of C. elegans Ced-3 Central executioners of apoptosis Made as inactive pro-enzymes Activated by upstream caspases or other proteases Cleave critical cell substrates to trigger cell demise and involution. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology Taylor RC et al 2008, 9:231-24, Copyright 2008.

15 Regulating Caspases Inhibitors of Apoptosis (IAP): Family of functionally-related proteins that bind to and inhibit active caspases through a BIR domain Smac/DIABLO: Mitochondrial protein that upon release promotes apoptosis by counteracting IAPs through physical interactions. Reprinted by permission from Macmillan Publishers Ltd., The EMBO Journal Adrain C et al 2001, 20: , Copyright 2001.

16 Apoptosis BH3-only BID Multi-domain Pro-apoptotic - Death receptors, Caspase-8 Apoptosome Effector Caspases BAD BIM NOXA/ PUMA / - Survival factors, glucose - Cytokines BAX BAK BCL-2 BCL-X L Sentinels for Death/Survival Signals MCL-1 Cyto c release (Apaf-1, - DNA Damage, p53 induced Active BAX/BAK Caspase-9, Cytochrome c) Multi-domain Anti-apoptotic Mitochondrial Gateway

17 Intrinsic Death Stimuli Converge on BAX/BAK Plasma Membrane Growth Factor Withdrawal Cyt c Release BAX/BAK Proteasome BAX/BAK Inhibitor Nucleus DNA Damage Apoptosome Formation Endoplasmic Reticulum Unfolded Proteins Altered [Ca 2+ ] Apoptosis Caspase Activation

18 The Unfolded Protein Response an ancient stress signaling pathway Current Molecular Medicine, Vol.6 no Figure 1 reprinted with permission from Bentham Science Publishers Ltd.

19 A Closer Look at the IRE1 Life/Death Switch Adaptation Apoptosis Initiator Caspases BH3 Only Proteins BAX/BAK Mito Perm. Effector Caspases cell death Adapted by permission from Macmillan Publishers Ltd., Nature Reviews Molecular Cell Biology Ron D et al 2007, 8: , Copyright 2007.

20 Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Immunology Thome M, et al 2001, 1:50-58, copyright Death Receptor Pathways: Signaling Apoptosis from the Outside (or Trail or TNF)

21 Death Receptor Pathways Fas Pathway: T-cell homeostasis, cytotoxic T-cell activity, tumor cell resistance (upregulation of Fas ligand) Tumor Necrosis Factor-alpha (TNFa): inflammatory processes TRAIL (TNF-related apoptosis-inducing ligand): member of TNF superfamily that binds to DR-4 and DR-5 receptors, which are often upregulated by tumor cells.

22 What happens when stressed out cells refuse to die? Cancer is an imbalance in proliferation vs cell death, resulting in the improper expansion of tumor cells. Ductal Carcinoma of the Breast with Comedo Necrosis Kumar et al Robbins and Cotran Pathologic Basis of Disease 8/E (Fig ) Copyright 2010 by Saunders, an imprint of Elsevier Inc. All rights reserved.

23 The many stresses of cancer cells that tip the balance towards cell death Cell autonomous a. Oncogenic stress b. Increased metabolic and biosynthetic demands c. DNA damage/genomic instability d. Endoplasmic reticulum stress Environmental a. Hypoxia b. Nutrient deprivation c. Loss of integrin-mediated cell-matrix contact d. Changes in microenvironment. e. Iatrogenic (chemotherapy, etc).

24 Many strong oncogenes activate apoptosis a condition termed oncogenic stress Myc can only efficiently induce islet cells tumors if apoptosis is blocked (e.g., BCL-XL overexpression) Reprinted from Cell, Vol.109 no.3: Pelengaris S et al, Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression Copyright 2008 with permission from Elsevier. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology Adhikary C, et al 2005,6: , Copyright 2005

25 Most tumor cells have an increased metabolic demand for glucose.. This increase in glucose metabolism is what allows positron emission tomography with 18 fluorodeoxyglucose (FDG-PET) to function as such a powerful imaging technique to detect cancer PET scan of the head of a 62-year-old man with brain cancer. National Institute of Standards and Technology. NIST Tech Beat. Accessed on Dec 1, 2009.

26 Cancers frequently show genomic gain/loss, which should normally activate DNA damage response pathways to trigger apoptosis. Spectral karyotyping (SKY) of pancreatic carcinoma cell line, illustrating extreme genomic instability. Ghadimi BM, et al Am J Pathol 1999, 154: with permission from the American Society for Investigative Pathology.

27 A buildup of misfolded proteins in the ER lumen, a condition known as ER stress, is common in solid tumors secondary to hypoxia and changes in intracellular ph. Reprinted by permission from Macmillan Publishers Ltd., Szegexdi E et al The EMBO Reports 2006, 7: , Copyright Reprinted with permission from Feldman et al 2005, 3: Figure 3 Copyright 2005 by American Association for Cancer Research

28 Cancer cells disable apoptotic pathways in order to survive. Fas mutations in ALPS. Bax is mutated in 50% of MSI-positive colorectal cancers BCL-2 overexpressed in lymphomas and solid tumors. XIAP and survivin overexpression Caspase-8 mutations in gastric and hepatocellular carcinoma Reprinted by permission from Macmillan Publishers Ltd., Nature Reviews Cancer Fesik S et al 2005, 5: , Copyright 2005.

29 ABT-737: A BH3-mimetic BCL-X L bound to BAD BH3 peptide (green) BCL-X L bound to BH3 mimetic ABT-737 BH3 mimetic Reprinted by permission from Macmillan Publishers Ltd., Nature Reviews Cancer Fesik S et al 2005, 5: , Copyright 2005.

30 Cancer cells are primed to die. Reproduced with permission from the American Society for Clinical Investigation. Moore V et al Journal of Clinical Investigation 117:

31 Anti-tumor activity of ABT-737 in small cell lung carcinoma xenograft mouse model Reprinted by permission from Macmillan Publishers Ltd., Nature Reviews Cancer Fesik S et al 2005, 5: , Copyright 2005.

32 Agents in clinical development that directly target apoptosis. Reprinted from The Lancet, Vol. 9 No.10: ,2008 Call J et al Targeted manipulation of apoptosis in cancer treatment Copyright 2008 with permission from Elsevier.

33 Recombinant Apo2L/TRAIL is in clinical trials as an anti-cancer agent Reprinted by permission from Macmillan Publishers Ltd., Nature Reviews Cancer Ashenazi A et al 2002, 5: , Copyright 2002.